PROGRAM AT A GLANCE
Friday, November 21
7:30-8:30 PM
Keynote address
Salon III,
7th Floor
8:30-9:30 PM
Wine & cheese
reception
Foyer,
7th Floor
Saturday, November 22
9:00 AM- Noon Symposium I: Metabolism and immunity
Salon III,
7th Floor
12:30-1:30 PM
Workshop:Getting
into your inner cell;
flowcytometryof
proteins and mRNA
Lincolnshire I/II,
6th Floor
2:00-4:15 PM
AbstractPresentations
forWorkshops1-9
6th Floor
MeetingRooms
4:15-6:15 PM
PosterPresentations
forUndergradsand
Workshops 1-9 and
Reception
Salon I/II,
7th Floor
Sunday, November 23
9:00 AM- Noon Symposium II: Resident Salon III,
memory
7th Floor
12:30-1:30 PM
LEGENDPlex Workshop
–Newmultiplexassay
fortheSimultaneous
QuantificationofCytokinesandChemokinesbyFlow
Great America
I/II, 6th Floor
12:30-1:30 PM
NIH Workshop–Weath- Lincolnshire I/II,
ertheStorm:Howto
6th Floor
Establish and Sustain
an Independent ResearchCareerinanEra
ofLimitedFunds
12:30-1:30 PM
Workshop:Careers
inImmunology
(Undergraduatesonly)
Salon III, 7th Floor
2:00-4:15 PM
AbstractPresentations
forWorkshops10-19
6th Floor
MeetingRooms
4:15-6:15 PM
PosterforWorkshops
10-19andReception
Salon I/II,
7th Floor
Monday, November 24
8:30-11:30 AM
Symposium III: Immunostimulation
Salon III,
7th Floor
Cover Art
Thecoverartisarepresentationofinflammasomeactivationand
subsequentIL-1βprocessingandrelease.
Don Cohen, Ph.D., Professor, Dept. of Microbiology, Immunology
andMolecularGenetics,UniversityofKentucky.
Program Booklet
DesignandassemblybyCalebShannon.
43rd Annual Autumn
Immunology Conference
Table of Contents
Awards
3
Friday – Keynote Address
5
Saturday – Symposium I
7
and Poster Session
Sunday – Symposium II
11
and Poster session
Monday – Symposium III
15
Saturday Abstracts (1-112)
17
Sunday Abstracts (113-213)
105
Author Index
185
th
Floor Hotel Floor Plan
7th
and Exhibit Floor Plan
1
204
TABLE OF CONTENTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
Notes:
2
AIC 2014 • Chicago, IL
Congratulationstoallawardwinnersatthe2014Autumn
ImmunologyConference
Please be present at the symposium in which your award is
planned to be recognized
FundedbyAbbottLaboratories,AncellCorporation,
Bio-Techne,andanNIHgrantR13-074121.
Shanna Ashley
Jesus Banuelos
AntiaCain-Veal
SebastianCarrasco
Hadijat Makinde
UniversityofMichigan
NorthwesternUniversity
UniversityofFlorida
IndianaUniversity
RushUniversityMedical
Center
GiovannyMartinez- UniversityofMichigan
Colon
Kevin Muite UniversityofChicago
Susan Olalekan RushUniversityMedical
Center
Samantha Simon GeorgiaStateUniversity
NMSS - AIC Travel Award Recipients
KatherineEichinger UniversityofPittsburgh
Marie Bockenstedt IowaStateUniversity
Shannon Rapovy CincinnatiChildren’s
Hospital
EricShifrut WeizmannInstituteof
Science
Recipients
Zachary Benet
SebastianCarrasco
RacquelDomingo-
Gonzalez
StephenGurczynski
Grant Jones
Jared Klarquist
DouglasKline
JonathanKurtz
JeffreyLiu
Nurbek Mambetsariev
Jaclyn McAlees
Rachael Philips
Kathryn Pothoven
Adam Scheid
AldoVacaflores
HaiguangWang
Zuoan Yi
XiaofengZhou
Erin Zook
UniversityofMichigan
IndianaUniversity
UniversityofMichigan
UniversityofMichigan
UniversityofKentucky
UniversityofCincinnati
UniversityofChicago
TulaneUniversity
NorthwesternUniversity
UniversityofIowa
CincinnatiChildren’s
Hospital
MayoClinic
NorthwesternUniversity
MayoClinic
UniversityofIowa
UniversityofMinnesota
UniversityofIowa
UniversityofMichigan
UniversityofChicago
3
Autumn Immunology Conference 2014
Friday, November 21, 2014
4:00 PM
5:00 PM
4:30 - 7:00
7:00 PM
8:00 PM
Keynote Address
7:30 - 8:30
Notes:
©williamburnettphotography.com
4
AIC 2014 • Chicago, IL
Keynote Address
7:30 – 8:30 PM
Salon III, 7th Floor
Sponsored by:
Luke O’Neill
TrinityCollegeDublin
Dr. Luke O’Neill is Professor of Biochemistry and
Immunology at Trinity College of Dublin, Director
of the Trinity Biomedical Sciences Institute, a member of the Royal Irish Academy, and a co-founder
and director of Opsona Therapeutics. Since his pioneering work on IL-1 signaling in the 1980’s, Dr.
O’Neill led the fields of inflammatory diseases and
innate immunity. His enthusiastic and dynamic lectures in immunology are as renowned as his many
scholarly contributions to biomedical research.
The major focus of Dr. O’Neill’s group is to provide
a molecular understanding of innate immunity
and inflammation. He is interested in receptors involved in innate immunity, such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs – including Nlrp3), and also signaling mediators activated
by them, including NF-kappaB, IRF family transcription factors and MAP kinases. Some current
projects include the role of the adapter Mal in the
epithelial barrier in the gut, control of TLRs and
NLRs by microRNAs, the role of Nlrp3 in Type 2 diabetes, novel proteins in the TLR and NLR systems,
the role of Btk in TLR signaling, the control of trafficking of TLR4, genetic variation in innate immune
genes and inflammatory diseases and the role of IL36 in inflammation.
Please join the AIC Organizers in welcoming Luke
O’Neill to Chicago as Keynote Speaker for the 43rd
Autumn Immunology Conference!
8:30 – 9:30 PM
7th Floor Lobby
5
Autumn Immunology Conference 2014
Saturday, November 22, 2014
8:00 AM
Symposium I
Noon
1:00 PM
proteins and mRNA
12:30 - 1:30
2:00 PM
2:00 - 4:15
4:00 PM
Notes:
©williamburnettphotography.com
6
AIC 2014 • Chicago, IL
Symposium I: Metabolism and
immunity
9:00 AM – Noon
Salon III, 7thfloor
ConvenedbyErikaPearce
1.
AjayChawla
UniversityofCalifornia,SanFrancisco
2.
immunity
NavdeepChandel
NorthwesternUniversity,Chicago
JohnWallaceFellowship
NMSS–AICTravelAwards
Award recipients are requested to remain in Salon III during coffee
break for group pictures.
3.
Erika Pearce
WashingtonUniversity,St.Louis
4.
JonathanPowell
JohnsHopkinsUniversity,Baltimore
Preregistration is required for all lunchtime workshops.
and mRNA
12:30 - 1:30 PM
Lincolnshire I/II, 6th Floor
By:eBioscience,AnAffymetrixCompany, PeggyJust,Ph.D.,VicePresidentofResearchand
Development
6thfloorworkshoprooms
2:00 - 4:15 PM
4:15 - 6:15 PM
Salon I/II, 7thfloor
PostersfromWorkshop1-9(abstracts1-87) PostersfromUndergraduates(abstracts88-110)
7
Autumn Immunology Conference 2014
Notes:
©williamburnettphotography.com
8
AIC 2014 • Chicago, IL
2:00 – 4:15 PM
Workshop rooms are on the 6thFloor.
Pleaserefertothe6thFloorHotelFloorPlanneartheendofthis
bookletforroomlocations.
1. Allergy and asthma I
Wisconsin Room
WorkshopChair:IanLewkowich,Ph.D.
2. Autoimmunity
NorthwesternRoom
WorkshopChair:BrianFife,Ph.D.
3. B cell development
Indiana Room
WorkshopChair:KayMedina,Ph.D.
4. Immune response to bacteria and parasites I
Great American I/II Room
WorkshopChair:SarahD’Orazio,Ph.D.
5. Immune response to viruses I
Lincolnshire I/II Room
WorkshopChair:KevinLegge,Ph.D.
Purdue Room
WorkshopChair:EmmaTeixeiro-Pernas,Ph.D.
7. Innate immunity I
Ohio State Room
WorkshopChair:KerryEmprey,Pharm.D.,Ph.D.
8. T cell subsets
Michigan/MichiganStateRoom
WorkshopChair:VladimirBadovinac,Ph.D.
IowaRoom
WorkshopChair:BryonJohnson,Ph.D.
Saturday Poster Session
Postersshouldbepostedbetween8:00-9:00AMonSaturdayandremaininplaceforviewinguntil6:15PMSaturday.
WorkshoppresenterswillbeavailableforposterpresentationanddiscussionduringtheSaturdaypostersession.
ThePosterPresentationscheduleisasfollows:
4:15 - 5:15 PM – Odd numbered posters
5:15 - 6:15 PM – Even numbered posters
PleaserefertothePosterandExhibitFloorplanattheend
ofthisbookletforposterlocations.
Undergraduate Posters
Abstracts90through112arepresentedintheSaturday
afternoonpostersessionaccordingtothesameschedule
asposterpresentationsfromWorkshops1through9.
9
Autumn Immunology Conference 2014
Sunday, November 23, 2014
8:00 AM
Symposium II
Noon
BioLegend
1:00 PM
Careers in
Immunology
the Storm
12:30-1:30
12:30 - 1:30
12:30 - 1:30
2:00 PM
4:00 PM
2:00 - 4:15
Notes:
©williamburnettphotography.com
10
AIC 2014 • Chicago, IL
Symposium II: Resident Memory
9:00 AM – Noon, Salon III, 7th Floor
ConvenedbyDavidMasopust
1.
memory T cells
FrankCarbone
UniversityofMelbourne
2.
memory T cells in mice and humans
Donna Farber
ColumbiaUniversity,NewYork
AAI–AICYoungInvestigatorAwards
UndergraduatePosterCompetition
Award recipients are requested to remain in Salon III during coffee
break for group pictures.
3.
and protect
Susan Kaech
YaleUniversity,NewHaven
4.
David Masopust
UniversityofMinnesota,Minneapolis
Preregistration is required for all lunchtime workshops.
Cytokines and Chemokines by Flow Cytometry
12:30 PM – 1:30 PM
Great America I/II, 6th Floor
Establish and Sustain an Independent Research
12:30 – 1:30 PM
Lincolnshire I/II, 6th Floor
12:30 – 1:30 PM
Salon III, 7th Floor
Sponsoredby:AmericanAssociationof
Immunologists
2:00 – 4:15 PM
Workshops 10 - 19
6thfloorworkshoprooms
4:15 – 6:15 PM
Salon I/II, 7th Floor
PostersfromWorkshops10-19(abstracts113–213)
11
Autumn Immunology Conference 2014
Preregistration is required for all lunchtime workshops.
Cytokines and Chemokines by Flow Cytometry
RebeccaBultema,MS.TechnicalApplications
Specialist
12:30 – 1:30 PM
Great America I/II, 6th Floor
Current multiplex assay technologies are frequently
associatedwithhighreagentand
instrumentcost,inconsistentassayperformance,and
time-consumingdatahandlingprocesses.
LEGENDPlex™ multi-analyte flow bead-based assays
offeramoreeconomicalalternativefor
multiplexassaysofsolublebiomarkers.
Establish and Sustain an Independent Research
LawrenceJ.Prograis,Jr.,M.DSeniorScientist,Special
ProgramsandBioethics,NIH/NIAID
AlisonDeckhutAugustine,Ph.D.
Chief,ImmunoregulationSection,NIH/NIAID
TinaMcIntyre,Ph.D.
ScientificReviewOfficer,NIH/CSR
David Winter, PhD
ScientificReviewOfficer,NIH/CSR
Zhuqing“Charlie”Li,Ph.D.
ScientificReviewOfficer,NIH/NIAID
12:30 – 1:30 PM
Lincolnshire I/II, 6th Floor
Careers in immunology – undergraduate workshop
HeatherA.Bruns,Ph.D.
12:30 PM – 1:30 PM
Salon III, 7th Floor
Sponsoredby:AmericanAssociationof
Immunologists
The Careers in Immunology Workshop is an event
designed for undergraduate students to learn about
scientific career opportunities in immunology from
scientistsworkingindiversesettings.Studentswillbe
abletoaskquestionsandcommunicatewithimmunologistsfromamedicalschool,largeresearchuniversity,asmallcollege,andindustry,aswellasagraduate
studentcurrentlystudyinginanimmunologyprogram.
Undergraduatestudentswillalsohavetheopportunity
tomeetwithparticipatinggraduateprogramrepresentativesandobtaininformationabouttheprograms.
12
AIC 2014 • Chicago, IL
Workshop rooms are on the 6thFloor.
Pleaserefertothe6th Floor Hotel Floor Plan near the end
ofthisbookletforroomlocations.
10. Allergy and asthma II
Wisconsin Room
WorkshopChair:KathrynHulse,Ph.D.
11. Immune response to bacteria and parasites II
Great America I/II Room
WorkshopChair:StevenTempleton,Ph.D.
12. Immune response to bacteria and parasites III
I Lincolnshire I/II Room
WorkshopChair:JerodSkyberg,Ph.D.
13. Immune response to viruses II
Purdue Room
WorkshopChair:RandySacco,Ph.D.
Purdue Room
WorkshopChair:EdithJanssen,Ph.D.
15. Innate immunity II
Ohio State Room
WorkshopChair:DaveFeola,PharmD.,Ph.D.
Illinois Room
WorkshopChair:VirginiaShapiro,Ph.D.
17. T cell signaling
Indiana Room
WorkshopChair:AdamSchrum,Ph.D.
NorthwesternRoom WorkshopChair:RebeccaShilling,M.D.
IowaRoom
WorkshopChair:RyanTeague,Ph.D.
Sunday Poster Session
Postersshouldbepostedbetween8:00-9:00AMon
Saturdayandremaininplaceforviewinguntil6:15PM
Sunday.
WorkshoppresenterswillbeavailableforposterpresentationanddiscussionduringtheSundaypostersession.
ThePosterPresentationscheduleisasfollows:
4:15-5:15 PM – Odd numbered posters
5:15-6:15 PM – Even numbered posters
PleaserefertothePosterandExhibitFloorplanattheend
ofthisbookletforposterlocations.
13
Autumn Immunology Conference 2014
Monday November 24, 2014
8:00 AM
Symposium III
8:30 - 11:30
11:00 AM
Notes:
©williamburnettphotography.com
14
AIC 2014 • Chicago, IL
8:30 AM – 11:30 AM, Salon III, 7thfloor
ConvenedbyPhilippaMarrack
1.
AkikoIwasaki
YaleUniversity,NewHaven
2.
John Kappler
NationalJewishHealth,Denver
3.
Philippa Marrack
NationalJewishHealth,Denver
4.
Tom Mitchell
UniversityofLouisville
15
Autumn Immunology Conference 2014
Notes:
16
1. Allergy and asthma I
1
Erin Zook
Committee on Immunology, Department of Pathology,
Department of Medicine, University of Chicago, Likoping
University, Likoping, Sweden
Natural killer (NK) cells and innate lymphoid cells
(ILCs) are important components of the innate
immune system that act as a first line of defense
against viruses, bacteria and parasites. Despite
their critical functions, the developmental pathways and transcriptional regulatory programs that
control their emergence are just beginning to be
revealed. We previously showed that the transcription factor ETS1 was required for the development
of mature NK cells but not their upstream progenitors (NKP). Here we demonstrate that ETS1 is required for the development of ILC2s but not for the
development of the common progenitor of ILC1, 2,
and 3s (ChILP). In vivo administration of IL33 led
to a blunted accumulation of ILC2s in the lungs of
ETS1-deficient mice as compared to controls. A
small number of ILC2-related progenitors could
be expanded in vitro from ETS1-deficient mice
and these cells expressed genes that are associated
with ILC2s, alternative ILC subsets, and ChILPs. Our
data reveals a possible convergent requirement for
ETS1 in the NK cell and ILC2 lineages for the proper emergence of immature progenitors from their
multipotent or newly specified progenitors.
2
Author’s request:
Do not include my abstract
in the online version of the
abstract book
17
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
ulate IL-33 expression and release from these
marrow derived mast cells from WT and
-GFP mice were primed with αOVA-IgE and
3 IL-33 is rapidly elevated during innate lung
mechanism
Laura Johnston,KrishanChhiba,PaulBryce
Northwestern University, Chicago
Along with IL-25 and TSLP, IL-33 is described as an
epithelial-derived cytokine associated with Th2-biased immunity. While IL-33 likely plays a role in allergy, it is also expressed in non-epithelial cells and
may play a role in innate responses to bacteria.
Consistent with prior work, macrophages and dendritic cells expressed IL-33 upon stimulation with
lipopolysaccharide (LPS). Interestingly, mast cells
expressed IL-33 upon IgE but not LPS stimulation.
Using the LPS-induced model of acute lung injury, IL-33 was rapidly expressed at high levels and
sustained over several days. This contrasted with
several other highly induced cytokines, which resolved within hours. While initial expression was
unaffected, sustained expression of IL-33 was dependent on its receptor, ST2. Using immunohistochemistry for IL-33 protein along with a reporter
mouse in which GFP is expressed on IL-33 transcription, we found the presence of IL-33+GFPcells in the lung parenchyma of naïve mice. Upon
LPS exposure, GFP expression was increased in the
18
endothelium and epithelium, but still not observed
in parenchymal cells. Thus, our data suggests that
IL-33 is present in and expressed by multiple cells
in response to innate bacterial signals. Furthermore, the dependency on ST2 for sustained IL-33
expression implies that IL-33 may act in an autocrine/paracrine manner.
4
dependent Th2-type responses
1
,CaraHrsuch1,NoraBarrett2, Anne
Sperling1
1
University of Chicago, 2Harvard University, Boston
While allergic sensitization can be generated
against various allergens, it is unknown how such
a diversity of antigens is able to promote type 2
helper cell (Th2)-mediated inflammation leading
to atopy. Our previous studies demonstrated that
allergen-specific IgG immune complexes (ICs) and
house dust mite (HDM) extract both induced dendritic cells (DCs) to drive Th2-mediated inflammation, but the mechanism by which these diverse
stimuli produce similar responses are unknown.
Our findings indicated that two distinct Th2 stimuli, ICs and HDM, both utilized FcRγ-associated
receptors, FcγRIII and Dectin-2 respectively, to
promote Th2-mediated lung inflammation. In this
study we demonstrated that both ICs and HDM induced expression of IL-33, a critical mediator in
asthma pathogenesis and the differentiation of Th2
cells, in DCs. Upregulation of IL-33 in DCs was dependent on FcRγ, toll-like receptor 4 (TLR4), and
phosphoinositide 3 (PI3)-kinase. Notably, exogenous IL-33 was sufficient to restore development
of Th2 responses in FcRγ-deficient mice. Finally,
adoptive transfer of allergen-pulsed FcRγ+/- BMDCs restored development of Th2-type inflammation in FcRγ-deficient mice, demonstrating the
necessity of this signaling pathway in DCs for allergen-induced inflammation. These data identify
a mechanism whereby Th2 stimuli signal through
FcRγ-associated receptors on DCs to upregulate IL33 production and induce Th2-mediated allergic
airway inflammation.
19
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
5
SATURDAY ABSTRACTS
wall remodeling
, Scott A. Hoselton, Sumit Ghosh,
GlennP.Dorsam,JaneM.Schuh
North Dakota State University, Fargo
Asthma is a clinical respiratory syndrome that affects more than 25 million individuals in the United States and over 300 million worldwide. Allergic
asthma that is triggered or exacerbated by fungal
exposure often presents a particularly difficult disease course to control that is marked by extensive
eosinophilic inflammation and airway wall fibrosis.
The purpose of the work presented here is to provide tools to begin to elucidate the role of eosinophils in this disease course. Previous work in our
laboratory supports the hypothesis that eosinophil
migration through the extracellular matrix breaks
down large molecules of hyaluronic acid to produce
a smaller immunologically active inflammatory
mediator. Therefore, characterization of the eosinophil is essential to our understanding of this process. Eosinophil differentiation from bone marrow
cells has allowed an in vitro representation that
mimics various pulmonary stimulations for a phenotypic characterization of fungal allergic asthma.
This complements the murine in vivo inhalational
fungal allergic asthma model that we are using to
follow the development of respiratory disease after
repeated waves of eosinophilic inflammation in response to fungal exposure.
endothelial cell transglutaminase 2
Frank Soveg1,HiamAbdala-Valencia1, Jackson
Campbell2,JoanCook-Mills1
1
Northwestern University, Allergy/Immunology Division,
Chicago, 2University of the Pacific, Stockton
Tissue transglutaminase 2 (TG2) is a multifunctional enzyme that is involved in a variety of inflammatory processes. In the context of allergic inflammation, previous research indicates that TG2
is upregulated in human asthma and in the lung
endothelium of OVA-challenged mice. We hypothesized that endothelial cell TG2 was required for
20
allergic inflammation. To test this hypothesis, we
generated mice in which TG2 was specifically deleted in the endothelium using the Cre-Lox recombinase system. To induce allergic inflammation,
mice were sensitized and challenged with OVA.
Compared to wild type mice challenged with OVA,
OVA-challenged knockout mice exhibited a significant reduction in airway resistance, the number
of eosinophils in the BAL, and the number of eosinophils in the airway spaces. Taken together,
these findings indicate that endothelial cell TG2 is
required for allergic inflammation. This suggests
that, during allergic asthma, TG2 regulates the recruitment of eosinophils into the lung. Future studies will focus on identifying mechanisms by which
TG2 regulates signals for leukocyte recruitment
during allergic lung inflammation.
7
-
,SergejsBerdnikovs
Northwestern University Feinberg School of Medicine,
Division of Allergy/Immunology, Chicago
While murine models of asthma are well-established, few studies have contrasted eosinophil
recruitment between lung tissue and the airway.
Moreover, phenotypic differences in eosinophils
between these two compartments are not well
understood. Using a kinetics mouse model of asthma with six consecutive ovalbumin challenges, we
performed multi-panel flow cytometry on lung
tissue digests and bronchoalveolar lavage (BAL)
fluid harvested at either 6 or 24 hours post-challenge. We identified two populations of eosinophils
in the lung tissue, Siglec-FmediumCD11c- and Siglec-FhighCD11clow, while the BAL contained only
one of these populations, Siglec-FhighCD11clow.
Upregulation of Siglec-F and CD11c by eosinophils
recruited to lung tissue became more pronounced
with more challenges and longer harvest times,
which coincided with more dominant eosinophil
presence in the BAL. We observed bimodal expression of cytokines IL-1β, IL-5, IL-33 and chemokines CCL2, CCL5 and CCL24, regulatory for eosinophils. Specifically, we detected two peaks matching
recruitment kinetics: early onset of eosinophil
21
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
recruitment (challenges 1-2) and dominant expansion of the Siglec-FhighCD11clow eosinophil
population in the BAL (challenges 5-6). Upregulation of integrin CD11c and increase in Siglec-F
expression by recruited eosinophils suggests a tissue-specific mechanism involved in targeting these
cells to the mucosal compartment, which merits
further investigation into their function in mucosal
surveillance.
8 The cardiac protein alpha-T-catenin contributes
Stephen Sai Folmsbee,CaraGottardi
Northwestern University, Chicago
10-25% of adult asthma is occupational-induced,
a subtype caused by exposure to irritants in the
workplace. A recent genome-wide association
study (GWAS) identified single nucleotide polymorphisms (SNPs) in the cardiac protein α-T-cat
that correlated with the incidence and severity
of toluene diisocyanate (TDI) occupational asthma. α-T-catenin (α-T-cat) is an essential mediator of cell-cell adhesion and is almost exclusively
expressed in cardiac cells. Interestingly, we have
found that α-T-cat is indeed expressed in lung within the cardiac cell sheath of pulmonary veins (PV).
To examine changes in lung physiology due to α-Tcat KO, we mechanically ventilated the mice using
the Flexivent system. There was no difference in
resistance, compliance, or elasticity in the KO mice
when compared to WT, but KO mice had a significantly increased pressure-volume curve area. To
test α-T-cat’s role in TDI-asthma, we used a murine
model with intranasal sensitization and nebulized
challenge of TDI. From this, TDI-exposed α-T-cat KO
mice show increased airway hyperresponsiveness
by plethysmography when compared to WT mice.
Interestingly, bronchoalveolar lavage revealed only
a mild macrophage-dominant inflammation that
was not significantly different between WT and
KO mice. Based on these data, we suspect cardiac
dysfunction may contribute to asthma through a
mechanism independent of inflammation, specifically due to decreased cardiac cell contractility and
increased airway edema.
22
Anju Peters, Robert Kern, Robert
Schleimer
Northwestern University, Chicago
AERD is the clinical triad of asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and sensitivity to aspirin. AERD patients have more severe
sinus disease than CRSwNP patients, but the mechanisms that account for this are unclear. This study
examined the prevalence of AERD at Northwestern
and investigated whether variations in gene expression profiles could distinguish AERD and CRSwNP.
Electronic health records of patients with AERD
or CRSwNP+Asthma were reviewed. Nasal polyp tissue (NP) was obtained during routine sinus
surgery from patients. NP RNA and protein were
isolated for microarray analysis, RT-PCR, and Luminex bead array. Compared to CRSwNP+Asthma,
patients with AERD underwent significantly more
sinus surgeries (p<0.01) and were more likely to be
dependent on oral corticosteroids (p<0.01). Eosinophil cationic protein in NP from AERD was elevated compared to CRSwNP (p<0.001). There was no
significant difference in type 2 cytokines or CCR3
ligand expression between the groups. Microarray
analyses revealed a differential gene expression
pattern that is under further study. In summary,
AERD appears to be more than a collective sum of
individual diseases but instead is a distinct clinical
process with unique molecular mechanisms.
23
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
2. Autoimmunity
10
signaling-1 (SOCS1) expressions by
lipopolysaccharide (LPS) in a SOCS1
,BhagyaLaxmiSukka-Ganesh,
HowardJohnson,JosephLarkin
University of Florida, Gainesville,FL
Suppressor of Cytokine Signaling-1 (SOCS1) is a
crucial cytokine-induced negative regulator of cytokine signaling. However, little is known about the
modulation and mitigation of SOCS1 under excessive cytokine induced conditions. This study aims
to investigate and characterize SOCS1 expression
in SOCS1+/+ (WT) and SOCS1+/- (HT) mice. We
cultured immune cells from the spleen (SP) and
lymph nodes (LN) of SOCS1 deficient mice under
LPS stimulation and harvested cells at different
time points. SOCS1 gene and protein expression
was subsequently analyzed using Real-time PCR
and Western Blot analysis respectively. Our results
show that SOCS1 protein expression is elevated in
the lymph nodes when compared to spleen. However, this expression was not dose dependent. The
present study aims with the future direction of
investigating different cell types in murine lupus
prone models to suggest the role of SOCS1 regulation under varying stimuli.
11
Author’s request:
Do not include my abstract
in the online version of the
abstract book
l center (GC) B cells
inflammatory chemo-
24
of CCL3/4 in the regulation of humoral immunity
within the GC. Our preliminary data suggest that
tes to
portrican
12 Predominant IL-12 versus IL-23 expression
1, 2
,HeatherGrifka-Walk1, Amanda
Huber3,BenjaminSegal3
1
Graduate Program in Immunology, 2Medical Scientist
Training Program, 3Department of Neurology, University of
Michigan, Ann Arbor
Experimental autoimmune encephalomyelitis
(EAE) is a demyelinating disease of the central nervous system induced by the adoptive transfer of
myelin-specific CD4+ T cells into naïve mice. It is
widely used as an animal model of multiple sclerosis (MS). IL-23-polarized Th17 cells have been
portrayed as the critical effector cells in EAE. However, our recent findings indicate IL-12 and IL-23
may promote EAE through parallel pathways.
IL-12-polarized and IL-23-polarized T cells were
both capable of inducing disease, independent of
IL-23 or IL-12 signaling, respectively. Both forms
of EAE were dependent on monocyte recruitment
by the chemokine CCL2; however, IL-12-polarized
cells induced inflammation dominated by monocytes with higher expression of MHC II and iNOS.
MS patients also fell into distinct subsets based on
immune profiling. Multivariate analysis of sera revealed that patients with relatively high IL-23 or
25
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
IL-12 expression demonstrated divergent cytokine
and chemokine patterns. These data suggest IL-12
and IL-23 may promote distinct forms of immunopathology in MS and EAE. Supported by NINDS,
NIH and Department of Veteran’s Affairs, Veteran’s
Health Administration.
13
Emily Thompson,VaivaVezys
Department of Microbiology, Center for Immunology, University of Minnesota- Twin Cities, Minneapolis
Elucidating the induction, maintenance, and reversal of self-tolerant CD8 T cells is imperative for
understanding the development and progression
of autoimmunity, as well as cancer. We have developed a mouse model that allows us to evaluate
endogenous polyclonal self-specific T cells, where
self-Ag expression results in tolerance induction.
However, this established CD8 T cell tolerance can
be reversed with repeated antigen stimulation to
generate large numbers of activated, functional
endogenous self-specific CD8 T cells. One reason
we postulate multiple rounds of antigen are necessary for breaking self-specific tolerance is that the
membrane composition of self-specific CD8 T cells
is different than that of nonself-specific cells which
could impact ability to bind and respond to cognate
antigen. We therefore hypothesized that self-specific CD8 T cells have an altered membrane composition compared to nonself-specific CD8 T cells.
Our confocal analyses have shown that self-specific
CD8 T cells exhibit a heterogeneous pattern of tetramer binding that is different than that observed
in nonself-specific cells. Furthermore, CD8 does
not co-localize completely with tetramer binding in
self-specific CD8 T cells, which suggests that binding to cognate antigen could be impacted. Future
studies will dissect whether the membrane composition of self-specific CD8 T cells changes through
out repeated antigen stimulation and if so what factors contribute to this process.
14
Author’s request:
2
Do 1not
include
my1, abstract
, Kristen
Pauken
,JustinSpanier1,
in the
online version
of the 1
1
1
JamesHeffernan
, Nathanael
abstract
book Sahli ,BrianFife
26
Programed Death (PD)-1 and its interaction with
PD-ligand 1 (PD-L1) is important for tolerance
15 Leukocyte Associated Immunoglobulin-like
Receptor 1 inhibits Th1 but enhances Th17
responses
1
, Melissa Keller2, Lynn Haynes1,Ewa
Gan1, Jeremy Sullivan1,WilliamBurlingham1
1
Department of Surgery/Transplant Division, School of
Medicine and Public Health, University of Wisconsin,
Madison, 2School of Medicine, Tulane University, New
Orleans
Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR1), is a trans-membrane receptor
expressed by multiple cells of the immune system.
It contains two immunoreceptor tyrosine-based inhibitory motifs (ITIM’s) in its cytoplasmic tail, making it an inhibitory receptor. Crosslinking of LAIR1
on cells is known to inhibit cellular functions of NK
(Natural Killer) cells and Cytotoxic T cells. Recently,
LAIR1 knock out (KO) mice were developed, which
we used in a minor mismatch model of heterotrophic heart transplantation. Results demonstrated
27
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
that the LAIR1 KO mice had less cellular infiltration and, vasculopathy as compared to wild type
(WT) mice. The T helper 17/Interleukin 17 (Th17/
IL17) dependent, immune response against Collagen Type V was also lower in KO mice as compared
with, WT mice, with no significant differences in
IL4 or Interferon γ production. In order to confirm
these results we utilized a trans-vivo delayed type
hypersensitivity assay that also revealed opposing
effects of LAIR1 on Th1 (Tetanus) vs Th17 (Col V or
Col V peptide) responses. Ongoing experiments in
the lab are designed to determine the mechanism
by which the discrepancy between Th1 and Th17
responses occur.
Farah R. Itani1, 2, Sushmita Sinha2,LeciaEpping3, John
T.Harty1, 2, 3,NitinJ.Karandikar1, 2
1
Interdisciplinary Graduate Program in Immunology,
Department of Pathology, 3Department of Microbiology,
University of Iowa, Iowa City
2
Multiple sclerosis (MS), a debilitating immune-mediated demyelinating disease of the central nervous
system (CNS), afflicts ~500,000 individuals in the
US. Experimental autoimmune encephalitis (EAE)
is a murine model of MS, induced by immunization
of mice with myelin-related antigens. Our lab has
previously demonstrated a novel and unexpected
regulatory role for CNS-specific CD8 T cells in both
MS and EAE. Transfer of “autoregulatory” CD8 T
cells resulted in attenuation of clinical disease in
immunization-induced EAE as well as adoptively transferred EAE using pathogenic CD4 effector
T cells. In the models thus far, CNS-specific CD8 T
cell responses are induced by active immunization
with exogenous antigens. We have now engineered
recombinant strains of Listeria monocytogenes to
encode myelin peptides, thus allowing endogenous
processing and presentation of peptides following
infection. We have confirmed the generation of
peptide-specific CD8 T cells seven days post infection. Interestingly, preliminary data suggests that
these endogenously generated neuroantigen-specific CD8 T cell responses are regulatory in nature.
28
The cellular and molecular interactions that determine their regulatory potential are currently under
investigation.
17
Susan Olalekan,YanxiaCao,AlisonFinnegan
Rush University Medical Center, Chicago
Autoimmunity is often associated with a deficiency in functional CD4+CD25+Foxp3+ T regulatory
cells (Tregs). In our mouse model of rheumatoid
arthritis (RA), proteoglycan induced arthritis
(PGIA); we have shown that B cell depletion using
a monoclonal antibody against CD20 suppresses
disease along with an increase in functional Tregs.
In an effort to enhance these Tregs we combined
anti-mCD20 with administration of IL-2/anti-IL-2
mAb complexes and investigated the effects of the
combination on PGIA. We show in G1 primed mice
that IL-2/anti-IL-2 mAb complexes increase the
percentage and numbers of Tregs higher than anti-mCD20 or control ab. To determine the effects of
the complex on arthritis, we induced PGIA in BALB/c mice. Mice treated with the complex displayed
an accelerated severe disease as compared to B cell
depleted or control Ab treated mice. This was likely
due to an early increase in antigen specific CD4+T
cells. Mice that received anti-mCD20 together with
IL-2/anti-IL-2 mAb however displayed a less severe disease as compared to the group that only
received the complex. Anti-mCD20 treated mice
displayed the least arthritic severity. To determine
the suppressive capacity of Tregs from the different treatment groups we setup Treg suppression
assays. Tregs isolated from B cell depleted mice
displayed the highest suppressive capacity. Our
findings demonstrate that B cell depletion generates effective Tregs and ameliorates PGIA better
than IL-2/anti-IL-2 mAb complexes.
18
1
,JevgenijsLusciks2, Hubert
Dolubizno2, Janet Zayas2, Frederick Kohlhapp2,
AndrewZloza2, 3
1
Departments of General Surgery,
29
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
2
SATURDAY ABSTRACTS
Immunology/Microbiology, 3Internal Medicine, Rush
University Medical Center, Chicago
Background: We have discovered that influenza
infection results in decreased anti-tumor/self antigen CD8+ T cell responses in the tumor microevironment. This is in part due to the migration of
anti-self CD8+ T cells to the site of the infection
(i.e., lungs). Based on this finding, we investigated
the ability of influenza infection to decrease autoimmunity in a CD8+ T cell-driven model of autoimmune diabetes. Methods: NOD mice were treated
via influenza infection (A/PR/8, 8,000 EID50 in 40
µl via intranasal injection) starting at week 10 of
life, and repeated at least twice thereafter weekly.
Mouse weights and blood glucose were recorded
at least weekly until animals became diabetic. Results: Influenza infection decreased autoimmune
diabetes formation in NOD mice. Specifically eight
weeks after the initiation of infections, only 25%
of mice that were infected with influenza developed diabetes (average blood glucose, 198 mg/dL),
while 75% of uninfected mice developed diabetes
(average blood glucose, 348 mg/dL). Conclusions:
These striking findings suggest that viral infections
may affect anti-self antigen CD8+ T cell responses
and that therapies utilizing viral infections may
have a role in the treatment of autoimmune diabetes. Ultimately, we aim to understand the mechanisms involved in these findings and to test the
general applicability of various infections agents to
autoimmunity control.
in bullous pemphigoid
Samantha Aust1,KellyMessingham1, Janet Fairley1, 2
1
University of Iowa, Iowa City, 2VA Medical Center, Iowa
City
Bullous pemphigoid (BP) is an autoimmune
disease targeting epidermal attachment proteins
resulting in inflammation and blistering of the
skin. Interestingly, BP manifests exclusively in
the skin, despite the fact that the target antigen
is expressed in many other tissues. Studies
suggest that responses to epidermal antigens are
generated largely within the skin itself through
antigen presenting cell (APC)-mediated activation
of regulatory T cells (Treg). It is our hypothesis
30
that differences in DC subset, frequency and/or
function play a role in the loss of tolerance to skin
antigens in BP. To address this, we evaluated the
number of APCs and Treg in BP vs. control skin.
Skin biopsies were cryosectioned and stained
with fluorescent antibodies specific for APC’s and
T cells, then visualized using confocal microscopy.
Staining for MHC class II and langerin revealed
a decrease in the overall number of APC and
Langerhans cells in BP skin, compared to control.
The average percent area of MHC class II positive
staining was 1.15% in control skin and .269% in BP
skin. The average percent area of langerin positive
staining was .427% in control skin and .0246% in
BP skin. In addition, a decreased number of Treg
(CD3+FoxP3+) was also observed in BP skin. The
average percent area of FOXP3 positive staining
was .316% in control skin, and .022% in BP skin.
These studies suggest alterations in tolerance
mechanisms within the skin may contribute to the
tissue specific immune response in BP.
31
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
3. B cell development
20
by adipocytes and myeloid-derived
suppressor cells
,KatherineLKnight
Loyola University Chicago Microbiology and Immunology,
Maywood
B lymphopoiesis declines with age in humans, mice,
and rabbits. In rabbits, loss of B lymphopoiesis occurs as early as two months of age and correlates
with an increase in adipose tissue in the bone marrow (BM). Increased adipose tissue has also been
observed in the BM of aged humans and mice. We
showed previously that adipocyte-derived factors inhibit B lymphopoiesis in vitro; however,
the mechanism by which this occurs is unknown.
Through co-cultures of mouse BM cells with OP9
stromal cells, we found that adipocyte conditioned
medium (ACM) induces the generation or expansion of CD11b+ Gr1+ myeloid cells. Further, we
found that these cells inhibit B cell development in
vitro. The ACM-induced CD11b+ Gr1+ cells express
arginase (Arg1) and iNos (Nos2), and functionally
suppress CD4+ T cell proliferation, suggesting that
these cells are myeloid-derived suppressor cells
(MDSCs). Examination of BM from rabbits greater
than two months-of-age revealed large fat depots
and high numbers of MDSC-like arginase-expressing cells. These findings lead us to suggest that the
decline in B lymphopoiesis in BM is due to adipocyte-induced MDSCs which in turn, secrete soluble
molecules that inhibit B lymphopoiesis.
21 EZH2 in B lymphocyte commitment
1
,JenniferWoodard1, Barbara
Kee1, 2
1
Committee of Immunology, 2Department of Pathology,
University of Chicago
EZH2 is the histone methyltransferase subunit of
the Polycomb Repressive Complex 2 that catalyzes
the repressive histone modification, H3K27me3.
Loss of EZH2 in hematopoietic cells leads to blocks
in B and T cell development (Su 2003, Su 2005).
Here we use an Il7racre mouse model to delete Ezh2
32
at the common lymphoid progenitor (CLP) stage
to study the role of EZH2 in B lymphocyte commitment. We hypothesize that EZH2 is required
to repress alternative lineages during B cell development. Similar to previously reported models, we
confirm that Il7racreEzh2fl/fl mice have a cell-intrinsic block at the pro-B cell stage of development.
Although there are normal numbers of pro-B cells
in Il7racreEzh2fl/fl mice, CLPs and pro-B cells from
these mice fail to expand in vitro. Gene expression
studies reveal that genes upregulated in Ezh2-deficient pro-B cells are enriched for hematopoietic
stem cell genes. By qPCR we find that there is minimal upregulation of Gata3 and Nfil3, which regulate innate lymphocyte development. Future studies will investigate the developmental plasticity of
Ezh2-deficient pro-B cells.
22
Sophiya Karki, SarahPowers,MarcusClark
University of Chicago, Chicago
During B-cell development, cells capable of rearranging and expressing the immunoglobulin heavy
(Igμ; pro-B cells) and then the light chain (Igκ; small
pre-B cells) genes of the B-cell receptor are clonally expanded. In order to preserve their genomic
integrity, cells that have successfully rearranged
their heavy chain first undergo clonal expansion,
before undergoing light chain rearrangement, attained by coordinating signals from the IL-7R and
the pre-BCR, respectively. Our lab has shown that
during clonal expansion, STAT5, downstream of
the IL-7R, while targeting the expression of cyclin
proteins also recruits the histone methyl-transferase, EZH2, at Eκi that directly represses the Jκ-Cκ
region. However, STAT5 mediated repression does
not apply to the 2.8 Mb variable (Vκ) region, which
is also devoid of other canonical repressive histone
marks. Previous microarray results from Ccnd3/and WT pro-B cells suggest that cyclin D3 may
have a role in Vκ repression. Among ~200 genes
that were up-regulated in Ccnd3-/-pro-B cells, multiple Vκ gene segments, but not Jκ-Cκ, increased by
3-8 fold. Interestingly, Vκ repression was found to
be mediated by a large fraction of cyclin D3 that
33
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
bind the nuclear matrix (NM-D3). Based on preliminary 3D-FISH studies, we hypothesize that the
absence of NM-D3 in Ccnd3-/- pro-B or WT small
pre-B makes Vκ gene segments more accessible to
the transcription and recombination machineries,
which may also contribute to Vκ usage and allelic
exclusion.
23
mice
1
, Kimberly Gwin1, Fan-Chi Hsu1, 2,
VirginiaShapiro1,KayL.Medina1
1
Mayo Clinic College of Medicine, Department of
Immunology, 2Mayo Graduate School, Rochester, MN
Flt3-ligand deficient (FL-/-) mice have reduced
bone marrow (BM) B cell output and reductions
in total splenic B cells. It is unclear if reductions in
splenic B cells in these mice are related to reduced
BM immature B cell output, or due to a Flt3-dependent component in the splenic microenvironment.
To examine, we compared the peripheral B cell
compartments in WT and FL-/- mice and found significant reductions in numbers of transitional (TS)
and follicular (FO), but not marginal zone (MZ) B
cells. To determine if immature B cells were being
diverted into the MZ compartment, we evaluated
WT and FL-/- mice crossed to a RAG1-GFP reporter. There was no enrichment of GFP+ cells within
the MZ. Mixed radiation chimeras together with FL
replacement therapy established that reductions
in TS and FO B cells were cell extrinsic. Importantly, normalization of the TS and FO B cell compartments was accompanied by restoration of BM
immature B cells. In addition to B cell progenitors,
dendritic cells in BM and spleen are dependent on
FL and provide cytokines important for peripheral
B cell biology. DCs were restored in the radiation
chimeras and with FL replacement. To address if
cDC deficiency contributed to reductions in TS and
FO B cells in FL-/- mice, we performed depletion
experiments in CD11c-DTR mice. Loss of splenic
cDCs had no impact on TS or FO B cells. Taken together, these data show that numbers of TS and FO
B cells are directly impacted by output of immature
B cells from the BM.
34
AIC 2014 • Chicago, IL
Jackson Turner,IrinaGrigorova
University of Michigan, Ann Arbor
A hallmark of the T cell dependent humoral immune response is increased affinity for antigen,
which is accomplished by selective expansion of
higher-affinity B cell clones in germinal centers
(GCs). Recent studies have suggested that competition for T cell help among GC B cells drives their
selection. However, whether BCR signaling could
act as an additional selection mechanism remains
unknown. To address this question, GC B cells were
exposed to antigen that provided BCR signaling but
no additional peptides to present for T cell help.
This antigen did not promote GC B cells’ survival
or effector differentiation, whereas antigen that
allowed for both additional BCR signaling and T
cell help did. These results are consistent with the
hypothesis that competition for T cell help is the
mechanism by which GC B cells are selected.
TRAF3 inhibits CREB-mediated survival
25 Nuclear
Author’s request:
and metabolic reprogramming in B cells
Do not include
1, 3
1 3
, Linmy
Waiabstract
, ,BrettHanson1,
Nurbek Mambetsariev
in
the online 1version of the
1, 5
1, 2, 3, 4, 5
LauraStunzabstract
, Tina Arkee
book, Gail Bishop
1
Department of Microbiology, 2Internal Medicine, 3Immunology Graduate Program, 4Holden Comprehensive Cancer
35
SATURDAY ABSTRACTS
24
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
tion. The human TRAF3 mutant LP1 isolated from
a myeloma tumor and lacking a TRAF-C domain,
failed to localize to the nucleus or associate with
CR
elluthe
ges
Epstein-Barr virus Latent Membrane Protein
2A (LMP2A) increases Fas expression and
Ryan Incrocci1, Samira Hussain2, Michelle
Swanson-Mungerson1
1
Chicago College of Osteopathic Medicine, 2College of
Health Sciences, Midwestern University, Downers Grove, IL
Epstein-Barr virus Latent Membrane Protein 2A
(LMP2A) is expressed in EBV-infected B cells in
the germinal center, a site of significant apoptosis
induced by engagement of Fas on activated B cells.
Previous studies have shown that B cell receptor
cross-linking can protect B cells from Fas-mediated
apoptosis and since LMP2A acts as a BCR mimic,
we hypothesized that LMP2A would protect B cells
from Fas-mediated apoptosis. The A20 murine B
cell lymphoma line was retrovirally transduced
to generate cell lines that stably express the empty VECTOR backbone, wildtype LMP2A, or LMP2A with a mutation in the Lyn binding site or the
ITAM motif that binds Syk. Cells were exposed to
a Fas-specific monoclonal antibody for four hours
and apoptosis was assessed by increases in annexin V staining and PARP cleavage. Surprisingly,
LMP2A-expressing B cell lines demonstrated an
increased sensitivity to Fas-mediated apoptosis,
due to an LMP2A-dependent enhancement in Fas
expression. Additionally, results using the LMP2A
cell lines with mutations in Lyn and Syk binding
sites showed that activation of both of these protein kinases are required for the LMP2A-dependent increases in Fas expression and sensitivity to
Fas-mediated apoptosis. These findings have impli-
36
cations for EBV latency as well as the treatment of
EBV-associated malignancies.
27
B cells
Rosemary Morman,StaciePoston,Elizabeth
Taparowsky
Department of Biological Sciences and Purdue
University Center for Cancer Research, Purdue University,
West Lafayette
BATF, an immune specific AP-1 transcription factor,
functions together with IRF4 and JUNB to play an
important role in the B cell process of class switch
recombination (CSR). Previous work has shown
that transcription complexes containing BATF mediate both the activation and repression of gene
expression. To further investigate the role of BATF
as an inducer of a B cell gene expression program
that leads to the production of class switched Abs,
RNA-Seq was performed and the profiles of gene
expression from BatfΔZ/ΔZ (KO) and WT mouse B
cells compared. We established that the induction
of BATF in B cells following treatment with LPS
and IL-4, but prior to CSR, occurs at 6 hours, and
used RNA from that time point for the comparison.
Bioinformatic analysis of the data obtained identified 47 genes which were either over-expressed
or under-expressed in the BatfΔZ/ΔZ B cells. Many
of these genes have been previously implicated in
B cell biology; others have not. Experiments to investigate how the products of these genes function
within a BATF-dependent network to regulate B
cell growth and the steps leading to CSR and antibody production are underway.
28
individuals
Allison Nipper,BasileSiewe,AlanLanday
Rush University Medical Center, Chicago
Although it has been well established aged individuals exhibit an impaired humoral response, few
studies have focused on basic characterization of
B cells of aged subjects. We hypothesize that the
impaired humoral response of aged individuals is
multifactorial and can be attributed to dysregulation in multiple aspects of B-cell signaling, specifi-
37
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
cally at the levels of the Toll-like receptors (TLRs),
inhibitory and co-stimulatory molecules, and perturbations in B-cell subsets. Thus, we performed
a comprehensive characterization of peripheral
B cells in young and aged subjects to identify potential factors contributing to the dysregulation of
B-cell response with age. Our results identify significant decreases in the frequency of total B cells
and resting memory B-cells of aged subjects. Additionally, dysregulated expression of multiple inhibitory receptors was seen in aged subjects. Finally,
examination of TLR expression revealed TLR4 and
TLR5 were significantly decreased in aged subjects.
Thus, we present novel data suggesting factors underlying attenuated B-cell responses with age are
multifactorial, and include significantly decreased
expression of TLRs, dysregulated expression of
molecules inhibiting BCR signaling, and perturbation of total B cells and B-cell subsets.
malignant B-1 cells
Sara Alhakeem1,KatieMcKenna1, Beth Gachuki1,
Natarajan Muthusamy2, John Byrd2,
Subbarao Bondada1
1
Dept of Microbiology, Immunology and Molecular Genetics, Markey Cancer Center, University of Kentucky, Lexington, 2Dept of Internal Medicine and Comprehensive Cancer
Center, Ohio State University, Columbus
The most common human leukemia is a B-cell
chronic lymphocytic leukemia (B-CLL), which is
characterized by the proliferation and accumulation of surface IgM+/CD5+ B-cells (B-1 cells). EμTcl1 mice, which express the Tcl1 oncogene in a B
cell specific manner, spontaneously develop B-CLL.
We show that Eμ-Tcl1 mouse B-CLL cells produce
Interleukin-I0 (IL-10) constitutively, which is further increased upon stimulation with LPS or anti-CD40. This study describes a novel role of B cell
receptor (BCR) signaling pathway in constitutive
IL-10 secretion by normal and malignant B-1 cells.
We found that inhibition of Src or Syk family kinases reduces the constitutive IL-10 production
in a dose dependent manner by both normal and
malignant B-1 cells. In addition, we found that EμTcl1 CLL cells exhibit clonal variation in their IL-10
38
production in response to BCR cross-linking. Further studies are being performed to understand the
mechanisms by which BCR signaling affects IL-10
production. (Supported in part by NIH grants and
Edward P. Evans Foundation)
30
nanovaccines
1
, Jonathan Goodman1,JuliaVela
Ramirez1, Rajarshi Roychoudhury2, Nicola Pohl2,
Michael Wannemuehler1
1
Iowa State University, Ames, 2Indiana University,
Bloomington
There is a need to develop improved adjuvants or
vaccine delivery vehicles that induce long-lived,
high titer and high avidity IgG responses to recombinant proteins. It is known that TLR ligands
modulate IgG responses but there is little information regarding the effect of ligands targeting
C-type lectin receptors (CLRs) to modulate B cell
or IgG responses. Previous data from our lab indicated that polyanhydride nanoparticles modified
with di-mannose, a CLR ligand, activated APCs in
vitro. In this study, BALB/c and C57BL/6 mice were
immunized with either di-mannose-modified or
non-functionalized (NF) nanovaccines containing
ovalbumin (Ova). The nanovaccine formulations
consisted 90 µg of soluble Ova and 10 µg of Ova
loaded (2 % w/w) into 20:80 CPTEG:CPH nanoparticles. Serum was collected bi-weekly for 10 weeks
post-immunization and the anti-Ova IgG responses were characterized. The data indicated that the
di-mannose-modified nanovaccine induced higher
anti-Ova IgG1 than was induced by the NF nanovaccine. There was no difference in the anti-Ova IgG1
responses measured in the C57BL/6 mice. The
di-mannose-modified nanovaccine induced higher
IgG1 anti-Ova titers in BALB/c mice compared to
C57BL/6 mice.
39
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
4. Immune response to bacteria and
parasites I
31
Borreliosis
Carrie Lasky,RachelOlson,CharlesBrown
University of Missouri, Columbia
Infection of susceptible mouse strains with the
causative agent of Lyme disease, Borrelia burgdorferi, reliably produces an infectious arthritis
and carditis that peaks and spontaneously resolves
within 60 days. The exact mechanisms that drive
the development and resolution of inflammation
during infection with B. burgdorferi are not well
understood. Macrophage polarization has been
suggested to drive inflammation, the clearance of
bacteria, and tissue repair and resolution in a variety of infectious models. During Lyme disease it is
well understood that macrophages are capable of
clearing Borrelia spirochetes as well as exhausted
neutrophils. However, to our knowledge macrophage phenotype has not been analyzed in a murine model of Lyme disease. Using flow cytometry,
we show there are significantly more macrophages
in both the joints and hearts of infected C3H/HeJ
mice, as compared with uninfected controls. Using
classical (M1) and alternative (M2) specific markers, we set out to describe the phenotype of macrophages throughout the infection time course.
We found that throughout the infection M2 macrophages dominate, but M1 macrophage numbers
become elevated during the peak of inflammation. Interestingly, we see a significant increase of
resolution phase macrophages (rM) that possess
characteristics of both M1 and M2 macrophages
once both inflammation and spirochetes have been
cleared.
32
Author’s request:
Borrelia burgdorferi
phagocytosis
Do not include my abstract
in the online
version of the
1
, Bryan Troxell2,YouyunYang1,
abstract book
Stephanie Brandt1,HenriqueSerezani1,FrankYang1
Carolina State University, Raleigh
40
Lyme disease, the most common vector-borne illness in the United States, is a multisystem inflam-
lls showed
33
Author’s request:
Do not include my abstract
in the online version of the
Jill Ippolito,MaryBrown,LuisRamirez,Elizabeth
abstract book
Kovacs
Alcohol intoxication is involved in 50% of all burn
injuries, resulting in an increased risk of lung in-
,
-
41
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
tion. Mice were given ethanol 30 minutes prior
to a 15% total body surface area dorsal scald injury, followed by an i.t. of 2,000 CFUs of PA. At 24
hrs
ned
JK),
].
34 CXCR2 is an important mediator
Carolyn Lacey,LaurenKeleher,CharlesBrown,Jerod
Skyberg
University of Missouri Veterinary Pathobiology, Columbia
Brucella spp. are facultative intracellular bacteria
that cause brucellosis. This bacteria may be employed as a biological weapon infecting humans and
a wide range of agricultural species. Brucellosis is
chronic in some individuals and displays manifestations such as endocarditis and osteoarticular inflammation. It has been found that IFN-γ-deficient
mice infected with Brucella melitensisdisplay arthritis and swelling in joints similar to human brucellosis. The purpose of this study is to understand
the primary mediators of joint inflammation. Here,
we report Brucella-induced arthritis is not initiated
through adaptive responses. Rag1-/- mice, deficient
in B and T cells, along with µMT-/- mice, deficient
in B cells, developed inflammation at the same
rate and severity as wild type (WT) animals. Mice
infected with B. melitensis and depleted of IFN-γ
42
displayed elevated levels of neutrophils and macrophages in their joints. Also, joints from B. melitensis infected IFN-γ-/- mice expressed increased
levels of the CXCR2 ligands, MIP-2 and KC and the
CCR2 ligand MCP-1. CXCR2-/- mice had delayed onset of inflammation and did not develop severe
focal swelling as compared to IFN-γ depleted WT
mice. In contrast, CCR2-/- mice did not show a reduction in inflammation or incidence. Collectively,
these results demonstrate CXCR2 is necessary for
a complete innate response in the joints and targets of CXCR2 could be used as potential therapies
when treating chronic articular brucellosis.
35
(MHC) class II alleles in rhesus
macaques
Trey Gilpin1,CourtneyDow1, Patrick Bohn2, Julie
Karl2,RogerWiseman2, Bianca Mothe1
1
California State University, San Marcos, 2Wisconsin
National Primate Research Center, University of
Wisconsin-Madison
One leading cause of death from infectious disease
is Tuberculosis (TB) due to the pathogen Mycobacterium tuberculosis (MTB). One third of all humans
(~2 billion people) are infected with latent MTB,
resulting in 2 million deaths, and 9 million reported new infections annually. The Bacille Calmette
Guérin (BCG) vaccine is currently the only licensed
TB vaccine, but protection against TB is incomplete and variable. In regions common with HIV/
TB co-infection, the vaccine is useless as immuno-compromised individuals are unable to mount
the proper immune response upon vaccination. It
is clear more preventative and effective measures
are crucial, making new vaccine research extremely important. To address this issue, we evaluated
the Major Histocompatibility Complex (MHC) class
II genes present in a cohort of 37[wa1] rhesus macaques by deep sequencing. The most common DRB
haplotype included the alleles Mamu-DRB1*03:03
and Mamu-DRB1*10:07 and was expressed by 43%
of our cohort. Likewise, the most common DQ haplotype included Mamu-DQA1*26:01 and MamuDQB*18:01 alleles which were expressed by 49%
of this cohort. The identification of these alleles
will provide a platform for the characterization of
43
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
natural or vaccine-induced immune responses.
SATURDAY ABSTRACTS
induces a robust immune response to the
Pneumocystis
murina
Heather Evans, Beth Garvy
University of Kentucky, Department of Microbiology,
Immunology, and Molecular Genetics, Lexington
Pneumocystis species are opportunistic fungal
pathogens that cause severe pneumonia in immunocompromised hosts. The life cycle of Pneumocystis species consists of single-nucleated trophozoites
and ascus-like cysts. We demonstrate that infection
of adult or neonatal mice with both life forms elicits
a more robust lymphocyte response than infection
with trophozoites alone. Infection of neonates with
trophozoites alone leads to proliferation of lung
resident antigen-presenting cells, without infiltration of non-resident cells during the first three
weeks post-infection. Bone marrow-derived dendritic cells (BMDCs) stimulated with mixed P. murina express more IL-1β and induce greater CD4+
T cell interferon-γ production than cells stimulated
with trophozoites alone. We demonstrate that the
addition of trophozoites suppresses IL-1β production by BMDCs in response to the β-1,3-glucan
curdlan. β-1,3-glucan is a predominant structural
component in the cyst wall, but is not produced by
trophozoites. We propose that dendritic cells stimulated by cysts direct a robust pro-inflammatory
response, while trophozoites suppress β-1,3-glucan-induced cytokine production.
37 Cryptoccocus neoformans
response in murine host
Ana Pamela Torres Ocampo,YafengQiu,Antoni
Malachoswki,WoosungCho,EnzeXing,Alison
Eastman,MichalOlszweski
Division of Pulmonary and Critical Care Medicine,
Department of Internal Medicine University of Michigan
Medical School & VA Ann Arbor Health System, Research
Service, Ann Arbor
C. neoformans is the major fungal opportunistic
pathogen worldwide. Successful clearance of cryp-
44
tococcosis relies on Th1 immune response, defined
by high expression of IFN-γ, TNF-α, IL-12 and classical activation of macrophages. This contrasts
with non-protective Th2 response defined by high
expression of IL-10, IL-4 and lung eosinophilia. C.
neoformans cultures grown to late logarithmic
phase are typically used for experimental mouse
infections. Less is known about properties of cultures in stationary phase and their effects on the
immune response are unknown. We hypothesize
the virulence of C. neoformans from different stage
of growth cultures would be different because of
differential modulation of the immune response.
Mice were infected with 104 cells at late-log or stationary phase of growth. Fungal loads in lung and
brains, inflammatory cells characteristics, survival
rate and cytokine expression were evaluated weekly for three weeks. Mice were infected with late-log
phase cultures showed 100% mortality in 45 days
and mice Infected with stationary cultures survived longer (>70 days) and showed minimal fungal burden. The profiles of cytokines were different
and lung eosinophilia was present only in the mice
infected with late-log growth cultures. Our preliminary data suggests that the changes in C. neoformans properties between logarithmic and stationary phase decreases virulence by promoting more
protective Th1 polarization in the infected host.
38 C. neoformans lysosome damage supports
virulence
3, 4
, Michael Davis1, 2, Lori Neal1, 4, Ali-
son Eastman1, 3,YafengQiu1, 4, 5,MichalOlszewski1, 3, 4
1
Division of Pulmonary & Critical Care Medicine, University
of Michigan Health System, Ann Arbor, 2National Institute
for Allergy and Infectious disease Laboratory of Clinical
Infectious Disease, Bethesda, 3University of Michigan,
Ann Arbor, 4VA Ann Arbor Healthcare System, Ann Arbor,
5
Shanghai Veterinary Research Institute, Chinese
Agricultural Academy of Science, Shanghai, China
Cryptococcus neoformans is an opportunistic fungal pathogen that has been shown to survive and
replicate within lysosomes in macrophages. Previous studies have reported that C. neoformans
induces lysosome damage in macrophages, which
we hypothesized may contribute to fungal growth.
Our lab has developed new microscopy methods
to quantitatively measure lysosome damage by C.
45
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
neoformans in bone marrow-derived macrophages.
The magnitude of lysosome damage correlated
with increased cryptococcal growth. Experimentally induced lysosome damaged also increased fungal growth. Activation of macrophages with IFN-γ
limited lysosome damage and enabled killing of
C. neoformans within the cells. We also measured
lysosome damage in vivo by flow cytometry. Increased lysosome damage was found in lung cells
containing C. neoformans compared to non-infected cells. We observed that recently recruited myeloid cells displayed lower damage than resident
myeloid cells, consistent with a protective role for
recently recruited macrophages in containing fungal growth in the lung. We conclude that crytococcal induced lysosome damage is crucial for fungal
growth and classical activation of macrophages
inhibits damage to limit fungal replication and promote host protection.
Notch signaling contributes to pulmonary
Lori M. Neal1, 2,YafengQui1, 2,JoohoChung1, Woo
SungCho1, 2, Ivan Maillard1,MichalA.Olszewski1, 2
1
University of Michigan, Ann Arbor, 2Veterans Affairs Ann
Arbor Healthcare System, Ann Arbor, MI
Cryptoccocus neoformans is a major opportunistic
fungal pathogen prevalent in hosts with impaired T
cell-mediated immunity. Optimal defenses against C.
neoformans depend on T-cell Th1/Th17 polarization
that promotes fungicidal pathways in leukocytes recruited into the lungs. Notch is a key conserved signaling pathway in T cell development but the role
of Notch signaling in T cells function during fungal
infections is unknown. To examine the role of Notch
signaling during C. neoformans infection we used
mice with dominant negative MAML1 in T cells (DNMAML). Equivalent fungal burdens were found in
the lungs, brains, and spleens of DNMAML and WT
mice at 3 wks but at 6 wks post infection, DNMAML
mice had 50 fold greater fungal burdens in the lungs
and brains and 10 fold greater burdens in the spleen.
Interestingly, there was no difference in lymphocyte
accumulation. However, inhibition of Notch signaling in DNMAML mice resulted in decreased pro-
46
duction of Th1 cytokine IFN-γ, but remarkably also
diminished Th2 cytokines IL-4 and IL-13 in lung leukocytes at 3 and 4 weeks post infection. There was
a reduction in the frequencies of IFN-γ, IL-17 and
TNF-α producing T cells. DNMAML mice also had increased mortality following infection, showing that
Notch signaling contributes to infected host survival. Collectively, these results show that Notch signaling in T cells promotes anti-fungal host defense by
enhancing T-cell cytokine production, fungal clearance and containment in the lungs.
40
eosinophil recruitment in response to the
human pathogen Aspergillus fumigatus
Nansalmaa Amarsaikhan, Evan O`Dea, Hongtao Li,
Steven Templeton
Department of Microbiology and Immunology, Indiana
University School of Medicine Terre Haute, Terre Haute
In humans and experimental animals infected with
the opportunistic fungal pathogen Aspergillus fumigatus, Th1 responses are considered protective,
while Th2 responses are associated with increased
morbidity and mortality. How host-pathogen interactions influence the development of these protective or detrimental immune responses is not well
understood. We have demonstrated that increased
surface chitin exposure in A. fumigatus promotes
airway eosinophil recruitment, Th2-skewed airway
immune responses and eosinophil-mediated pathology. In this study we examined innate immune
cell and cytokine regulation of chitin-mediated eosinophil recruitment. Gene expression screening
revealed increased IL17A expression in response
to single aspiration of high chitin isolate Af 5517 as
well as increased Th-2 associated cytokines after repeated exposure. RAG1-/- mice did not exhibit decreased eosinophil recruitment in response to a single aspiration of Af5517 conidia, suggesting a role
for γδ T cells, a TCR+ subset capable of rapidly producing IL-17A in response to infection. In support
of this, we observed decreased eosinophil recruitment in γδ T cell-deficient mice after aspiration of
Af5517 conidia. Furthermore, although our results
in IL-17A-deficient mice suggest global production
of this cytokine is dispensable for eosinophil recruitment, the specific role of cytokine producing γδ
T cells is currently being explored.
47
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
5. Immune response to viruses I
41 Cell-Intrinsic Responses to Incoming
Andrew Burrage1, Shauna Marvin1,Edward Campbell1, Harald Wodrich2,ChristopherWiethoff1
1
Dept. of Immunology and Microbiology, Loyola University
Chicago Stritch School of Medicine, Maywood, 2MCMP
CNRS UMR 5234, Inst. for Molecular and Cellular Microbiology and Pathogenicity, University Bordeaux, France
Virus entry into host cells represents the first opportunity to detect infection and restrict viral
replication. Although many studies focus on the
detection of incoming virions by various pattern
recognition receptors, much less is known regarding the cellular mechanisms employed to restrict
viruses before they establish infections and replicate. We demonstrate that endosomal membrane
damage induced by adenoviruses (Ads) to cross
cell membranes results in the induction of autophagy. Infection-induced autophagy is blocked by
the chelation of intracellular Ca2+, suggesting that
transient increases in intracellular Ca2+ when Ad
ruptures endosomal membranes triggers autophagy. While inhibiting autophagy does not alter Ad
infectivity, we have identified a mutant that fails to
escape endosomes after rupturing them. Mutating
a conserved PPxY motif in the capsid protein VI
(Ad5-WT) to PGAA (Ad5-M1) results in increased
sequestration of virus by autophagy. We show that
microtubule depolymeration or chemical inhibition of the motor protein dynein inhibits Ad5-WT
from efficiently escaping ruptured endosomes, but
has no effect of Ad5-M1 infection. Thus, we have
uncovered a novel mechanism for host cell intrinsic immunity to detect incoming virions, resulting
in the induction of autophagy machinery to limit
infection, as well as a mechanism of viral evasion
of this response.
42
in neonatal and adult mice
, Nathan Pincus, William Muller,
RichardLongnecker
48
AIC 2014 • Chicago, IL
The increased severity of HSV infection in the neonate compared to the adult is thought to be due
to differences in the immune response between
the developing and mature brain, but the precise
reasons remain unknown. The HSV-1 protein γ34.5
is required for neuropathogenesis and counters
several host signaling responses to infection. We
investigated the host phosphatase PP1α binding
function of γ34.5, important in reversal of host
translational arrest after infection. Neonatal and
adult mice were inoculated intracranially with either a virus deleted for PP1α binding or its marker
rescue. Both neonatal and adult mice had significantly delayed mortality after inoculation with the
PP1α-mutant virus compared to the rescue virus.
The PP1α-mutant virus also had slower replication
in the brain compared to rescue virus. Furthermore, inoculation of type I interferon (IFN) receptor knockout mice restored the virulence of the
PP1α-mutant virus to wild-type levels, suggesting
that host translational arrest is a type I IFN-dependent response. Our findings show that host translational arrest slows viral replication and time to
mortality, and PP1α binding by HSV-1 is important
for disease in both the adult and neonatal murine
brain.
43 Th17 responses mediate the pathologic relung post-bone marrow transplant
,HillaryLoomis-King,CarolWilke,
Bethany Moore
University of Michigan, Ann Arbor
Bone marrow transplanted (BMT) patients are at
high risk of pulmonary infections and development
of fibrosis and pneumonitis within the lung. We
have established a mouse model involving syngeneic BMT and subsequent infection with murine
gammaherpesvirus 68 (γHV-68), which develops
pneumonitis and fibrosis that mimics some of the
complications in BMT patients. We found that BMT
mice were impaired in initial restriction of viral
lytic replication and their T cell differentiation was
skewed toward Th17 response, while T cell differentiation in non-transplanted mice is Th1 biased.
Those Th17 cells are essential for development
49
SATURDAY ABSTRACTS
Northwestern University, Feinberg School of Medicine
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
of pathology, because BMT mice treated with anti-IL-17A neutralization antibodies or mice given
bone marrow from Il-17a-/- donors did not develop pneumonitis and fibrosis post γHV-68 infection.
Lung dendritic cells of BMT mice expressing higher
levels of pro-Th17 cytokines IL-6, IL-23 and TGF-Β1
than those of controls. Adoptive transfer of lung
dendritic cells of non-transplant mice partially corrected the skewing of Th cell differentiation postBMT. Thus, after γHV-68 infection, lung dendritic
cells in BMT mice appear to induce Th17 response
which in turn causes lung pathology. Supported by
NIH T32 and NIH HL AI065543.
44
Kirsten Eberle1, 2, Jodi McGill3, Randy Sacco1, 2
1
National Animal Disease Center, 2Iowa State University,
Ames, 3Kansas State University, Manhattan
There is conflicting evidence to suggest the expression of type III IFNs (IFN-λs or IL-29/IL-28A/IL28B) may be induced by type I IFNs, and that the
role of these two cytokines may be homologous.
Unlike the global expression of IFNAR, the IFN-λR1
is more restricted, but is expressed on epithelial
cells. In the human bronchial epithelial cell line,
BEAS-2B, poly I:C and PIV-3 can induce significant
IL-29 mRNA and protein. Vero cells were used as
a tool to examine type III IFN responses independent of type I IFNs. In Veros, recombinant IFNα/β
is unable to drive IL-29 gene expression to levels
similar to that produced during PIV-3 infection. Although IL-29 expression is significantly increased
during PIV-3 infection in Veros, downstream IFN
signaling molecules are not upregulated. However,
prophylactic treatment of Veros with rIL-29, rIL28A, and/or rIL-28B significantly reduced PIV-3
titers. Our results suggest that PIV-3 infection may
interfere with signaling downstream of type III IFN.
Through an unexplained mechanism, it is clear that
type I IFNs are not necessary to induce type III IFNs
in Veros. However, as seen in BEAS-2B, an intricate
signaling pathway may exist allowing TLR3 ligation
to drive type III IFNs with the aid of type I IFNs.
45
responses
50
AIC 2014 • Chicago, IL
Peterson1
1
Center for Immunology, 2Department of Obstetrics,
Gynecology, and Women’s Health, Division of
Maternal-Fetal Medicine, University of Minnesota,
Minneapolis
PTPN22 promotes type 1 IFN production after Pattern Recognition Receptor (PRR) engagement on
myeloid cells. An autoimmune disease-associated
variant of PTPN22 encodes an R620W substitution (PTPN22-W). PRR-induced Type I IFN is necessary for optimal viral immunization responses,
including CD4 T cell expansion and neutralizing
antibody production. The magnitude of T cell and
humoral vaccine responses correlates with protection against influenza virus infection. Since viral
vaccination triggers PRR-induced type I IFN, and
since PTPN22-W carriers show reduced capacity
for PRR-driven type I IFN production, we hypothesized that PTPN22-W carriers would show diminished immune response to the influenza vaccine.
Healthy PTPN22-W carriers and age-matched controls were immunized with the 2013-2014 trivalent inactivated influenza vaccine, and their T cell
and antibody responses were assessed. PTPN22-W
carriers exhibited significantly decreased induction of influenza-specific CD4 T cells expressing
TNFa, IL-2, and IFNg, Moreover, carriers displayed
reducedfold-induction of neutralizing antibody titers post vaccination. These data strongly suggest
that PTPN22-W carriers have defective capacity
to mount protective immune responses to influenza vaccination. Further studies should determine
whether PTPN22-W carriage predisposes to increased incidence of influenza infection in immunized individuals.
Zahra Olson1, 2,MatthewSandbulte1, 2,CarineSouza1,
DanielPerez3,AmyVincent1,CrystalLoving1
1
USDA, ARS, National Animal Disease Center, Ames, 2Iowa
State University, Ames, 3Department of Veterinary Medicine, University of Maryland and Virginia-Maryland Regional College of Veterinary Medicine, College Park
Live-attenuated influenza virus (LAIV) vaccines
51
SATURDAY ABSTRACTS
Juliet Crabtree1, Shelly Tien2, Weihua Guan1, Erik
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
provide broader cross-protection than whole-inactivated virus (WIV) vaccines. However, standard
correlates of protection, such as serum hemagglutination inhibition (HI) titers, do not accurately
predict cross-protective efficacy. While contribution of T cells to IAV immunity is appreciated, data
comparing methods assessing T cell responses is
limited. To understand the differential immunogenicity between LAIV and WIV vaccines as it relates
to T cell responses, IFN-γ production by peripheral
T cells was assessed post-vaccination. ELISpot assays used to enumerate IFN-γ secreting cells (SC)
in peripheral blood 42 days post-vaccination (dpv)
indicated that WIV-vaccinated pigs had a greater
number of IFN-γ SC compared to LAIV vaccinated pigs, regardless of recall IAV used. There was
an increase in IFN-γ SC to homologous virus with
PBMC from LAIV vaccinates from 42 to 77 dpv. At
77 dpv an increase in IFN-γ SC to homologous IAV
was appreciated for LAIV as compared to dpv 42.
ELISA assays used to quantify secreted IFN-γ from
peripheral T cells 42 dpv indicated no significant
difference when using homologous IAV as recall
antigen. Collectively, these data indicate that peripheral IFN-γ recall responses to IAV may not be
the best predictor of cross-protection associated
with LAIV vaccines; however, additional cytokines
are being evaluated to further evaluate differential
immunogenicity of WIV and LAIV vaccines.
47
HollyRHughes1, Susan L Brockmeier1,DanielRPerez2,
Crystal L Loving1
1
USDA-ARS-National Animal Disease Center, Ames,
University of Maryland, College Park
2
In North America there are six antigenically distinct
H1 subtypes currently circulating in pigs. Live-attenuated influenza virus (LAIV) vaccines provide
broader cross-protection than whole-inactivated
virus (WIV) vaccines; however, a defined immune
correlate or standardized assay has not been identified to predict cross-protection following LAIV
vaccination. Hemagglutination-inhibiting (HI) immunoglobulin (Ig) in serum has long been the gold
standard correlate of protection following WIV vac-
52
cination; however, LAIV does not elicit a robust serum HI Ig titer. Oral fluids (OF) have become a rapidly developing diagnostic specimen for a variety
of animal pathogens. In order to evaluate mucosal
immunogenicity and identify a potential correlate
of protection, groups of pigs were vaccinated with
different LAIV vaccines encoding pandemic surface
genes and subsequently challenged with heterologous IAV. Following vaccination, serum, nasal wash
(NW) and OF were collected to evaluate Ig levels
against a panel of H1 viruses. Both NW and OF had
detectable levels of IAV-specific Ig when measured
by whole-virus ELISA. Higher IAV-specific IgA endpoint titers were associated with reduced virus
shedding following challenge with heterologous
IAV. However, serum IAV-neutralization titers were
predictive of cross-protection even when HI titers
were undetected. Collectively, these data suggest
that OF or serum neutralization titers may be useful for predicting cross-protection.
48
Holly Hughes1, Zahra Olson1,PhillipGauger2,
AmyVincent1, Susan Brockmeier1,CrystalLoving1
1
Virus and Prion Research Unit, Agricultural Research
Service, National Animal Disease Center, U.S. Department
of Agriculture, Ames, IA
2
Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State
University, Ames
Vaccine associated enhanced respiratory disease
(VAERD) has been described in pigs vaccinated
with whole-inactivated influenza virus (WIV) following infection with heterologous influenza A
virus (IAV). WIV vaccination elicits production of
non-neutralizing antibody that is cross-reactive
to the challenge IAV strain suggesting immune
complexes or antibody dependent cell-mediated
cytotoxicity could contribute to VAERD pathology.
Moreover, pathological findings in VAERD lungs
include significant lymphocytic infiltration and
increased levels of proinflammatory cytokines. A
characterization of lymphocytes in lungs following
IAV challenge has not been completed and such
information will contribute to understanding the
53
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
mechanism of VAERD versus cross-protection. WIV
vaccinated pigs had a transient increase in IAV-specific IFNγ secreting cells (SC) in peripheral blood
and numbers were heightened after challenge.
Post-challenge IAV-specific IFNγ SC in the lungs
were increased in pigs with VAERD compared to
non-vaccinated, challenged controls. CD8 T cells
and NK cells were significantly higher in the lungs
of pigs protected from homologous challenge, suggesting these cells may play a role in protection.
Interestingly, pigs with VAERD had a significant increase of CD4/CD8 double-positive T cells that was
not observed in pigs without VAERD. These data
further describe the lymphocytic pathology seen
in VAERD and suggest double-positive memory T
cells may contribute to disease pathology.
Elizabeth Johnson,VitalyGanusov
University of Tennessee, Knoxville, Knoxville
As HIV evades the T-cell response, it accumulates
mutations that may compromise the structural integrity and function of the virus, decreasing viability. Therefore, the viral population must negotiate
a tenuous path between fitness pressures and host
pressures as it evolves. Upon transmission to a naive host, however, mutated viral residues are free
to revert back to their wild form. Host wide transitions back to wild residues post transmission are
termed ‘reversions’. Since revertant positions are
both vulnerable to immune attack and costly to
mutate, they make attractive vaccine targets. Unfortunately, it is difficult to quantify the in vivo fitness cost a mutation inflicts on the virus due to the
following confounding factors: 1) immune pressure and 2) co-linked mutations. We will present a
tentative sketch of the in vivo fitness landscape of
the HIV poly-protein Gag. This landscape is extracted by dissecting viral population kinetics obtained
from longitudinal surveys of viral diversity within
patients via nested ODE systems. We will also show
how the entire protein reverts over the timescale
of 500 days post transmission and address population level convergence and divergence in wild type
composition.
54
50
in vivo
1, 2
, Sharmila Manoj1, Robert Zie-
mann1, 2
1
Biologics Discovery and Design and 2Antibody Research,
Abbott Diagnostics Research and Development, Abbott
Laboratories, Abbott Park
DNA immunization offers the advantage of allowing
for the initiation of animal immunogenicity studies
while work to produce and purify the protein of
interest is completed. Here, we sought to evaluate
in vivo electroporation (IE) as a means to enhance
the antigen specific immune response from DNA
immunization. Mice were immunized three times
with DNA encoding the protein of interest via intramuscular (IM) or intradermal (ID) injections. Test
animals were administered an electrical pulse into
the muscle or dermis at the site of injection immediately following immunization. Additionally, cardiotoxin was injected into the muscle of a subset of
test animals five days prior to each DNA injection.
Nine weeks following the final DNA immunization
mice were immunized with the protein of interest
emulsified in Freund’s adjuvant. Sera samples taken following the final DNA immunization show a
significant enhancement in immune response for
IE mice. Specifically, those mice pre-dosed with
cardiotoxin, immunized IM and given IE showed
the strongest response. This response was only observed vs. solid phase and not solution phase protein, suggesting the resulting antibody was of low
titer and affinity. Additional testing following protein immunization revealed a significantly higher
affinity response. Our results suggest IE can effectively help stimulate a stronger immune response,
thereby aiding in the use of DNA immunization as a
means to initiate animal immunizations.
51
Xuequn Xu
Department of Medical Microbiology & Immunology,
University of Wisconsin School of Medicine and Public
55
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
Health, Madison
SATURDAY ABSTRACTS
Natural killer T (NKT) cells are innate T lymphocytes that are among the first immune cells to
traffic to sites of inflammation, and that have been
implicated in recruiting neutrophils that are critical for promoting host defense. The mechanisms
through which NKT cells carry out these functions
are not well understood. We have developed a
simple experimental model in which human NKT
cells are co-injected with inflammatory dendritic
cells (DCs) into the footpad of an immune deficient
mouse, inducing edema and infiltration of local tissues by activated murine neutrophils that results
in enhanced resistance to a sub-dermal challenge
with Candida albicans. The NKT cells initiate the
protective inflammatory response by stimulating
P2X7-dependent calcium signaling in the DCs that
leads to the biosynthesis of eicosanoid lipid mediators. Since neither foreign antigens nor microbial
compounds are required for NKT cells to activate
the DCs, this interaction constitutes a novel pathway of protective sterile inflammation mediated
by the endogenous induction of inflammatory lipid
mediators.
52
1, 2
, Lisa Abernathy1, 2, David
Hoogstra ,ChristopherYunker2, Fulvio Lonardo3,
2
Gilda Hillman1, 2
1
Department of Immunology and Microbiology, Wayne
State University School of Medicine, 2Department of
Radiation Oncology, Karmanos Cancer Institute,
Detroit,3Department of Pathology, Wayne State University
School of Medicine, Detroit
Our previous findings demonstrated that soy isoflavones mitigate lung pneumonitis and fibrosis
induced by radiotherapy for lung cancer patients.
Radiation also causes esophageal tissue damage resulting in pain and difficulties swallowing. We have
now investigated radiation-induced esophagitis
and effects of soy isoflavones on this adverse event.
C57BL/6 mice were treated with 10 Gy thoracic irradiation and soy isoflavones given daily at 1 mg/
mouse. Esophagi were resected and processed for
histological analysis (H&E, Masson’s Trichrome,
56
anti-αSMA, anti-CD45). Radiation-induced esophagus damage was observed as tissue edema, inflammatory leukocyte infiltration, and connective tissue
disruption with smooth muscle cell hypertrophy.
However, irradiated mice treated with soy isoflavones showed reduction in tissue damage based on
morphometric measurements of subepithelial tissue layers. Multiple histological changes indicative
of radiation-induced esophagitis were mitigated by
soy isoflavones, suggesting their protective role to
modulate various aspects of the inflammatory process.
53
macrophage and neutrophil responses in
Lisa Abernathy1, 2,MatthewFountain1, 2, John David2,
ChristopherYunker2, Gilda Hillman1, 2
1
Department of Immunology and Microbiology, Wayne
State University School of Medicine, Detroit, 2Department
of Radiation Oncology, Karmanos Cancer Institute, Detroit
Radiation therapy for lung cancer causes normal
lung tissue injury, including pneumonitis and fibrosis. Our lab has previously shown in preclinical
mouse models that treatment with soy isoflavones
mitigates radiation-induced lung injury. This study
investigates the role of macrophages and neutrophils in radioprotection of lung tissue by soy isoflavones. BALB/c mice received a single 10 Gy dose
of thoracic irradiation and up to 18 weeks of soy
isoflavones given orally at 1 mg/mouse daily. Bronchoalveolar lavage fluid (BALF) and lungs were
harvested at different time points post-radiation
and differential cell counts on BALF cytospins,
immunophenotyping of lung cells and cytokine
measurements in lung tissue were performed. Our
data indicate that infiltration and activation of macrophages and neutrophils induced by radiation in
both lung compartments are inhibited by soy isoflavones. These findings suggest that radioprotection by soy isoflavones may occur via inhibition of
damaging inflammatory responses to radiation in
normal tissues. Supported by the American Institute for Cancer Research.
54
57
SATURDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
Rene Gonzalez, David Feola
SATURDAY ABSTRACTS
Pharmacy Practice and Science, University of Kentucky,
Lexington
Nuances in macrophage polarization states play
a critical role in regulating inflammatory damage.
Our group has previously shown that Azithromycin
(AZM) can polarize macrophage to an alternatively
activated-like phenotype and that this polarization
is beneficial in mice infected with Pseudomonas.
However, the genetic signature associated with this
phenotype is not well defined and there remain
a number of potential targets for therapeutic exploitation. We set out to characterize the expression of a more robust set of M1 and M2 genes induced by AZM and determine differences between
resident macrophage and infiltrating monocyte
lung populations. Our infection model consists of
intra-tracheal instillation of Pseudomonas followed
by fluorescence activated cellular sorting. We then
utilized microarray analysis for assessing trends in
global genetic expression between immune subsets. AZM served as a manipulated variable used
as a polarizing stimulus for alternative activation.
Results showed that AZM up-regulated several
M2-assocated genes including Stat6, Chi313, Mrc1,
& Trem2 with notable gene expression differences
between infiltrating monocyte and tissue macrophage populations. Additionally, the up-regulation
of Trem2 was an interesting finding given the substantial interest in this molecule in the setting of
inflammatory neurodegeneration. We believe this
warrants further investigation into Trem2s’ potential association with AZM and its role in mitigating
lung inflammation
55
paracellular diapedesis in a human model
1
,JenniferKoblinski1, 2, William Muller1
1
Northwestern University Feinberg School of Medicine,
Chicago, 2Virginia Commonwealth University School of
Medicine Richmond
Leukocyte transendothelial migration (TEM; diapedesis) is a critical event in immune surveillance
and inflammation. Most TEM occurs at endothelial
cell borders (paracellular). However, there is indirect evidence to suggest that, at the tight junctions
58
(TJ) of the blood-brain barrier (BBB), leukocytes
migrate directly through the endothelial cell body
(transcellular). Why leukocytes migrate through
the endothelial cell body rather than the cell borders is unknown. To test the hypothesis that the
tightness of endothelial cell junctions influences
the pathway of diapedesis, we developed an in vitro model of the BBB that possessed 10-fold higher
electrical resistance than standard culture conditions and strongly expressed the BBB tight junction
proteins claudin-5 and claudin-3. We found that
paracellular TEM was still the predominant pathway (≥98%) and TEM was dependent on PECAM-1
and CD99. We show that endothelial TJ expressing
claudin-5 are dynamic and undergo rapid remodeling during TEM. Membrane from the endothelial
lateral border recycling compartment is mobilized
to the exact site of tight junction remodeling. This
preserves the endothelial barrier by sealing the intercellular gaps with membrane and engaging the
migrating leukocyte with unligated adhesion molecules (PECAM-1 and CD99) as it crosses the cell
border. These findings provide new insights into
leukocyte-endothelial interactions at the BBB and
suggest that TJ are more dynamic than previously
appreciated.
Joshua Stoolman1, 2, Patrick Duncker1, 2, Amanda
Huber2, 3,BenjaminSegal2, 3
1
Program in Immunology, 2Holtom-Garrett Program in
Neuroimmunology, 3Department of Neurology, University
of Michigan, Ann Arbor
Multiple Sclerosis (MS) is a heterogeneous inflammatory demyelinating disease of the CNS characterized by the development of lesions in the white
matter of the brain, spinal cord and optic nerves.
Lesion distribution varies widely between individual patients, however, little is known about the
mechanisms that regulate immune cell infiltration
into different regions of the CNS. Clinical and histopathological features of MS are simulated by the
animal model, experimental autoimmune encephalomyelitis (EAE), induced by the adoptive transfer
of myelin-reactive CD4+ T cells into naïve mice. We,
59
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and others, have found that donor T cells induce
an ascending paralysis, associated with spinal cord
inflammation, in WT hosts, referred to as conventional EAE. However, the same population of donor
cells induces ataxia and/ or imbalance, associated
with brainstem inflammation, in IFNγR -/- hosts
(atypical EAE). We found that inflammatory infiltrates in WT mice are enriched with monocytes,
which correlated with higher levels of CCL2, while
infiltrates in IFNγR -/- mice are enriched in neutrophils and CXCL2. Additionally, CCR2 -/- mice had
attenuated conventional, but not atypical disease.
Conversely, CXCR2 antagonism attenuated atypical
but not conventional disease. Our study illustrates
how regional differences in chemokine expression
shape the spatial pattern and composition of immune infiltrates, leading to disparate clinical outcomes.
57
, Elkin Galvis, Richard Gomer
Texas A&M Biology, College Station
Monocytes play a key role in both wound healing
and fibrosis by entering a tissue and differentiating into pro-fibrotic M2a macrophages and fibroblast-like cells called fibrocytes. M2a macrophage
and fibrocyte differentiation are strongly inhibited
by the plasma protein Serum Amyloid P (SAP), and
healthy tissues contain very few fibrocytes. A central question regarding the innate immune system,
wound healing, and fibrosis is what overrides the
SAP inhibition to trigger monocytes to differentiate
into fibrocytes and M2a macrophages. In wounds
and fibrotic lesions, mast cells degranulate to release tryptase, and in early wounds thrombin mediates blood clotting. Here we report that tryptase
and thrombin potentiate fibrocyte and pro-fibrotic
M2a macrophage differentiation at biologically relevant concentrations and exposure times, even in
the presence of concentrations of serum and SAP
that normally completely inhibit fibrocyte differentiation. The fibrocyte potentiation by these proteases is mediated by protease-activated receptors
1 and 2. Tryptase and thrombin also potentiate
pro-fibrotic M2a macrophage differentiation from
60
M1 and M2 macrophages. Together, these results
suggest that tryptase and thrombin are an initial
trigger to override SAP inhibition to initiate monocyte-mediated scar tissue formation, and present
a novel mechanism to regulate wound healing and
fibrosis.
58
-
Shuang Zhang1, XinYi Yeah1,WilliamFrazier2,Edward
Thorp1
1
Northwestern University, Chicago, 2Washington University,
St. Louis
Recent discoveries linked optimal healing of cardiac reperfusion injury with macrophage-mediated inflammation resolution in the heart. During
myocardial reperfusion, phagocytic clearance of
dying cardiomyocytes by resident/recruited macrophages (i.e. efferocytosis) leads to inflammation
resolution and tissue repair in the heart. Compare
to other tissues, efferocytosis is inefficient in the
ischemia/reperfusion injured myocardium. We
propose that this leads to delayed inflammation
resolution and tissue repair. An anti-phagocytic
ligand (i.e. “don’t-eat-me” signal) CD47 was found
increased on injured cardiomyocytes. We hypothesize that elevated myocardial CD47 prevents cardiomyocyte efferocytosis and CD47 blockade promotes tissue repair through enhancing efficient
phagocytic removal of dying cardiomyocytes. In
this study, we found that CD47 blockade in vitro
rescued the low efferocytic efficiency of cardiomyocytes compared to cardiac fibroblast and neutrophils via interrupting CD47/SIRPα axis between
cardiomyocytes and macrophages. We also showed
that acute CD47 blockade in vivo specifically enhanced efferocytosis of cardiomyocyte debris by
macrophages and this led to reduced infarct size
and enhanced cardiac function during myocardial
reperfusion. Supported by NIH and BD biosciences.
dying cells
Jared Klarquist1,CassandraM.Hennies1,MariaA.
Lehn1,RachelA.Reboulet1, Sonia Feau2,EdithM.
61
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AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
Janssen1
SATURDAY ABSTRACTS
1
Division of Immunobiology, Cincinnati Children’s Hospital
Medical Center and the University of Cincinnati College of
Medicine, Cincinnati, 2Division of Developmental Immunology, La Jolla Institute for Allergy and Immunolog
Adaptive immune responses to antigens released
by dying cells play a critical role in the development of autoimmunity, allograft rejection, and
spontaneous as well as therapy-induced tumor
rejection. Although cell death in these situations
is considered sterile, various reports have implicated type I IFNs as drivers of the ensuing adaptive immune response to cell-associated antigens.
However, the mechanisms that underpin this type
I IFN production are poorly defined. Here we show
that dendritic cells (DCs) can uptake and sense nuclear DNA released by dying cells to induce type I
IFN. Remarkably this molecular pathway requires
STING but not TLR or NLR function and results in
the activation of IRF3 in a TBK1-dependent manner. DCs are shown to depend on STING function
in vivo to efficiently prime IFN-dependent CD8+ T
cell responses to tumor antigens. Furthermore, loss
of STING activity in DCs impairs the generation of
follicular helper T (Tfh) and plasma cells as well as
anti-nuclear antibodies in an inducible model of
systemic lupus erythematosus (SLE). These findings suggest that the STING pathway could be manipulated to enable the rational design of immunotherapies that enhance or diminish anti-tumor and
autoimmune responses, respectively.
62
7. Innate immunity I
-
Alison Eastman,NicolePotchen,JakeCarolan,Antoni
Malachowski,IllonaKryczek,SteveKunkel
University of Michigan, Ann Arbor
TNFα is required for protective Th1 immunity to
Cryptococcus neoformans (Cneo) and these effects
are linked to the stable, early classical activation of
dendritic cells (DC1), preventing alternative (DC2)
activation. We hypothesized that TNFα stabilizes
DC1 DC by inducing epigenetic modification of key
DC1 genes. We modeled TNFα-stabilized DC (TSDC)
in vitro by treating DC with DC1-skewing IFNγ +/TNFα followed by a switch to DC2-skewing IL-4,
and we assessed transcriptional and epigenetic
signatures. Epigenetic studies focused on histone
3 methylation at lysines 4, 9 and 27 (H3K4, 9 or
27, respectively). H3K4 methylation is associated
with activation of proximal genes, but H3K9 and
27 methylations are associated with repression.
TSDC showed resistance to induction of DC2 genes
and increased transcript levels of the key histone
methyltransferases G9a (H3K9), EZH2 (H3K27)
and MLL1 (H3K4). Further, we performed ChIP on
TSDC and found that IL12b and CD40 promoters
are associated with the activating H3K4 methylation while non-stabilized DC1 and DC2 were not.
We next assessed EZH2, G9a, and MLL1 transcript
levels and the global methylation state of H3K4 in
Cneo-infected lungs and found that both mRNA
transcript levels and H3K4 methylation increased
significantly in control but not TNFα-depleted
mice. We conclude that histone modifications in
DC are significantly altered by TNFα, and that this
correlates with increased DC1 stability during protective responses to Cneo infection.
homeostasis
, Yasmina Laouar
63
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Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
Department of Microbiology and Immunology, University
of Michigan Medical School, Ann Arbor
Recent studies have discovered that intestinal
CD103+CD11b+ dendritic cells (DCs) are important
for Th17 differentiation and also for group 3 innate
lymphoid cells (ILC3s) to secrete IL-22. ILC3 cells
are indispensable IL-22 producer of innate immunity against bacterial infection in mucosal tissues.
Here we demonstrate that TGF-βR-Smad2 signaling is essential to develop CD103+CD11b+DCs and
this DC subset serves suitable cytokine milieu to
maintain ILC3 cells in gut. CD11cdnR mice lacking
TGF-βR signaling showed dramatically reduced
CD103+CD11b+ DCs, supported by bone marrow
transplantation model. Impaired generation of
CD103+CD11b+ DC subset led microenvironmental
changes in CD11cdnR mouse intestine by decreasing IL-6 and increasing IL-1β. Interestingly, ILC3
population were dramatically decreased in in vitro
culture with IL-6 deletion or IL-1β addition, suggesting that CD103+CD11b+ DCs contribute to set
up environment for ILC3 homeostasis by producing adequate cytokine levels. Indeed, CD11cdnR
mice harbored decreased ILC3 cells compared to
littermate and succumbed to Citrobacter rodentium infection unlike wild type. Taken together, our
finding revealed novel role of TGF-βR signaling in
gut CD103+CD11b+ DC development and its ability
to control ILC3 maintenance.
Cryptococcus neoformans
Jacob Carolan1, 2, Alison Eastman1, 2, Nicole
Potchen1, 2,EnzeXing1, 2,AntoniMalachowski1, 2,
MichalOlszewski1, 2
1
University of Michigan, Ann Arbor, 2Veterans Affairs Health
System, Ann Arbor
Robust Th1 immune polarization and TNFα production are required for clearance of opportunistic
fungus C. neoformans (C.neo), responsible for over
600,000 deaths, annually.
To study the mechanism, by which TNFα promotes
cryptococcal clearance, CBA/J mice treated with
TNFα neutralizing (αTNF) or control antibodies
were infected with C.neo 52D. Immune polariza-
64
tion was assessed by qPCR and flow cytometry of
isolated DC and CD4+ T-cells.
Single administration of αTNF resulted in persistent
pulmonary C.neo infection in mice that otherwise
cleared the fungus. We also found increased alternative activation (DC2) hallmarks (IL-4, Arg1) and
decreased co-stimulatory molecule (CD86,CD40),
MHC Class II, and the node-homing receptor CCR7
expression by DCs. Consistently, αTNF treatment
decreased IFNy expression and increased IL-4 and
IL-10 by CD4+ T-cells in the infected mice, indicating non-protective Th2.
To determine, if TNFa directly contributed to DC1
polarization, the bone marrow-derived (BM)DC
cultures were stimulated with IFNy ±TNFα for
24hrs, then washed and treated with proDC2 cytokine, IL-4 and evaluated for key DC1 and DC2
gene expression. Following IL-4 exposure, BMDCs
pretreated with TNFα/IFNy continued to show
high DC1 (iNOS and IL-12b) and low DC2 (Gal3 and
Fizz) gene expression, while BMDC pretreated with
IFNγ rapidly converted to DC2 phenotype.
We conclude that TNFα promotes stable DC1 polarization, thereby promoting robust Th1 immunity
and clearance of the infection.
Pseudomonas aeruginosa
Halide Tuna1,TheodoreCory2, Brian Murphy1, David
Feola1
1
University of Kentucky, Lexington, 2University of Tennessee
Health Science Center, Memphis, TN
Cystic Fibrosis (CF) patients infected with Pseudomonas aeruginosa are commonly treated with Azithromycin (AZM) for its immunomodulatory properties. Previously, our lab has shown that AZM can
shift macrophage (MΦ) phenotype in vitro from inflammatory (M1) towards the alternatively activated (M2) phenotype. During P. aeruginosa infection,
mice treated with AZM have increased percentage
of M2-like MΦs, resulting in blunted inflammation and decreased production of IL-6 and TNFα
in the lung. Here, we examined the effect of M2
cells in bacterial pneumonia by adoptively transferring wildtype T cells with or without wildtype
bone marrow (BM) cells, as a source of MΦs, into
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IL-4Rα-/- recipient mice. Mice were infected intratracheally with 1X105 CFU P. aeruginosa embedded
in agarose beads. Lungs were harvested 4, 7 and 14
days post infection and separated into digest and
lavage portions to analyze interstitial and alveolar
cell populations. Results show that mice receiving
wildtype BM cells had less severe disease marked
by decreased weight loss compared to the control
group. Adoptive transfer of wildtype BM resulted in
decreased PMN influx into the lungs and decreased
TNFα+ CD11b+ cells compared to the control
group. Lung sections stained for collagen showed
decreased collagen deposition in the lungs of mice
receiving wildtype BM. These data suggest that M2
MΦs are necessary for the control of inflammation
during the P. aeruginosa infection in the lung.
Arginase-1 expression in the immune
response to Pseudomonas aeruginosa
(PA) pneumonia
1, 2
, David Feola1
1
Department of Pharmacy Practice and Science,
Department of Pharmaceutical Sciences, University of
Kentycky, Lexington
2
Background: Azithromycin induced alternative
macrophage polarization is associated with increased Arginase-1 (Arg-1) activity. Arg-1 is believed to play an essential role in decreasing injury
and promoting repair.
Objective: To determine the importance of Arg-1
for azithromycin control of inflammation in response to PA pneumonia.
Methods: Arg1flox/flox;LysMcre and wild-type mice
were infected with PA intra-tracheally. Analysis of
weight loss and characterization of inflammatory
cells via flow cytometry was performed. Cytokine
production was measured by intracellular staining.
Inflammatory markers were then assessed in Arg1flox/flox;LysMcre and wild-type mice dosed with azithromycin daily via oral gavage starting four days
prior to infection versus control Arg1flox/flox;LysMcre.
Results: Azithromycin treatment was not associated with significant advantage in terms of weight
loss in Arg1flox/flox;LysMcre mice. Moreover, azithromycin treated Arg1flox/flox;LysMcre mice had de-
66
creased neutrophil, macrophage, and lymphocyte
infiltration. Lastly, azithromycin treated Arg1flox/
flox
;LysMcre mice had decreased numbers of CD4+
T-cells, activated CD4+ T-cells, and INFγ producing
CD4+ T-cells.
Conclusion: Arg-1 deficiency was associated with
a greater inflammatory response along with increased T-cell activation which was not completely
resolved with azithromycin treatment. This re-emphasizes the protective role of Arg-1 in azithromycin control of PA pneumonia.
1
,StevenHuang2,
Yasmina Laouar3,GregoryYanik5,JeffreyCurtis4,
Bethany Moore2, 3
1
Graduate Program in Immunology, 2Pulmonary and
Critical Care Medicine Division, Department of Internal
Medicine, 3Department of Microbiology and Immunology,
University of Michigan Medical School, 4Research Service,
VA Ann Arbor Healthcare System, 5Department of Pediatrics, Division of Hematology-Oncology, University of
Michigan Medical School, Ann Arbor
Hematopoietic stem cell transplantation (HSCT) is
complicated by pulmonary infections that manifest
post-transplantation. Despite engraftment, susceptibility to infections persists long after reconstitution. Previous work using a murine bone marrow
transplant (BMT) model implicated increased cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2)
in promoting impaired alveolar macrophage (AM)
responses. However, mechanisms driving COX-2
overexpression remained elusive. Previously, TGFβ
signaling post-BMT was shown to promote hypomethylation of the COX-2 gene. Here, we provide
mechanistic insight into how this occurs and show
that TGF-β induces microRNA (miR)-29b while decreasing DNA methyltransferases (DNMT) 1, DNMT
3a and DNMT 3b in AMs post-BMT. De novo DNMT
3a and 3b were decreased upon transient transfection of miR-29b, resulting in decreased methylation of the COX-2 promoter, and induction of COX-2.
As a consequence, miR-29b-driven upregulation of
COX-2 promoted AM dysfunction, and transfection
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of BMT AMs with a miR-29b inhibitor rescued the
bacterial killing defect. MiR-29b-mediated defects
in BMT AMs were dependent on increased levels
of PGE2 as miR-29b-transfected AMs treated with
a novel EP2 antagonist abrogated the impaired
bacterial killing. We also demonstrate that HSCT
patients exhibit increased miR-29b; thus, these
studies highlight miR-29b in driving defective AM
responses and identify this miRNA as a potential
therapeutic target.
Author’s request:
inhibits the STING pathway
Do not include my abstract
,Seng-RyongWoo,ThomasGajewski
in the online version of the
University of Chicago
abstract book
Spontaneous T cell responses against tumors frequently occur, despite the fact that most tumors
lack an infectious etiology. We have identified
f pTBK1 and
t that the inthe AIM2 inNG pathway
nces
t tuThe ER stress sensor IRE1alpha licenses
bacterial killing through sustained oxidant
68
AIC 2014 • Chicago, IL
MaryO’Riordan1
1
Department of Microbiology and Immunology, University
of Michigan School of Medicine, Ann Arbor, 2Department of
Biological Sciences, University of New England, Biddeford,
3
Department of Molecular, Cellular, and Developmental
Biology, University of Michigan, Ann Arbor
The inositol-requiring enzyme 1-α (IRE1) sensor of
the unfolded protein response (UPR) is activated in
macrophages during bacterial infection. The IRE1
arm of the UPR contributes to immune clearance
of bacterial infection in vivo, but the mechanisms
driving IRE1-mediated innate immunity are not
fully understood. Here we use methicillin-resistant Staphylococcus aureus (MRSA) as an infection
model to probe the contribution of IRE1 to innate
immune effector function. MRSA infection induced
IRE1 activation through TLR and MYD88 signaling, which led to bacterial killing. IRE1-dependent
bactericidal activity required ROS, and occurred in
a sustained manner over hours of infection. The
SNARE protein, SEC22B, which regulates ER-phagosome trafficking, was dispensable for IRE1-driven global ROS production but necessary for late
accumulation of ROS in bacteria-containing phagosomes. These data suggest a model where infection-induced IRE1 promotes microbial killing
through sustained ROS production, and highlights
the role of ER-phagosome trafficking in host anti-microbial effector function.
RacquelDomingo-Gonzalez1, Giovanny
1
,CarolyneSmith1, 4, Mariana Kaplan4,
Bethany Moore2, 3
1
Graduate Program in Immunology, 2Pulmonary and
Critical Care Medicine Division, Department of Internal
Medicine, 3Department of Microbiology and Immunology,
University of Michigan Medical School, 4National Institute
of Arthritis and Musculoskeletal and Skin Diseases, Ann
Arbor
Neutrophils (PMNs) are among the first types of
immune cells to be recruited to the site of infection. These cells combat infection by utilizing different pathogen trapping and killing mechanisms.
A particular activity of the PMNs is their ability to
69
SATURDAY ABSTRACTS
Basel Abuaita1,KristinBurkholder2, Blaise Boles3,
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
release neutrophil extracellular traps (NETs). NETs
are extracellular strands of DNA bound to antimicrobial neutrophil-derived peptides and proteins
that can trap and kill invading microbes. Research
has shown that inhibition of the mammalian target
of rapamycin induces formation of NETs likely via
the induction of an autophagy program. However, little is known about physiologic regulation of
NETosis. We have previously shown that mice are
more susceptible to bacterial infection following
bone marrow transplantation (BMT). PMNs recruited into the lungs of mice post-BMT overexpress cyclooxygenase (COX)-2, leading to increased
secretion of prostaglandin E2 (PGE2). Additionally,
these BMT PMNs are defective in bacterial killing.
Here we show that PMNs from BMT mice exhibit an
impaired ability to form NETs compared to PMNs
from untransplanted mice. NETosis is regulated by
PGE2 as exogenous addition of PGE2 impairs NETosis in control PMNs. Furthermore, antagonism of
the PGE2 receptor, EP2, or inhibition of COX-2 restores NET formation in BMT PMNs. These studies
explore the role of PGE2 as a negative regulator of
NETosis and provide new information about why
innate immune responses are impaired post-BMT.
homeostasis
Bongkum Choi, Yasmina Laouar
Department of Microbiology and Immunology, University
of Michigan Medical School, Ann Arbor
It is well known that interleukin-15 (IL-15) promotes NK cell proliferation and survival. Our previous study has showed that TGF-β is negative
regulator of development and maturation of NK
cells. Here we demonstrate that TGF-β suppresses
IL-15-mediated NK cell homeostasis in periphery
using CD11cdnR mice lacking TGF-βR signaling in
NK cells. We found that CD11cdnR splenic NK cells
showed incredibly faster incorporation of BrdU
compared to littermate. In splenocyte culture, IL15 induced expansion of NK cell population in both
CD11cdnR mice and littermate, but TGF-β suppressed
wild-type NK cell in vitro proliferation only. Moreover, TGF-β decreased expression of anti-apoptotic
molecule Bcl-2 and IL-15 receptor subunits such as
CD122 (IL-2Rβ) and CD132 (γc), indicating TGF-β
70
down-regulates NK cell sensibility to proliferation
factor IL-15 and even NK cell survival in periphery.
In addition, we found reduced Annexin V expression in CD11cdnR splenic NK cells compared to littermate. Thus, our results suggest that TGF-β controls
IL-15-participating NK cell homeostasis in periphery not even NK cell generation and maturation.
71
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Autumn Immunology Conference 2014
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8. T cell subsets
70
response
,YouJeongLee,KristinHogquist
Center for Immunology, Univ. of Minnesota, Minneapolis
iNKT cells can be rapidly activated by α-galactosylceramide (α-GalCer), and α-GalCer or analogues of
it are being tested for clinical use against certain
cancers and autoimmune diseases. Recent studies
in our lab showed that iNKT cells are comprised of
NKT1, NKT2 and NKT17 effector subsets. In the current study, we asked whether these different iNKT
subsets respond equivalently to α-GalCer using
Nur77GFP transgenic mice to quantify the antigen
specific response. We observed a robust response
by NKT1, but not NKT2, in the spleen, despite in vitro stimulation showing they could be equivalently
activated. Using an in vivoantibody-labeling technique, we found that NKT1 possess significantly
higher accessibility to blood than NKT2, suggesting that NKT1 cells are preferentially localized to
red pulp. This was confirmed by tetramer-based
immunofluorescence studies. As expected, i.v. labeled iNKT cells showed a greater response than
unlabeled cells. Thus, iNKT cells with accessibility
to blood are the predominant cells to respond to
lipid. Consistent with this, we observed a robust
response of liver iNKT cells, and no response by
lymph node iNKT cells, regardless of the subset.
Because iNKT subsets produce distinct cytokines,
their differential response to i.v. lipids is a critical
consideration in therapeutic applications.
71
hematopoiesis
Elizabeth Bobeck,XuequnXu,JennyGumperz
University of Wisconsin-Madison, Madison
Prior studies of human subjects and murine models have established that natural killer T cells (NKT
cells), a subset of innate lymphocytes that can be
found in bone marrow, play important roles in promoting hematopoietic stem cell (HSC) engraftment
after transplantation; however, the mechanisms
remain unclear. Our preliminary data indicate that
72
NKT cells not only secrete GM-CSF, but can also
promote prostaglandin E2 (PGE2) production by
monocytic cells. Since GM-CSF promotes HSC differentiation into myeloid cells types and PGE2 has
recently been identified as a factor that improves
transplanted HSC survival, homing, and proliferation, here we investigate whether these pathways
are mechanisms by which human NKTs influence
hematopoiesis. The effects of NKT cells on human
umbilical cord blood engraftment were modeled
through transplantation into immunodeficient
(NSG) mice and also tested using in vitro co-culture
systems. Since HSC transplantation is central to the
treatment of many hematopoietic malignancies but
is limited by high risks of engraftment failure and
other serious problems, we expect that the determination of the cellular and molecular pathways
by which NKT cells influence HSC engraftment will
allow for their use as a cellular immunotherapy to
improve this therapeutic modality.
72
to an immunosuppressive tumor
microenvironment
1
,YanZheng1,ThomasGajewski1, 2
1
Departments of Pathology and 2Medicine, University of
Chicago
Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous anti-tumor response, immune regulatory pathways can
subvert the effector phase and enable tumor escape. Negative regulatory pathways include extrinsic suppression mechanisms but also T cell-intrinsic anergy. Recently, we have shown that the
transcription factor Egr2 is critical in controlling
the anergic state in vitro. In the current study we
examined whether the Egr2-driven cell surface
proteins, 4-1BB and Lag3, might identify dysfunctional tumor-reactive CD8+ T cells. Flow cytometric analysis revealed a major population of CD8+
TILs co-expressed Lag3, 4-1BB and PD-1 in multiple tumor models. This triple-positive (TP) population appeared over time only in the tumor and
showed the most severe dysfunction as reflected
by defective IL-2 production and proliferation.
Further, Egr2GFPhigh TILs also showed defective
73
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SATURDAY ABSTRACTS
IL-2 production ex vivo. TCRβ repertoire analysis
indicated significant skewing in the TP population
and Egr2GFPhigh TILs. SIY-Kb pentamer staining
revealed a majority of these Ag-specific TILs were
found in the TP fraction. Despite this dysfunction,
the TP population produced high levels of IFN-γ,
Gzmb, CCL1 and IL-10 and suppressed Tconv cell
proliferation in vitro. Our results suggest that
co-expression of Lag3, PD-1, and 4-1BB may identify a critical subpopulation of dysfunctional TILs
specific for tumor antigens that contribute to an
immunsuppressive tumor microenvironment.
73
Amanda Contreras, Andrew Tatar, Siddhartha
Sen, Justin Meyers, Prakrithi Srinand, Clifford Cho
University of Wisconsin-Madison, Madison
We have found that adoptive cell transfer (ACT) of
melanoma-specific memory T cells (TM) results in
a more potent local and systemic T cell response
than ACT with melanoma-specific effector T cells
(TE). In vitro, we have seen that TM are not more
cytotoxic than TE. We hypothesized that a combination of TE and TM ACT would have an additive
effect compared to TE- and TM-based ACT alone.
Mice bearing B16GP33 tumors received GP33-specific TE, TM, or TE + TM ACT. Combinatorial ACT resulted in the most durable suppression of in vivo
B16GP33 melanoma growth and resulted in higher
populations of CD8+ TILs compared with TM ACT.
Combination ACT resulted in a profound induction
of endogenous TILs and the strongest systemic T
cell response to tumor antigen. In vitro, TE and TM
were comparable in their ability to inhibit melanoma growth, and the combination was synergistic.
This effect was reproduced by adding conditioned
media derived from activated TM to wells containing TE, suggesting that cytokines released by -stimulated TM may augment the local cytotoxicity of TE.
These data suggest that a synergistic interaction
between TE and TM may promote combinatorial
ACT’s superior anti-tumor efficacy.
74
74
AIC 2014 • Chicago, IL
John Harty1, 3, 4
1
Interdisciplinary Program in Immunology, 2Carver College
of Medicine, 3Dept. of Microbiology, 4Dept. of Pathology,
University of Iowa, Iowa City
The widespread implementation of vaccines has
been one of the most successful medical interventions in public health. With the growing incidence
of chronic diseases, there is considerable interest
in designing therapeutic vaccines to stimulate natural host defenses against illnesses such as malignancy. Cytokine therapy and dendritic cell (DC) immunization offer potential treatment options, but
at the risk of severe side effects or variable disease
outcome. Here, we propose a short-term DC prime
followed by stabilized IL-2/Ab boost (DC+IL-2/Ab)
to robustly stimulate tumor antigen (TA)-specific
cytotoxic T lymphocytes (CTLs). This amplification
strategy greatly enhances the magnitude of CTLs.
DC+IL-2/Ab also selectively upregulates expression of 4-1BB and GITR, sustains high granzyme
B expression, and increases antigen sensitivity for
IFNg production by CTLs. Furthermore, the percell killing capacity of CTLs is enhanced following
DC+IL-2/Ab treatment. DC+IL-2/Ab did not induce
expression of inhibitory receptors, but enhanced
CTL/TReg populations in treated groups. Importantly, this amplification strategy enhanced endogenous TA-specific CTLs to provide a marked
reduction in tumor burden in multiple models of
pre-existing malignancy. Notably, human IL-2/
Ab complex treatment of aCD3-stimulated human
CTLs resulted in higher proliferation, number and
granzyme B production, supporting the translational potential of this strategy for CTL-mediated
immunotherapy of human malignancy.
75
Shaniya Khan1, Emily Hemann1,KevinLegge1, 3, Lyse
Norian1, 2,VladimirBadovinac1, 3
1
Interdisciplinary Graduate Program in Immunology, 2Department of Urology, 3Department of Pathology, University
of Iowa, Iowa City
The extent to which obesity compromises the differentiation and maintenance of protective mem-
75
SATURDAY ABSTRACTS
Marie Kim1, 2, Martin Richer3, Valdimir Badovinac4,
Autumn Immunology Conference 2014
SATURDAY ABSTRACTS
ory CD8 T cell responses and renders obese individuals susceptible to infection remains unknown.
Here, we show that diet-induced obesity did not impact the maintenance of pre-existing memory CD8
T cells including acquisition of a long-term memory phenotype (i.e. CD27hi, CD62Lhi, KLRG1low)
and function (i.e. cytokine production, secondary
expansion, and memory CD8 T cell-mediated protection). In addition, obesity did not influence the
differentiation and maintenance of newly evoked
memory CD8 T cell responses in inbred and outbred hosts generated in response to different types
of systemic (LCMV, L. monocytogenes) and/or localized (influenza virus) infections. Interestingly,
the rate of naïve-to-memory CD8 T cell differentiation after a peptide-coated dendritic cell (DC)
immunization was similar in lean and obese hosts
suggesting that obesity associated inflammation,
unlike pathogen- or adjuvant-induced inflammation, did not influence the development of endogenous memory CD8 T cell responses. Therefore,
our studies reveal that the obese environment does
not influence the development or maintenance
of memory CD8 T cell responses that are either
primed before or after obesity is established, a
surprising notion with important implications for
future studies aiming to elucidate the role obesity
plays in host susceptibility to infections.
,VladimirBadovinac
University of Iowa Interdisciplinary Graduate Program in
Immunology, Iowa City
While it has been shown that memory CD8 T cells
(mCD8Ts) are activated in an Ag-dependent manner, activation also can be induced by pathogen
derived inflammatory cytokines in an Ag-independent fashion. While recent studies have suggested that early activation (expression of IFNg and
GnzB) of mCD8Ts is independent of cognate Ag
recognition, others have indicated that functions
of mCD8Ts are dependent upon Ag recognition. In
order to resolve these discrepancies, we examined
activation of LCMV-specific mCD8Ts following heterologous infection with L. monocytogenes (LM)
either expressing cognate Ag or not. We showed
76
in vitro that Ag and inflammation acted synergistically to induce mCD8Ts activation, indicating that
both Ag and inflammation contribute to mCD8Ts
activation in response to pathogen encounter. In
vivo, mCD8Ts activation was dependent upon antigen clearance. In situations where mCD8Ts did not
contribute to clearance of infection, Ag-dependent
and –independent mCD8Ts activation was similar.
However, when mCD8Ts contribute to clearance of
infection, mCD8Ts respond faster, and responses
wane as infection is cleared. Thus, early activation
is dependent upon cognate Ag recognition, while
continued responses of mCD8Ts are dependent
upon Ag clearance.
77
Jodi Gullicksrud1, John Harty1, 2, Hai-Hui Xue1, 2
1
Immunology Graduate Program, 2Department of
Microbiology, University of Iowa, Iowa City
In response to a pathogenic infection, antigen-specific CD4 T cells will become activated and undergo
differentiation and proliferation. This is followed
by contraction and memory T cell persistence. We
have previously demonstrated that TCF1, a Wnt
transcription factor, is required for memory CD8 T
cell generation and persistence. However, the role
of TCF1 in CD4 T cell responses has been largely
unexplored.
We generated a TCF1 reporter allele by “knockin” insertion of an “IRES-GFP” cassette into intron
2 of the TCF1 gene. GFP precisely reported TCF1
expression levels in thymic and peripheral T cell
subsets. Homozygous TCF1-reporter mice lack
the long isoform of TCF1 (p45) capable of binding
β-catenin. We crossed the TCF1-reporter to the
SMARTA CD4 TCR transgene, which specifically
recognizes the GP61 epitope of LCMV. By adoptive
transfer followed by LCMV infection, we observed
diminished TCF-1 expression in Th1 cells at effector phase, but retention of TCF1 in Tfh cells. In
the absence of p45, both Th1 and Tfh cells showed
reduced numbers at effector and memory phases.
Furthermore, p45-deficient Tfh cells failed to expand when re-challenged. This suggests unique
TCF-1 requirements for Th1 and Tfh cells during
77
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immune responses.
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78
apoptosis
Jesus Banuelos, Nick Lu
Allergy-Immunology, Northwestern University, Chicago
Glucocorticoids (GCs) are widely used in the treatment of inflammatory disorders. However, the GC
sensitivity of Th cells varies. We found that Th0 and
Th1 cells, but not Th2 or Th17 cells, were sensitive
to GC induced apoptosis. Even though Th subsets
had comparable GR levels, PCR array and qRT-PCR
revealed significant differences in the apoptotic
machinery between Th1 and Th17 cells. At baseline, Th17 cells had higher Bcl2 and lower Bim than
Th1 cells. Although GCs induced Bim in both Th1
and Th17 cells, the induction was higher in Th1
than in Th17 cells. In addition, GCs decreased Bcl2
in Th1 but not Th2 and Th17 cells. Overall ratio
of Bim to Bcl2 protein was elevated by GCs in Th1
but not in Th2 and Th17 cells. Bcl2 knockdown appeared to restore sensitivity to GCs in Th17 cells.
Selective inhibiton of Bcl2 protein by GCs in Th1
cells was likely posttranscriptional since GCs did
not suppress Bcl2 mRNA. Despite the resistance
of Th2 cells to GC killing, production of IL-4 along
with IFN-γ and IL-22, but not IL-17 (A or F) was
inhibited by GCs. These findings support that the
GC insensitivity of Th17 cells both in apoptosis and
cytokine production is mediated by Th cell subset-specific gene regulation by GCs. These studies
may provide a basis for improved treatment regimens for multiple Th17 mediated disorders.
T-cell receptor repertoires share a
,AsafMadi,HilahGal,IrunR.Cohen,Nir
Friedman
Department of Immunology, Weizmann Institute of
Science, Rehovot, Israel
The T cell receptor (TCR) repertoire is formed by
random recombination events of genomic precursor elements, facilitating protection against
78
a diverse collection of pathogens. The resulting
combinatorial diversity renders unlikely extensive
TCR sharing between individuals. Here, we studied
CDR3β amino-acid sequence sharing in a repertoire-wide manner, using high-throughput TCR-seq
in 28 healthy mice. We uncovered hundreds of public sequences shared by most mice. Public CDR3
sequences, relative to private sequences, are two
orders of magnitude more abundant on average
and feature high convergent nucleic-acid combinations. Functionally, public sequences are enriched
for CDR3 sequences that were previously associated with autoimmune, allograft and tumor-related reactions, but not with anti-pathogen-related
reactions. Public CDR3 sequences are shared between mice of different MHC haplotypes, but are
associated with different, MHC dependent, V genes.
Thus, despite their random generation process,
TCR repertoires express a degree of uniformity in
their post-genomic organization. These results, together with numerical simulations of TCR genomic
rearrangements, suggest that biases and convergence in TCR recombination combine with ongoing
positive selection to generate a restricted subset of
self-associated, public CDR3 TCR sequences.
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80
Author’s request:
models
Do not include my abstract
in the online version of the
Taylor Person
abstract book
University of Illinois Urbana Champaign, Urbana/Champaign, Mayo Clinic Summer Undergraduate Research
Genomic instability is one of the most studied hallse. Breast cancers espe-
81
immunotherapy
Christopher Parks1, Michael Hansen1,Andrew
Bordner2, Larry Pease1
1
Mayo Clinic, Rochester, MN, 2Mayo Clinic, Scottsdale, AZ
Immunity consists of a highly regulated system capable of discerning foreign from self. Recognition
of foreign antigens results in immune activation,
while recognition of self results in tolerance. The
fundamental mechanism regulating immune tolerance is the selection of a T cell repertoire in which
receptors with strong affinity for self ligands are
eliminated. We hypothesize that T cells bearing
80
weakly reactive receptors for self ligands are present in the naive T cell repertoire, and that given
sufficiently strong stimulation these cells can be
activated to become efficient killers. A problem in
generating effective cancer immunotherapy is that
the available T cell repertoire lacks receptors with
sufficiently strong reactivity for effective killing of
tumor cells bearing self antigens. Our approach
addresses this problem by targeting normally tolerant T cells with weak affinity for tumor antigens
and activating them with sufficiently strong stimulation, resulting in the generation of efficient killers. Using knowledge of the interaction between
Class I and the TCR, alterations were made in conserved domains of MHC class I that interface with
germline encoded loops of the TCR, increasing the
affinity of the overall interaction while maintaining contacts that determine antigen specificity. It
is possible that these altered MHC molecules are
capable of breaking tolerance for tumor associated
self antigens, generating killer T cells capable of effective cancer immunotherapy.
82
to cytotoxicity and altered vascular
permeability
,FangJin,IanParney,KevinPavelko,
Aaron Johnson
Mayo Clinic, Rochester, MN
Glioblastoma (GBM) is an aggressive malignancy of
the central nervous system that exhibits extensive
vascularization and invasiveness into surrounding brain parenchyma. Recent evidence in animal
models and clinical trials has demonstrated the
viability of immunotherapeutic strategies at selectively targeting tumor cells, with the generation of
tumor antigen-specific CD8+ T cells correlating to
reductions in tumor burden. We have utilized the
GL261 glioma model in immune-competent mice
to demonstrate the efficacy of a novel picornavirus
vaccination approach. Treatment of established gliomas with a picornavirus engineered to express tumor-specific antigens delayed tumor progression
and extended survival, outcomes accompanied by
increased tumor-specific CD8+ T cell responses in
the brain. Perforin, an immune effector molecule,
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is well characterized for its role in CD8+ T cell-mediated cytotoxicity. Our data demonstrate that, in
addition to this role in direct tumor cell lysis, perforin also contributes to alterations in tumor vascular permeability. Untreated perforin deficient
mice bearing GL261 gliomas demonstrated no
difference in tumor burden compared to controls.
However, the heterogeneity of tumor vascular permeability in mice lacking perforin was significantly
reduced. As such, we propose that perforin serves
dual functions in CD8+ T cell-glioma interactions,
contributing to both direct killing of tumor cells
and immune-mediated alterations to tumor vasculature.
83
tolerance
Lauryn Swier,MelissaBerrien-Elliott,RyanTeague
Department of Molecular Biology and Immunology, Saint
Louis University Medical School, Saint Louis
Interleukin-2 (IL-2) is a well-characterized T cell
growth factor that promotes proliferation and effector function of CD8+ T cells. These attributes
made IL-2 an early strategy to treat patients with
melanoma. Despite its promise, only limited therapeutic outcomes have been achieved, which require
large potentially toxic doses of IL-2. Efforts to enhance efficacy and safety of cytokine therapies have
recently focused on cytokine/antibody complexes.
These complexes have increased bioactivity in vivo
relative to IL-2 alone, making IL-2c an attractive approach to enhance anti-tumor T cell responses. One
of the major impediments to anti-tumor immunity
is CD8+ T cell tolerance, where T cells are either
eliminated by apoptosis or rendered dysfunctional.
The influence of IL-2c on tolerant tumor/self-reactive CD8+ T cells has not been explored. We utilized
a mouse model of T cell tolerance to determine if
treatment with IL-2c could overcome tolerance and
provide immunotherapy for mice with disseminated leukemia. Specifically, IL-2c was administered
daily starting 3 days after T cell transfer into tumor-bearing mice. The function and persistence of
transferred tumor-reactive T cells was assessed 5
days later. Treatment with IL-2c prevented deletion
of transferred CD8+ T cells and improved effector
function. These results suggest that IL-2c treat-
82
ment is sufficient to overcome peripheral T cell tolerance, and support further exploration of IL-2c as
an immunotherapeutic for cancer.
84
exogenous interleukin-33
Donye Dominguez, Siqi Chen, Lei Qin, Alan Long,
Jie Fan, Bin Zhang
Northwestern University, Chicago
IL-33 is associated with the induction of Th2-type
immunity, however it has recently gained attention for its role in boosting Th1-type immunity and
CD8+ T cell function. We have discovered that IL-33
can induce robust antitumor effect through a CD8+
T cell dependent mechanism. Systemic administration of recombinant IL-33 (rIL-33) alone was
sufficient to inhibit growth of established tumors
in multiple mouse tumor models. Notably, rIL-33
treatment promoted antitumor CD8+ T cell expansion and effector function. Mechanistically, rIL-33
therapy primarily and directly targets dendritic
cells in tumor bearing mice, thereby restores CTL
activity by increasing antigen cross-presentation
within the tumor microenvironment. Furthermore,
agonist activation of CD40, which is induced by rIL33 treatment, stabilized and induced regression of
established tumors. Our study has elucidated the
potential use of rIL-33 as a novel immunotherapy
option for the treatment of established cancers.
85
regression
Siqi Chen,MinghuiZhang,LeiQIn,JieFan,Donye
Dominguez,BinZhang
Northwestern University, Chicago
Agonist antibodies (Ab) directed against costimulatory molecule CD137 (4-1BB) on the surface
of antigen-primed T-lymphocytes are currently in
various stages of pre-clinical and clinical development, though therapeutic benefit has been limited
as single agents. Here, we examined the inhibitory
role of CD73 on the antitumor efficacy of agonistic anti-CD137 Ab therapy. CD73-deificient mice
treated with anti-CD137 Ab completely rejected
CD73-nonexpressing B16 melanoma growth when
compared with wild-type mice, highlighting the
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importance of an inhibitory role for host-derived
CD73. To explore the therapeutic relevance of
these observations, we combined anti-CD137 and
anti-CD73 Ab to treat mice with established B16SIY melanoma. This combination therapy induced
tumor regression associated with enhanced antitumor CD8+ T cell responses and decreased number of tumor-infiltrating CD4+Foxp3+ regulatory T
cells. In contrast, neither anti-41BB nor anti-CD73
monotherapy is effective to control the growth of
established melanoma. Our study demonstrates
that CD73 blockade has critical implications for
effective clinical targeting of CD137 and possibly
other costimulatory molecules.
tumor microenvironment
Brendan Horton1, Jason Williams2,StefaniSpranger3,
ThomasGajewski1, 2, 3
1
University of Chicago Committees on Cancer Biology
and 2Immunology, 3University of Chicago Department of
Pathology
We have found that dysfunctional CD8+ tumor infiltrating lymphocytes (TIL) express not only multiple inhibitory receptors, but paradoxically also
the co-stimulatory molecule 4-1BB. We therefore
investigated whether delivering positive signals
through 4-1BB, in combination with blockade of
either CTLA-4, PD-L1 or LAG-3, restored TIL function to induce tumor regression. Combinations of
an agonistic 4-1BB antibody +/- either CTLA-4,
PD-L1 or LAG-3 neutralizing antibodies induced
tumor regression of established B16.SIY tumors.
To test whether these combinations affect CD8+
T cells in the tumor microenvironment or in the
periphery we measured the number of SIY reactive CD8+ T cells in the tumor, the tumor-draining
lymph nodes, and in the spleen. We found that the
number of SIY-reactive CD8+ T cells increased in
both the periphery and the tumor after antibody
combinations. To test if newly primed cells from
the periphery were required for tumor regression,
we used the S1P1 inhibitor FTY720 to block T cell
egress from lymph nodes. FTY720 treatment giv-
84
en continuously beginning hours before antibody
combinations did not prevent tumor control, suggesting that the necessary effects of these combinations occur within the tumor microenvironment.
Effective combinations also restored IL-2 production by CD8+ T cells within the tumor site. Our data
suggest that reversing T cell dysfunction within the
tumor microenvironment may be a common mechanism of multiple immunotherapy combinations.
87
molecule expression in vivo and alters T cell
Samantha Simon,AnitaKumari,Charlie
Garnett-Benson
Georgia State University, Atlanta
The use of sub-lethal radiation has been shown
to alter tissue phenotype through the modulation of gene expression. OX40L (CD252), 4-1BBL
(CD137L), and their respective binding partners,
OX40 and 4-1BB, belong to the TNF superfamily
and are expressed on professional antigen presenting cells (APCs). Co-stimulatory signals through OX40:OX40L and 4-1BB:4-1BBL binding help induce
survival, maturation, and proliferation of effector T
cells. Data from our lab has shown that radiation
can modulate the expression of these co-stimulatory molecules in human tumor cells in vitro.
This study was designed to assess whether this
phenomena also occurs in vivo in tumor bearing
mice. We examined the in vivo effects of localized
low dose radiation on s.c. tumors 24- to 48-hours
post-irradiation. Our data indicates that the use of
sub-lethal radiation can be used to modulate the expression of these co-stimulatory molecules in vivo.
Moreover, these pathways have also been shown to
reduce T regulatory (Treg) cell activity and we have
also observed reduced Treg cell numbers following
irradiation of tumor bearing mice.
88
Author’s request:
responses against murine acute
myeloid
Doleukemia
not include my abstract
in1, 2the
onlineFosco
version
of the 2,
2
, Dominick
,XiufenChen
abstract book
JustinKline1, 2
,
University of Chicago
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In a recent study, we observed that leukemia-specific CD8+ T cells underwent abortive proliferation
and
cancer
1
,AmarapornWongrakpanich2,Aliasger
Salem2, Lyse Norian1, 3
1
Interdisciplinary Graduate Program in Immunology,
Division of Pharmaceutics and Translational Therapeutics,
3
Department of Urology, University of Iowa, Iowa City
2
Metastatic breast cancer is currently incurable,
and is a leading cause of cancer-related incidences
and mortalities in women worldwide. The therapeutic induction of adaptive anti-tumor immunity
is a promising approach for disseminated breast
cancer, as these responses are: i) systemic; ii) antigen-restricted; and iii) generate immune memory populations that protect against future tumor
86
reoccurrence. Pursuant with this approach, we
have recently reported the success of a heterologous prime/boost vaccination regimen that utilizes tumor lysates as a source of antigen. This vaccine protocol significantly reduces spontaneous
metastases arising from established murine 4T1
breast adenocarcinomas in a CD8-dependent manner. A distinguishing feature of our vaccine is the
use poly(lactic-co-glycolic) acid polymer microspheres as a lysate delivery vehicle. We are currently exploring variants of these microspheres
that incorporate surface-bound antibodies and/or
encapsulated immunomodulatory RNA. Our goal is
to selectively target lysate-loaded microspheres to
phagocyte populations predisposed to cross-present antigen, and to further enhance immunogenic
antigen cross-presentation with co-delivered RNA.
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Undergraduate Posters
on the humoral immune response in Mus
musculus
Amy Zawacki, Jeanne Minnerath
Swanson-Mungerson1
Saint Mary’s University of Minnesota, Winona
Artificial sweeteners are commonly found in a variety of foods and beverages including yogurts,
desserts, sodas, and juices. Splenda, containing
sucralose, is one of the five artificial sweeteners
approved by the FDA in the United States. Over
100 scientific studies have been completed on sucralose to examine the potential effects this artificial sweetener has on systems of the body. One area
that has had little focus is the effect of sucralose
on the immune system. The purpose of this study
was to determine whether sucralose consumption
impacted humoral immunity in an animal model
(Mus musculus). To do this, CD1 mice were provided with drinking water supplemented with varying
concentrations of Splenda, containing sucralose,
for a period of fourteen weeks. After eight weeks of
treatment, the mice were immunized with the foreign protein, ovalbumin. Serum samples were then
collected at 0, 2, 4, and 6 weeks post-immunization,
and anti-ovalbumin antibody titers were measured
by indirect ELISA. Statistical analysis (ANOVA) indicated there was no significant difference in the
anti-ovalbumin antibody titers between mice that
received Splenda, containing sucralose, and control
mice that did not. This suggests that sucralose does
not have an impact on antibody production (humoral immunity) in mice.
, Melanie Gubbels Bupp
Randolph-Macon College, Ashland
Malnutrition-related immunodeficiency increases the risk of infection and reduces the efficacy of
some vaccines. The homeostatic equilibrium of the
T cell population is clearly disrupted by malnutrition but the precise cues and mechanisms through
which it occurs are unknown. A better understand-
88
ing of how malnutrition contributes to the disruption of CD8+ T cell homeostasis has the potential to
enhance the efficacy of vaccinations and infectious
disease treatments in malnourished individuals.
Short-term malnutrition reduces the size of the peripheral CD8+ T population in mice; and perhaps
paradoxically, malnourished CD8+ T cells are less
sensitive to death-by-neglect. This study aims to
identify whether vitamin and mineral deficiency
or reduced caloric intake triggers these changes.
Therefore, we compared the overall number of
CD8+ T cells and their relative sensitivity to deathby-neglect in malnourished, calorie restricted, and
well-fed mice. Preliminary results suggest that reduction in total CD8+ T cell numbers and sensitivity to death-by-neglect are triggered by reduced caloric intake but not vitamin and mineral deficiency.
Thus, it appears that a molecular network, likely including the IL-7/IL-7R signaling pathway, links the
size of the CD8+ T cell pool with energy availability.
1
, Karen Hedin1
1
Mayo Clinic, Rochester, 2Medical College of Wisconsin,
Madison
SDF-1, also known as CXCL12, is a chemokine protein with a variety of functions including proliferation and trafficking of immune cells. It has also
been implicated in contributing to the growth and
metastasis of numerous different cancers. Surprisingly, SDF-1 has been observed to induce apoptosis in acute myeloid leukemia (AML) cells (Kremer
et al. 2013) by binding the chemokine receptor
CXCR4 and has also been shown to induce association of CXCR4 with the T Cell Receptor (Kumar et
al. 2006). For this work, we investigated the ability
of SDF-1 to induce apoptosis in AML cells, internalize CXCR4, induce CXCR4-TCR association, prolong
ERK activation, and induce cellular migration (results not shown) through the use of multiple different SDF-1 variants.
Leigh Smith
Lewis University, Romeoville
D-type cyclins are a family of proteins that medi-
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ate progression through cell cycle. There are three
types: cyclins D1, D2, and D3. As studied in mice, it
is specifically cyclin D3 that is needed for B and T
cell development. Models lacking Ccnd3 did not develop mature lymphocytes and replacing cyclin D3
with structurally similar cyclin D2 was not restorative. This suggests that there is something unique
to Ccnd3 that allows for the maturation of lymphocytes, which may be as a transcriptional regulator
in addition to its cell cycle role. Mutations of Ccnd3
have been reported in human cancers. It was previously assumed that these mutations lead to abnormal cell cycle regulation; however, it is possible
that abnormal cyclin D3 changes the transcriptional profile of the cell, resulting in a cancerous state.
Compiling mutated sequences from multiple human cancer databases has shown patterns that can
be linked to regions within the protein. Abnormal
proteins were qualitatively and quantitatively assessed to calculate structural changes induced by
mutations. The gene expression profiles were then
clustered into designated pathways correlating
with the mutation location. Interestingly, cancers
of hematopoietic origin containing mutant Ccnd3
contain mutations strictly linked to the C-terminus,
a region of the protein with a putative transcriptional role.
with Staphylococcus aureusinduce a pro-in, Mallary Greenlee-Wacker, William
Nauseef
Inflammation Program and Department of Internal
Medicine, Roy J. and Lucille A. Carver College of Medicine,
University of Iowa, and Veterans Administration Medical
Center, Iowa City
Diseases caused by Staphylococcus aureus
(SA) manifest both as mild skin infections and
life-threatening diseases, including endocarditis,
pneumonia and bacteremia. Initially, neutrophils
(PMN) are the predominant cell type involved in
the innate host defense against SA. Whereas PMN
kill the majority of the ingested inoculum, some
SA survive within the PMN phagosome. PMN laden
with viable SA resist engulfment by macrophages
and alter the macrophage inflammatory response.
90
Phagocytosis of SA by PMN also induces PMN production of ectosomes, particles 100 to 1000 nanometers in size that bud off of the plasma membrane
of many cells, including neutrophils. We hypothesize that ectosomes arising from PMN that have
ingested SA contribute to the amplication of local
inflammation that accompanies staphylococcal infections. To test our hypothesis, we used differential ultracentrifugation to isolate ectosomes from
human PMN fed SA and quantitated recovered protein using the bicinchoninic acid (BCA) assay. Using
flow cytometry and immunoblotting, we demonstrated that ectosomes expressed CD66b on their
surface and contained myeloperoxidase, markers
for PMN membranes and granules, respectively.
In addition, the ectosomes stimulated production
of the proinflammatory cytokine TNFα by human
macrophages. Together these data support the hypothesis that ectosomes from SA-laden PMN are
pro-inflammatory and may contribute to exuberant
inflammation during staphylococcal disease.
garden
1
,LawrenceLemke2
1
Wayne State University, Detroit, 2Calvin College, Grand
Rapids
This project evaluated the distribution of seven
metals (As, Cr, Cu, Mn, Ni, Pb, and Zn) in an urban
garden in northeast Detroit. It was postulated that
small-scale soil metal variability would be present and that higher metal concentrations would
be found in areas of the garden plots where anthropogenic sources of metal pollutants were suspected. If these metals had shared sources, then
covariance between their concentrations was also
hypothesized to be present. Composite samples
were collected following EPA protocol and nested
grid sampling methods were used to investigate
meter-scale variability. Soils were analyzed using
x-ray fluorescence (XRF). Variability on the order of
3-10m was evident in variograms and in ordinary
kriged concentration maps for each metal. The
unique concentration patterns observed indicated
that metals are not consistently present in greater
concentrations in particular regions of the plots.
Linear regression of collocated metal concentra-
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tions yielded statistically significant correlations
(p≤0.05) among 14/21 metal pairs. The strongest
correlation was between Pb and Zn (R2 = 0.63, p
< 0.001). With the exception of As, concentrations
of the metals investigated met MDEQ criteria for
direct soil contact. These results are promising for
urban gardening in Detroit. The protocols developed can be used for future soil studies in other
small urban gardens. Further inquiry on XRF accuracy and the risk assessment levels guiding As criteria is recommended.
on the cellular immune response: developing a
, Melanie Gubbels Bupp
Randolph-Macon College, Ashland
Malnutrition is the leading cause of immunodeficiency. Moreover, some vaccines are less effective
in the malnourished, further exacerbating the issue. The Gubbels Bupp lab has established a mouse
model of short-term malnutrition, which results in
reduced numbers of cytotoxic CD8+ T cells. This
study aims to assess the functional capabilities of
CD8+ T cells from malnourished vs. ad libitum mice
in an in vivo killing assay, in order to develop a system for testing improved vaccine strategies for the
malnourished. Malnourished and ad libitum-fed
mice were vaccinated with an irradiated cancer cell
line, B16-GMCSF. A mixture of target and control
cells was subsequently injected. Targets consisted
of splenocytes pulsed with a cancer cell-associated peptide and labeled with a low concentration of
CFSE, while control cells were labeled with a high
concentration of CFSE. After five hours, the frequencies of target and control cells remaining in
the spleens were determined. Unfortunately, there
were technical difficulties associated with either
the vaccine or the preparation of target cells. However, the conditions for labeling, injecting, and recovering target cells are now well established for
this model.
commercial Echinacea extracts
Lorrie Klutse, Joyce Doan
Bethel University, St. Paul
92
Echinacea is among the most popular herbal remedies in the United States. It is typically used at the
first sign of infection; some studies have shown it
to reduce symptom duration and severity. Others
use it prophylactically to prevent common viral
illnesses. The present study aims to assess the impact of echinacea treatment on activation of the
macrophage-like cell line RAW 264.7. To model the
most common uses of Echinacea, two commercially
available extracts were applied to RAW 264.7 cells
alone, then either preceding or following stimulation with mediators of classical (IFNγ and LPS alone
or together) or alternative (IL-4 and LPS alone or
in tandem) macrophage activation. Classical activation was assessed spectrophotometrically by
measuring nitric oxide production using the Griess
reagent. Preliminary results suggest that the two
Echinacea preparations tested neither enhance nor
attenuate nitric oxide production by RAW 264.7
cells when administered alone, or in the presence
of IFNγ and/or LPS. This effect is observed regardless of the timing of Echinacea administration.
Spectrophotometric analysis of arginase activity
in the presence of Echinacea administered alone
or in combination with IL-4 and/or LPS is ongoing.
Based on previous reports Echinacea treatment is
predicted to enhance arginase activity.
survival during malnourishment
Sarah Murphy,Dr.MelanieGubbelsBupp
Department of Biology, Randolph-Macon College., Ashland,
VA
Malnutrition is the leading cause of immunodeficiency worldwide.The total number of cytotoxic
CD8+ T cells is diminished in mice undergoing mild,
short term malnourishment. Paradoxically, the
remaining cytotoxic T cells in malnourished mice
are less sensitive to death-by-neglect and express
higher levels of CD127, a partial component of the
IL-7 survival protein receptor. It is unclear whether
cytotoxic T cells up-regulate CD127 or if CD127low
cells simply perish in a malnourished environment. Labelled cells expressing different levels of
CD127 (high vs. low) were adoptively transferred
into mice on ad libitum or malnourished diets. Preliminary data suggest that adoptively transferred
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cytotoxic T cells expressing high levels of CD127
exhibit the ability to further up-regulate CD127,
while CD127low cytotoxic T cells remain unchanged.
Our results indicate that cytotoxic T-cells have the
ability to up-regulate CD127, suggesting that the
increased expression of CD127 observed during
malnourishment is not exclusively the result of selective survival. Additionally, we have determined
that CD127 up-regulation likely precedes gross T
cell death during malnourishment.
Lonicera japonica
-
Michael Curry,AustinBrooks,HeatherBruns
Ball State University
L. japonica is a honeysuckle species commonly used
in traditional Chinese medicine for treating a variety of ailments. L. japonica has been shown to have
several medicinal properties such as anti-bacterial,
anti-viral and anti-inflammatory. Given these properties, L. japonica is hailed as an immune enhancer,
but previous studies on isolated compounds from
L. japonica have shown varying abilities to inhibit lymphocyte functions, which would oppose the
ability of L. japonica to serve as an immune enhancer. Furthermore, prior studies in our lab have
demonstrated that L. japonica inhibits T cell activation and functions. Given that both T cells and B
cells contribute to a productive immune response,
the goal of these studies is to examine the effect
of L. japonica on B cell functions to assess its capability as an immune enhancer. Our data demonstrates that L. japonica alters the expression of activation-induced B cell proteins and decreases IgM
production. These findings provide insight into the
immune effects of L. japonica on B cell responses
and suggest that further investigations into the effect of L. japonica on cell survival and proliferation
are needed to determine the ability of L. japonica to
serve as an immune enhancer.
100
bacteria belonging to a novel genus
Cephalotes ants
Jonathan Lin,JohnWertz
Department of Biology, Calvin College, Grand Rapids
94
Ants play an integral role in terrestrial ecosystems.
Yet, little is known about their behavior, feeding
habits, and source of nutrition. Bacterial symbionts
are known to play a key role in the diversification
and ecological adaptation of numerous organisms,
including ants. Previously, we successfully isolated
CV41 and CAG34, two novel bacteria belonging to
Verrucomicrobia from ant guts. In this study, we
physiologically characterized and determined the
roles of the bacteria in Cephalotes guts. Growth
was possible in an atmosphere of 0.5-20% O and
up to 5% CO2, pH 6.9-7.7, and 0.5-1.5% NaCl (w/v)
for CV41 and CAG34. CV41 could not grow in the
absence of CO2, making it a capnophile. Both isolates grew optimally at 37oC. While this temperature is higher than expected, it is possible that the
bacteria have adapted to the tropical habitat of the
ants. CV41 and CAG34 use sugars as well as organic
acids, suggesting that both are important in the gut.
CV41 and CAG34 also have different substrate preferences, perhaps reflective of long-term co-speciation and dietary preferences of the ants. From comparisons of their 16S rRNA gene sequences, CV41
and CAG34 are 98% similar, suggesting, along with
physiological differences, they are distinct species.
Both isolates share a 93% similarity with their closest cultivated neighbor Opitutus terrae, suggesting
that CV41 and CAG34 form a novel genus.
101
Mus musculus
Jacquelyn Bongard, Jeanne Minnerath
Saint Mary’s University of Minnesota, Winona
Sucralose, normally found in Splenda, is an artificial sweetener used to sweeten food and beverages without the added calories of sucrose. Studies have been completed to determine the effects
that sucralose has on body systems, but few studies have focused on the effects that sucralose has
on the immune system. The purpose of this study
was to determine the effects of sucralose consumption on cytokine production using mice (Mus musculus) as an animal model. More specifically, the
objective was to assess production of interleukin
(IL)-2 and tumor necrosis factor (TNF)-α secreted by splenocytes from mice treated with Splenda,
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which contains sucralose. This was done by providing CD1 mice with drinking water supplemented
with varying concentrations of Splenda. After 16
weeks, splenocytes from the mice were harvested and stimulated with Concanavalin A to induce
IL-2 production by T cells or lipopolysaccharide to
induce TNF-α production by macrophage cells. Supernatant was collected from the cell cultures, and
ELISAs were completed to determine the levels
of cytokines (IL-2 or TNF-α) secreted by the cells.
Statistical analysis (ANOVA) indicated that there
was no significant difference in IL-2 production
by splenocytes from mice treated with sucralose
compared to control mice that did not receive sucralose. However, it was found that splenocytes
from mice treated with sucralose produced significantly reduced levels of TNF-α compared to splenocytes from control mice.
102
porter 1
Sam Schuiteman, Larry Louters, Eric Arnoys, Brendan
Looyenga
Calvin College, Grand Rapids
Glucose transporter 1 (GluT1) is a universally expressed facilitative transporter responsible for
basal glucose uptake in mammalian cells. GluT1
has significant implications for both cancer and diabetes, where glucose uptake is either constantly
stimulated or hardly stimulated. It is believed that
multiple GluT1 proteins can oligermize to allow for
increased glucose uptake in cells; this study seeks
to determine if cell density (the amount of cells in
a given area) leads to the activation of GluT1 oligimerization. Supported by Calvin College and the
National Institutes of Health.
103
Brandon Larsen, Jeanne Minnerath
Saint Mary’s University of Minnesota, Winona
Nutrition impacts the body’s ability to mount an effective immune response to infection. The purpose
of the present study was to specifically examine the
effects nutrition has on humoral immunity using
mice (Mus musculus) as the animal model. CD1
mice were divided into four groups; the control
96
group received standard rodent food, the second
group received rodent food that contained high
concentrations of carbohydrates, the third group
received rodent food that contained high concentrations of protein, and the fourth group received
rodent food that contained high concentrations
of fat. The mice were fed the specialized diets for
5 months. After 3 months of treatment, the mice
were immunized with the foreign protein, ovalbumin. At weeks 0, 2, 4, and 6 post-immunization, serum samples were isolated from the mice and were
analyzed for anti-ovalbumin antibodies by indirect
ELISA. Statistical analysis of the data will indicate
whether diet impacts the humoral immune response in mice.
104
inhibitors in renal and lung cancer cells
Miriam Rienstra
Calvin College, Grand Rapids
Leucine-rich repeat kinase 2 (LRRK2) is protein
involved in vesicle trafficking. LRRK2 is overexpressed in renal and lung cancer cells. Previous experiments have shown that when LRRK2 is knocked
down the golgi apparatus is destroyed which causes cell death. It was hypothesized that a therapy
could be designed combining LRRK2 inhibitor
drugs with a chemotherapy drug. The combination
drug would be just as effective but more targeted
to cancer cells and therefore less toxic. The LRRK2
inhibitor drugs used were GNE, L2N1, PFE and the
chemotherapy drug used was Vincristine a cell
viability assay was used to test this hypothesis. It
was also hypothesized that LRRK2 inhibitor drugs
would affect cell growth and cell migration. This
hypothesis was tested by using a soft agar assay
and a scratch assay. From the cell viability experiments a concentration range and LD50 were found
for treatments of Vincristine on cancer cell. The
soft agar and scratch assays both showed LRRK2
inhibitors to have some effect on the tumorgenicity
and migration (respectively) of the renal and lung
cancer cells. Future experiments would include
more testing of the combination LRRK2 inhibitors
and Vincristine in cell viability assays. Further utilization of soft agar assays and scratch assays would
also be used to collect more data on the effects of
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LRRK2 inhibitor drugs on tumorgenicity and cell
migration in renal and lung cancer cells.
105
EllieHofer,MattMolenaar,AlexStangel,Tracie
Weber, Rachel Gibbons Johnson
University of Minnesota Morris, Morris
A promising approach for cancer therapy is to harness the power of the immune system to destroy
tumor cells. CD8+ T cells are capable of killing tumor cells throughout the body, but tumor cells employ a wide range of mechanisms to evade CD8+ T
cells. One such mechanism is the expression of B7H1 (PD-L1) on the surface of tumor cells. Signaling events downstream of B7-H1 interacting with
PD-1 on CD8+ T cells are well characterized and are
known to result in inhibition of cell proliferation
and induction of apoptosis. One important signaling event induced downstream of PD-1 ligation by
B7-H1 includes inhibition of Akt activation, allowing for stabilization of Bim protein levels, leading to
increased apoptosis. B7-H1 is also known to interact with CD80, which is expressed on the surface of
effector CD8+ T cells. The impact of B7-H1 interacting with CD80 expressed by CD8+ T cells is unclear.
We are currently investigating signaling events
downstream of B7-H1 interacting with CD80 on
effector CD8+ T cells that could contribute to the
induction of apoptosis. Our findings would provide
insights for targeting B7-H1 signaling in effector
CD8+ T cells to achieve protective immunity in the
treatment of cancer.
Emma Kusick,HannahRocholl,BreanneSteffan,
ScottA.Hoselton,SumitGhosh,JaneM.Schuh
North Dakota State University, Fargo
Allergic asthma with fungal sensitization is a clinical syndrome that is often difficult to adequately
control and accounts for increased medical intervention and hospital stays. It is characterized by
increased airway inflammation, mucus production,
and fibrotic airway remodeling that limit air ex-
98
change. Our model of fungal allergic asthma utilizes sensitization with Aspergillus fumigatus antigen
and multiple nose-only inhalational exposures to
live A. fumigatus conidia, allowing for a better representation of the development of the disease process. The data presented here shows the histology
of the nasal passage and lung after A. fumigatussensitization and challenge. Airway inflammation,
mucus production, and collagen deposition in the
lung tissue, as well as airway hyperresponsiveness
and a morphometric analysis of inflammatory cells
in the lumen, are presented.
107 Complement component C1q regulates
SeanO’Conner2,SuzanneBohlson2, Emily Gonser1
1
Drake University, Des Moines, 2Des Moines University
The inability to clear apoptotic cells is related to
the development of autoimmunity because dead
cells serve as a source of auto-antigens to which the
body mounts an immune response. Complement
component C1q enhances phagocyte clearance of
apoptotic cells while dampening inflammation, and
C1q-deficiency results in development of autoimmunity. The optimal concentration of C1q required
to modulate phagocytosis was identified and applied to investigate C1q-dependent regulation of
macrophage pro-inflammatory signaling. Mouse
bone marrow derived macrophages were stimulated with C1q and left untreated or were polarized to a pro-inflammatory M1 macrophage using
lipopolysaccharide (LPS) and interferon-γ. Pro-inflammatory cytokines TNF-α and IL-6 were measured by ELISA. M1 macrophages stimulated with
C1q produced less TNF-α when compared to control macrophages. Similar experiments were performed using CpG, a TLR-9 agonist. These studies
indicate that low concentrations of C1q stimulate
enhanced macrophage phagocytosis and inhibition
of pro-inflammatory cytokine production supporting the hypothesis that C1q-dependent regulation
of macrophage activation is important in the maintenance of normal tissue homeostasis.
108
,CarmelaPratt,CarrieLasky,Charles
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Brown
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University of Missouri Department of Veterinary
Pathobiology, Columbia
Borrelia burgdorferi (Bb) is the causative agent of
Lyme disease and is spread by the bite of the Ixodes scapularis tick. Subsequent arthritis and carditis can develop after Bb infection. Immunological
mechanisms that drive both the onset and resolution of inflammatory diseases are unknown. Eicosanoids are known to regulate inflammatory processes and have been shown to be critical in certain
infectious disease models. The enzyme 12/15 lipoxygenase (LO) is a component of the eicosanoid
pathway that is thought to produce primarily anti-inflammatory mediators. We hypothesize that
infection of 12/15 LO-/- mice with Bb will develop
exacerbated disease. There was not a significant
difference in swelling curves of arthritis-resistant
B6 or susceptible C3H 12/15 LO-/- mice compared
to their wild-type counterparts. Cell infiltrates into
the ankles determined using flow cytometry was
not different between B6 12/15 LO and wild-type
controls. Flow analysis of C3H 12/15 LO-/- ankles is
pending. Histology heart and ankle severity scores
were not significantly different between B6 12/15
LO and their wild-type controls. Assessment of the
C3H 12/15 LO-/- H&E stained slides are pending
pathologist’s review to determine heart and ankle
severity scores. In the future, we will assess tissue
protein and lipid concentrations of pro- and anti-inflammatory mediators. A better understanding
of this pathway may lead to the development of
treatments for Lyme and other inflammatory diseases.
Tran Nguyen,AndingShen
Calvin College
Resting CD4+ T cells have been found to be the
major targets of HIV infection. Their frequent interaction with endothelial cells (ECs) has been
concerned, since ECs appear to exchange certain
soluble factors that aid the virus in infecting the
cells. In this study, we attempted to further understanding the interaction between resting CD4+
100
T cells and ECs and the roles of specific cytokines
IL6, IL8 and CCL2 in infection of the cells. We performed different infection assays on resting CD4+
T cells isolated from donated blood. We found that
the amount of infection was the same, whether or
not direct contact between resting CD4+ T cells and
ECs happened. We successfully showed that among
the three cytokines, only IL6 played a significant
role in rendering infection of resting CD4+ T cells.
Our research provided more insights into our understanding of EC-stimulated resting CD4+ T cells
and the roles of IL6 as well as the importance of
soluble factors in HIV infection.
110
Ranu Sinniah1,MikeCatalano2, Kent Gates2
1
Calvin College, Grand Rapids, 2University of Missouri,
Grand Rapids
Conventionally, Mutagenesis has dealt with small
molecule damage to DNA base-pairs resulting in
lesions that lead to incorrect replication by error prone polymerase resulting in mutated DNA.
Pathways to repair said lesions often go through
an Ap (Apyrimidic/purinic) site formation which
is then cleaved and repaired. Previous research in
the Gates laboratory has shown that the ring open
form of an Ap site reveals an aldehyde moiety from
the anomeric carbon that can be attacked to form
an adduct. This adduct also results in incorrect replication past the site by error prone polymerase.
Arylamines are well known functionalities that are
used in pharmaceuticals to this day and have also
been shown to form stable adducts via the aldehyde moiety. Using dR(Deoxyribose), Aniline, and a
series of para-disubstituted Anilines, we were able
to observe in vitro adduct formation at the anomeric carbon for the aforementioned Anilines, using
various techniques to characterize structure, rate
of reaction, and equilibrium constants. Contradictory to source material, the dR ring also isomerized
under adduct formation from a 5 to 6 membered
ring. In sum, the potential for arylamines as mutagens in relation to Ap directed mutagenesis should
be heeded in regards to past and future developments of pharmaceuticals.
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111 Mice lacking ERK5 in their T cells have an
SATURDAY ABSTRACTS
Jaisel A Cervantes,ColinABill,CharlotteVines
University of Texas at El Paso
During an adaptive immune response antibodies
are produced against a particular antigen. It was
previously shown that a lack of C-C chemokine
receptor (CCR7) or inhibition of the CCR7 ligand,
CCL19, resulted in a delayed, but enhanced antibody
production during an adaptive immune response,
although the mechanism of this effect is unclear.
We hypothesize that by inhibiting downstream signaling from the normal CCR7/CCL19 response we
will be able to enhance antibody production during
a secondary immune response. To this end we targeted ERK5, which we have previously reported to
be phosphorylated after CCR7/CCL19 binding in
mouse T cells. Since ERK5-/- mice are embryonic
lethal, we used mice that contain an ERK5flox/flox
site, which upon crossing with syngeneic Lck-Cre
mice produce litter that are ERK5flox/flox/Lck-Cre
resulting in specific loss of ERK5 in the T cells only.
Upon a second challenge with dinitrophenol-keyhole limpet hemocyanin (DNP-KLH) we showed a
significantly elevated production of IgG1, IgG2, and
IgG3 in ERK5flox/flox/Lck-Cre mice compared to
ERK5flox/flox mice and wild type mice with normal CCR7/CCL19 pathways at days 10, 20 and 30
after re-challenge with DNP-KLH.
112
GLUT1 glucose transporter
CharlotteVines,BillColin,Olga Soto
UTEP, El Paso
Chemokines are involved in inflammatory and immune regulatory processes. C-C motif chemokine
receptor 7 (CCR7) plays a role in the trafficking of
T cells to, and T and B cells within, regional lymph
nodes. The ligand CCL19 regulates trafficking by
specifically binding to CCR7. We have shown that
CCR7/CCL19 induces expression of two transcription factors, Extracellular Regulated Kinase 5, and
Kruppel Like Factor-2 and sphingosine phosphate
receptor 1. Thus, we wanted to determine if CCL19
induced expression of other proteins associated
with an effector phenotype. Stimulation of CEM
acute lymphoblastic leukemia cells with CCL19
102
mediates a decrease in the expression of CCR7 over
72 hours. Levels of CCR7 remain steady during 0,
12, and 24 hour time points followed by a decrease
starting at 48 hours. We then examined levels of
GLUT1 glucose transporter, the major glucose
transporter in T cells, which is induced upon TCR
activation. We found that there is upregulation of
GLUT1 at 24 hours, which is sustained for at least
72 hours suggesting a metabolic shift toward glycolysis in CEM stimulated by CCL19. These studies
suggest that CCR7/CCL19 promote a change in T
cell metabolic state, which is marked by upregulation of GLUT1. We are currently investigating other
metabolic parameters.
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10. Allergy and asthma II
113
Phoebe Richgels,IanLewkowich
CCHMC, Cincinnati
Asthma is characterized by airway hyperresponsiveness (AHR), smooth muscle hypertrophy, and
goblet cell hyperplasia. While asthma susceptibility has a genetic component, its incidence is rising
too rapidly to have a solely genetic cause, suggesting that environmental factors play a role. Indoor
allergen sensitization, in particular to house dust
mite (HDM), is a major risk factor for asthma development. Advances in HVAC technology have improved our ability to maintain living spaces at temperatures both comfortable for us, and optimal for
HDM growth. As maternal asthma and prenatal environmental exposures influence asthma risk, we
hypothesized that prenatal HDM exposure (without powerful skewing adjuvants) was sufficient
to enhance asthma development in offspring. To
test this, we treated pregnant A/J dams (F0) with
HDM or PBS, and assessed asthma development in
offspring. F1 offspring of HDM-exposed mothers
showed increased HDM-induced AHR, lung inflammation, and IL-13 and IL-17A+ cells compared to
offspring of PBS-exposed mothers. Additionally,
IL-4, IL-5, and IL-13, asthma-associated Th2 cytokines were also increased in the F1 generation. F2
females, but not males, also showed increased AHR.
This animal model of multi-generational enhancement of asthma severity may help us to uncover
important mechanisms regulating inheritance of
asthma risk.
114 Amish children with low atopy risk
display regulatory blood leukocyte
phenotypes and are hyporesponsive to
Anne Sperling1,CaraHrusch1, Michelle Stein1,Katie
Igartua1, Mark Holbreich2, Peter Thorne3, Donata
Vercelli4,ErikaVonMutius5,CaroleOber1
1
University of Chicago, 2Allergy and Asthma Consultants,
Indianapolis, 3University of Iowa, Iowa City, 4University of
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AIC 2014 • Chicago, IL
The Amish community, a US population of Swiss
decent who utilize a traditional farming lifestyle,
have significantly decreased risk of developing
atopic diseases. To identify immune mechanisms
that contribute to reduced atopy risk, we compared peripheral blood leukocytes (PBLs) from
Amish children to the PBLs from Hutterite children, a genetically similar population from another
farming community in the US with a higher atopy
risk. Compared to the Hutterite children, Amish
children had a lower proportion of eosinophils,
but higher neutrophils. Interestingly, the Amish
children’s neutrophils displayed an immature phenotype characterized by low expression of CD11b
and CXCR4. Further, the Amish PBLs had increased
percentages of inhibitory monocytes and activated
CD45RO+ Tregs. Network analysis of gene expression revealed that the Amish have higher expression of the innate response genes IRF7 and TNFA,
which may be the result of the elevated levels of endotoxin found in Amish homes. Consistent with the
idea that microbial exposure could be promoting a
regulatory immune phenotype similar to endotoxin-induced tolerance, PBLs from the Amish were
hyporesponsive to LPS. These results indicate that
environmental exposure has a profound impact on
baseline immune function and may prevent the induction of atopic disease in children.
115 Lunasin alleviates allergic airway
Tregs
Chun-Yu Tung1,XiaoweiYang2, 4, Baohua Zhou2, 3,
Hua-ChenChang1
1
Department of Biology, Indiana University Purdue
University Indianapolis, 2Department of Pediatrics, HB
Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, 3Department of
Microbiology and Immunology, Indiana University School
of Medicine, 4Department of Veterinary Medicine,
Southwest University at Rongchang, China
Lunasin is a naturally occurring peptide isolated from soybeans and has been explored in cancer treatment. Lunasin inhibits NF-κB activation
and thus pro-inflammatory cytokine and mediator production in macrophages. In this study we
demonstrate that lunasin can effectively suppress
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Arizona, Tucson, 5University of Munich, Germany
Autumn Immunology Conference 2014
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allergic airway inflammation in two murine models of asthma. In an OVA+Alum sensitization model, intranasal lunasin treatment at the time of OVA
challenges significantly reduced total cells counts
in bronchoalveolar lavage (BAL) fluid and eosinophilia, peribronchiolar inflammatory infiltration,
goblet cell metaplasia and airway IL-4 production.
In an OVA+LPS intranasal sensitization model, lunasin treatment either at the time of sensitization
or challenge has similar effects in suppress allergic airway inflammation including significantly reduced total cell and eosinophil counts in BAL fluid,
inflammatory gene Fizz1 expression in the lung,
and IL-4 production by OVA re-stimulated cells
from mediastinal lymph nodes. We further show
that intranasal instillation of OVA+lunasin significantly increases OVA-specific regulatory T cell
(Treg) accumulation in the lung comparing to OVA
only treatment. Taken together, our results suggest
lunasin as an anti-inflammatory agent can be potentially used in asthma therapy or as an adjuvant
to enhance the induction of antigen-specific Tregs
and thus boost the efficacy of allergy immunotherapy.
IL-13 and IL-17A in severe asthma
,AdelaidevanLier,XueZhang,
UmasundariSivaprasad,MelindaButschKovacic,Ian
Lewkowich
CCHMC, Cincinnati
IL-17A is elevated in mice with severe asthma
and correlates with disease severity in humans.
Although the signaling pathways between IL-17A
(TRAF6-NFκB-C/EBPβδ-MAPK) and IL-13 (JAK1STAT6) differ, we report that IL-17A synergistically
enhances IL-13-driven airway responses, STAT6
phosphorylation (pSTAT6), and gene transcription.
Transcriptional synergy is absent in IL-17RA-/cells and is not rescued by co-culture with wildtype
cells, suggesting that synergy requires IL-17A signaling in IL-13-responsive cells. However, pSTAT6
is synergistically enhanced in TRAF6-/- cells, suggesting that enhanced STAT6 activation occurs independent of traditional IL-17A signaling. To interrogate pathways responsible for transcriptional
synergy, we inhibited NFκB, MAPK, or C/EBPβ. Erk
106
inhibition had no effect, while inhibition of NFκB
and C/EBPβ partially attenuated IL-13/IL-17A synergy. By contrast, p38 inhibition completely abrogated synergy, suggesting that NFκB and C/EBPβ
activation occur downstream of p38. Interestingly, p38 inhibition did not alter the enhancement
of pSTAT6, which implies that this feature of synergy may be regulated independently. Finally, we
demonstrate that synergy is conserved in primary
human cells, suggesting that similar mechanisms
may operate in severe asthmatics.
117 Th17 cell subsets in allergic asthma
Jaclyn McAlees,IanLewkowich
Cincinnati Children’s Hospital, Cincinnati
Allergic asthma is an inflammatory lung disease
classically characterized by airway eosinophilia,
airway hyperresponsiveness (AHR) and a maladaptive Th2 response to aeroallergens. Such responses, characteristic of mild asthma, are controlled
with steroids. In contrast, individuals with severe
asthma often display neutrophilic inflammation
and steroid refractory AHR. Although human studies demonstrate a correlation between severe asthma and IL-17A levels, many murine studies point to
a potentially protective role for Th17 cells. Recent
data define functionally distinct Th17 populations:
regulatory Th17 (rTh17) cells that exist in mucosal
tissues during homeostasis and pro-inflammatory
Th17 (pTh17) cells that arise under inflammatory
conditions. In a mouse model of asthma, we show
that A/J mice develop severe steroid refractory
AHR and IL-17A production, and demonstrate selective expansion of pTh17 cells. In contrast, C3H/
HeJ mice develop mild, steroid sensitive AHR, IL17A production and expansion of pTh17 cells.
Additionally, vitro-skewed pTh17 cells are steroid
resistant and factors from these cells demonstrate
increased capacity to synergize with IL-13. In contrast, in vitro-skewed rTh17 cells are steroid sensitive and demonstrate reduced synergism with
IL-13. Collectively, these data suggest that different
types of Th17 cells may differentially contribute to
asthma pathogenesis.
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Erik Anderson1, Takao Kobayashi2, Koji Iijima2, Gail
SUNDAY ABSTRACTS
Kephart2,Chien-ChangChen1, Hirohito Kita1, 2
1
Mayo Graduate School, Rochester, 2Mayo Clinic Division of
Allergic Diseases, Rochester
CD8+ T-cells represent a relatively understudied cell type in allergic lung inflammation despite
their presence in the mucosal tissues. To study
CD8+ T-cells, we used CD4-/- mice in an intranasal
OVA-specific memory response model, using IL-33
as an adjuvant. As expected, WT mice developed an
antigen-specific memory response with high lung
eosinophil infiltration and Th2 cytokines. CD4-/mice on the other hand also developed a similar
response against the antigen with high eosinophil
infiltration into the airways, higher IL-17 levels in
lung tissue, but lower levels of IL-5 and IL-13 compared to WT mice. Additionally, multiple chemokines increased in the lungs of CD4-/- mice. Lung
histology demonstrated CD8+ cells infiltrated in the
airways of WT mice, which was more pronounced
in knockout animals. Both strains produced similar amounts of OVA specific IgE. After sensitization
and challenge with antigen alone, lung single-cell
suspensions were stimulated with anti-CD3/28 in
vitro for 24 hours. Intracellular staining revealed
that WT CD8+ T-cells expressed IFNγ only, whereas
CD4-/- mice had CD8+ T-cells expressing IFNγ, IL-5,
and IL-17. These results showing the presence of
eosinophilic inflammation in CD4-/- mice suggests
a role of CD8+ T-cells in lung inflammation which
may also function in WT mice. Supported by the
Mayo Foundation.
Repeated challenge with oxazolone provokes
Author’s request:
Do not include my abstract
in the online version of the
abstract book
Charles J. Benck,AlyssaAshbaugh,TijanaMartinov,
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120
Shanna Ashley1,CarolWilke2, Bethany Moore2
1
Graduate Program in Immunology, 2Department of
Internal Medicine, University of Michigan, Ann Arbor
Periostin is a matricellular protein that regulates
cellular interactions with the extracellular matrix
components (ECM). In bleomycin-induced fibrosis,
loss of periostin by either structural or hematopoietic cells can limit the development of pulmonary
fibrosis. Periostin can increase the rate of wound
closure, collagen 1 expression and survival in lung
mesenchymal cells. However, these studies did not
separate fibroblasts from fibrocytes and therefore
it is unknown if periostin has similar effects on
both cell types; thus, we performed analyses on
sorted fibrocytes and fibroblasts from wild-type
and periostin-/- mice. Previous work suggested
that blockade of alpha-V beta 3 and alpha-v-beta
5 integrins partially blocked the effects of periostin on the rate of mesenchymal cell wound closure
and bleomycin-induced fibrosis. Characterization
of integrin profiles showed that fibrocytes express
lower integrin levels than fibroblasts, and periostin influences the expression of some integrin
subunits. Fibrocytes and fibroblasts appear to use
different integrins to signal via periostin. For instance, in fibrocytes, treatment with a beta 1 integrin blocking antibody decreased periostin-induced
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collagen 1 mRNA expression, but this was not true
in fibroblasts. Current studies are underway to determine the alpha chain that beta 1 is bound to on
the surface of fibrocytes as well as to assess how
the integrin profiles change during the evolution of
bleomycin-induced fibrosis.
110
11. Immune response to bacteria and
parasites II
121
S.
aureus
, Kyle Jensen, Bryan Hair, Jacob
Hatch,TylerWhite,BradfordBerges
Department of Microbiology and Molecular Biology
Brigham Young University, Provo
Staphylococcus aureus, a normally commensal bacteria, is an opportunistic pathogen that can cause
life-threatening disease. These infections are becoming increasingly dangerous and difficult to
treat due to the growth in cases involving strains
of methicillin-resistant Staphylococcus aureus
(MRSA), leaving vancomycin as the only effective
antibiotic. With the fear that MRSA strains may
also gain vancomycin resistance, new options are
needed to control S. aureus, especially in hospitals
where nosocomial infections are a very serious
concern. With this goal in mind, we have isolated
18 novel bacteriophages that can lyse MRSA. We
have isolated over 40 strains of S. aureus, many of
which are MRSA, from environmental samples. Lysogens were induced by mitomycin C treatment of
these strains, and lytic phages were isolated from
environmental samples. All strains were purified
by plaque purification and high-titer lysates were
produced. We are currently evaluating the hostrange and killing ability of these phages to identify
those with broad tropism as potential candidates
to be used in a phage cocktail that could kill diverse
S. aureus strains. Such a cocktail may be useful to
decontaminate surfaces and possibly to treat human infections.
122 Community-associated methicillin-resistant
Staphylococcus aureuspromotes RIP1-depen,WilliamNauseef
Inflammation Program and Department of Internal
Medicine, Roy J. and Lucille A. Carver College of Medicine
University of Iowa, and Veterans Administration Medical
Center, Iowa City
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Community-associated methicillin-resistant S. aureus (CA-MRSA) is a dangerous human pathogen.
Neutrophils (PMN) are the first responders during
a staphylococcal infection; however 10-30% of the
initial ingested inoculum survives within the PMN
phagosome. Despite extensive study of staphylococcal infections, the contribution of S. aureus-laden PMN to disease pathogenesis is unknown. We
hypothesize that CA-MRSA manipulates the immune response in two complementary ways, by
avoiding engulfment by macrophages (MΦ) and by
promoting programmed necrosis of PMN. In support of this hypothesis, we demonstrate that MΦ
ingested apoptotic PMN rapidly, whereas PMN harboring viable CA-MRSA strain USA300 LAC (PMNSA) upregulated the “don’t eat me” signal CD47, remained bound to the surface and were inefficiently
ingested by MΦ. Although PMN exhibited limited
hallmarks of apoptosis within 3h following ingestion of CA-MRSA, including increased exposure of
phosphatidylserine and mitochondrial membrane
depolarization, PMN failed to activate caspase 3,
upregulated PMN pro-survival factor PCNA, and
lysed within 6h. Lysis of PMN-SA was dependent on
RIP1, suggesting that PMN-SA die via necroptosis.
In the cytosol of resting PMN, PCNA was detected
within the ripoptosome complex, including RIP1,
RIP3, FADD, and caspase 8. Elucidation of how
phagocytosis of CA-MRSA initiates necroptosis and
modifies the ripoptosome in PMN may provide insight into novel CA-MRSA therapies.
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are not due to impaired systemic responses
Luqiu Chen,CarolineBartman,MichaelDavid,Susan
Boyle-Vavra,AnitaChong,RobertDaum,Maria-Luisa
Alegre
Dept of Medicine and Pediatrics, University of Chicago
MRSA causes skin and soft tissue infections (SSTIs)
in epidemic proportions among otherwise healthy
individuals. The prevalent strain of S. aureus isolated from CA-MRSA infections is USA300. Although
many healthy people are colonized with MRSA, few
individuals develop SSTIs prompting the hypothesis that a healthy immune system may limit MRSA’s
growth or virulence. Indeed, S. aureus can activate
112
immune cells via engagement of TLR and NOD-like
receptors. In this study we tested whether individuals with CA-MRSA SSTIs have a mild genetic defect in systemic innate or adaptive immunity that
makes them poor responders to MRSA antigens
and more susceptible to this infection. PBMCs from
71 patients and 142 controls were stimulated with
MRSA lysates, Pam3CSK4 (Pam3), or anti-CD3, and
supernatants were collected at 24h for detection
of cytokines by ELISA. The production of IFNγ and
IL-17 against anti-CD3 was similar in patients and
controls. MRSA and TLR2 agonist Pam3 stimulated
increased secretion of IL-1β, IL-6 and TNF in patients than controls, especially those infected with
USA300. Elevated serum cytokine levels of IL-6
and TNF in patients were also observed. No defect
was found in the ability of PBMCs from patients to
produce innate or adaptive cytokines in response
to MRSA lysate or anti-CD3 mAb suggesting that
susceptibility to infection is not due to impaired
systemic responses to MRSA antigens. Rather, patients displayed evidence of an ongoing immune
response to the infection.
124
Francisella tularensis
, K Laura del Barrio, Joseph
Reynolds, Fabio Re
Department of Microbiology and Immunology, Rosalind
Franklin University of Medicine and Science, North Chicago
The role of IL-1β and IL-18 during lung infection
with Francisella tularensis LVS has not been characterized in detail. Here, using a mouse model of
pneumonic tularemia, we show that both cytokines
are protective, but through different mechanisms.
Il-18-/- mice quickly succumb to the infection and
showed higher bacterial burden than C57BL/6J
wild type mice, and lower level of IFNγ in BALF and
serum. Administration of IFNγ rescued the survival of Il-18-/- mice. In contrast, mice lacking IL-1
receptor or IL-1β, but not IL-1α, appeared to control the infection in its early stages, but eventually
succumbed. IFNγ administration had no effect on
Il-1r1-/- mice survival. Il-1r1-/- mice were found to
have significantly reduced titer of Ft LPS-specific
IgM. The anti-Ft LPS IgM was generated in a IL-1β-,
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TLR2-, and ASC-dependent fashion and was shown
to be protective in passive immunization experiments. A significant decrease in the percentage of
B1a B cells was observed in the spleen and peritoneal cavity of infected Il-1b-/- mice, compared to
C57BL/6J mice, suggesting that these cells are responsible for production of the protective anti-Ft
LPS IgM. Collectively, our results show that IL-1β
and IL-18 activate non-redundant protective responses against tularemia and identify an essential role for IL-1β in the rapid generation of pathogen-specific IgM by B1a B cells.
125
B/Macrophage cells in vitro and in
pneumonic Francisella tularensis
, Jerod Skyberg, Carrie Lasky, Charles
Brown
University of Missouri Department of Veterinary
Pathobiology, Columbia
Biphenotypic B/Macrophage cells are a unique cell
type derived from B-cells that co-express both B
and macrophage cell surface markers (B220, IgM,
F4/80, and CD11b). Following treatment with
M-CSF and GM-CSF, splenic B-2 B cells can be induced to transition into biphenotypic cells. As the
cells transition, the expression of CD19 becomes
down-regulated, CD11b and F4/80 become up-regulated, and B220 and IgM expression remain static. mRNA microarray analysis of in vitro biphenotypic cells demonstrates the expression of several
monocyte and lymphocyte chemotactic receptors.
Biphenotypic cells are capable of phagocytosing
opsonized murine RBCs and Borrelia burgdorferi.
To evaluate the presence of these cells in an infectious model, B6 WT mice were intratracheally inoculated with the LVS strain of Francisella tularensis,
a pulmonary pathogen. Biphenotypic cells within
the pulmonary tissue were significantly elevated
as compared to uninfected mice on days 10 and
14 (p<0.0001, p<0.001, respectively) post-infection, and corresponded to the resolution of disease.
Preliminary data suggests intratracheal M-CSF administered post infection may alter recovery times
from pneumonic Tularemia. Further functional
studies are underway to elucidate the role of bi-
114
phenotypic cells in disease, which may provide the
premise for devising therapies to enhance or attenuate their development.
Bacillus
subtilis exopolysaccharides
Erica Fleming
Loyola University, Chicago
Commensal bacteria residing in the gastrointestinal tract are important modulators of immune
homeostasis. We have found that the commensal
bacterium, Bacillus subtilis, protects against disease induced by the murine enteric pathogen, Citrobacter rodentiumwhen administered orally one
day before infection. This protective effect is mediated by the exopolysaccharides (EPS) secreted
fromB. subtilis, and purified EPS administered i.p
is sufficient for limiting disease. Previous studies
demonstrated that EPS induces anti-inflammatory M2 macrophages in the peritoneal cavity three
days post-EPS. Additionally, the total peritoneal
cell population from an EPS-treated mouse can be
transferred into a naïve host and confer protection
after C. rodentium challenge. My goal is to identify
other innate immune cell types that contribute to
the anti-inflammatory effects of EPS. One cell type
closely associated with M2 macrophages is the eosinophil. Eosinophils can produce IL-4 and IL-13,
two cytokines that induce and sustain M2 macrophages. Additionally, M2 macrophages secrete YM1, a chemoattractant for eosinophils. We are testing if EPS treatment modulates the frequency and
function of eosinophils and if eosinophils contribute to EPS-mediated protection.
127 Mechanism by which commensal
Mallory L Paynich,SaraEJones,KatherineLKnight
Department of Microbiology and Immunology Loyola
University Chicago, Maywood
Trillions of bacteria live in homeostasis within
the gastrointestinal tract and provide a variety of
benefits to the host immune system. Many of these
commensal bacteria have been shown to limit colitis; however, little is known about the mechanisms
by which this occurs. We utilize a mouse model in
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which a single dose of the commensal bacterium,
B. subtilis, protects mice from acute colitis induced
by the enteric pathogen C. rodentium. Our goal is
to elucidate the mechanism by which B. subtilis
prevents inflammation. We determined that a single i.p. dose of exopolysaccharides (EPS) produced
by B. subtilis protects mice from disease. EPS binds
F4/80+CD11b+ peritoneal macrophages (MΦ), and
after i.p. injection of EPS, we identified peritoneal
F4/80+CD11b+ cells that are CD206+Arg1+YM-1+MHCII+, indicative of M2 MΦ. We further showed that
EPS-induced M2 MΦ suppress T cell proliferation
in vitro. CD4+ T cells drive much of the inflammation associated with C. rodentium infection; since
EPS-induced M2 MΦ suppress T cell proliferation
and activation in vitro, we hypothesized that EPS
suppresses CD4+ T cell responses in vivo. Accordingly, we measured levels of cytokines IFNγ (Th1),
IL-17 (Th17), and IL-13 (Th2) in splenic T cells following EPS treatment and found decreased levels
of these cytokines. We hypothesize that EPS-induced M2 MΦ mediate suppression of CD4+ T cell
responses in vivo and further, that these cells mediate protection from acute colitis induced by C.
rodentium.
128
Listeria monocytogenes
Grant Jones,TanyaMyers-Morales,SarahD’Orazio
University of Kentucky, Lexington
Listeria monocytogenes (Lm) is a facultative intracellular bacterium that invades the intestinal barrier and causes systemic infection in susceptible
individuals after the consumption of contaminated
food. We previously showed that intracellular Lm
were a minimal fraction of the total Lm burden in
the intestines, but were vital for spread to the mesenteric lymph nodes (MLN) after food borne infection in mice. Two days after infection with GFP-expressing Lm, a large proportion of GFP+ cells in the
intestinal lamina propria and MLN were Ly6ChiCD11bhiLy6G-, a phenotype consistent with inflammatory monocytes. Surprisingly, when these cells
were sorted from the MLN of infected mice, only
half of the cell-associated Lm was resistant to gentamicin, an antibiotic that kills only extracellular
116
bacteria, and, few Ly6Chi cells contained cytosolic
Lm as defined by the presence of actin tails. In vitro
infection of bone marrow-derived monocytes confirmed that Ly6Chi cells were significantly more resistant to Lm invasion compared to macrophages,
or differentiating monocytes that had up-regulated
FcγR1. Thus, Lm associates with Ly6Chi inflammatory monocytes in the gut mucosa, and adherent
Lm may be transported to the MLN by these cells,
but Lm is not efficiently taken up by Ly6Chimonocytes.
Listeria monocytogenes is not
,TanyaMyers-Morales,SarahD’Orazio
University of Kentucky, Lexington
Wild-type (WT) mice are significantly more susceptible to intravenous (IV) L. monocytogenes (Lm)
infection than are mice lacking the Type I interferon receptor (IFNAR1-/-), suggesting that IFNα/β
signaling promotes an environment favoring bacterial growth. One mechanism proposed to explain this is IFNβ-dependent silencing of IFNγR1
transcription in phagocytes, which results in fewer
activated cells able to restrict intracellular growth
of Lm. Here, we showed that during foodborne Lm
infection, WT mice were not more susceptible than
IFNAR1-/- mice. Surprisingly, there was no detectable difference in IFNα/β levels in the spleen
after IV or foodborne infection of WT mice; however, both routes yielded decreased IFNγR1 levels
on phagocytes. Furthermore, surface IFNγR1 was
reduced on macrophages and dendritic cells even
in IFNAR1-/- mice, indicating that IFNα/β-independent mechanisms can also modulate IFNγR1
expression during Lm infection. Together, these
results suggest that modulation of IFNγR1 expression on phagocytes does not account for differences
in susceptibility between IFNAR1-/- and WT mice
and, additionally, that Type I interferon may not be
a critical mediator of early innate immunity when
Lm infection occurs by the natural route.
130
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,AlbertJergens,Alexandra
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Proctor,GregoryPhillips,JesseHostetter,Michael
Wannemuehler
Iowa State University, Ames
LF82 is an adherent invasive E. coli (AIEC) strain
associated with Crohn’s disease. We have previously used C3H mice colonized with the Altered
Schaedler Flora (ASF) to demonstrate that adult
mice (F0) colonized with LF82 are more susceptible to DSS-induced colitis than control ASF mice.
For the current studies, we hypothesized that ASF
mice colonized with LF82 by vertical transmission
(F1) would be less susceptible to DSS-induced
colitis compared to F0 ASF mice colonized with
LF82. Adult mice were colonized with E. coli LF82
and bred to produce LF82-colonized F1 pups. At
8 weeks of age, F0 and F1 mice were exposed to
drinking water with 2.0% DSS for seven days. At
necropsy, gross and microscopic lesion scores were
more severe (i.e., diffuse, transmural colonic inflammation) in F1+DSS compared to F0+DSS mice.
Despite the more severe DSS-induced colitis, evaluation of serum antibody and T cell immunoreactivity revealed less robust ASF-specific responses in
F1+DSS mice. Results demonstrated that mice colonized with LF82 by vertical transmission develop
more severe DSS-induced colitis compared to mice
colonized with LF82 as an adult suggesting that
the age at which an individual is colonized with a
pathobiont adversely impacts the susceptibility to
subsequent colitic insults.
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12. Immune response to bacteria and
parasites III
131 Liver induced immunosuppression by
Jonathan Kurtz, James McLachlan
Tulane University School of Medicine, New Orleans
What role microenvironments play in dictating
immune outcomes during infection is largely unknown. Our lab combines a persistent S. Typhimurium infection model with recombineered
Salmonella strains and MHC-II tetramers to visualize endogenous CD4 T cell responses in various
organs. We hypothesize a stalemate between bacterial persistence and the host immune response is
determined by anatomical location, which dictates
T cell function and infection outcome. We show Salmonella-specific CD4 T cells transferred from lymphoid organs protect mice from challenge, while
CD4 T cells from infected livers increase susceptibility. Furthermore, lymphoid Salmonella-specific Th1 cells produce higher levels of IFN-γ, while
FoxP3- liver CD4 T cells produce larger amounts of
IL-10. Additionally, we show lymphoid Th1 cells are
potent activators of iNOS and reduce macrophage
bacterial loads, while liver-derived Tr1-like cells
induce an alternatively activated macrophage state
and fail to control bacterial replication, through attenuated iNOS activation. Finally, we show livers of
Salmonella infected mice induce immunosuppressive Tr1-like cells. These results demonstrate that
during persistent Salmonella infection different
immunological responses occur at different anatomical sites, which may dictate infection outcome.
132
permeability in vital brain regions during
experimental cerebral malaria
1, 2
,FangJin1, Aaron Johnson1
1
Mayo Clinic Department of Immunology, 2Mayo Graduate
School, Rochester
One of the most severe complications of Plasmodium falciparum infection in humans is cerebral ma-
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laria. Complications from cerebral malaria lead to
coma, neurological deficits, and potentially death.
The disease is characterized by blood-brain barrier
(BBB) disruption and vascular permeability. Due
to this association we investigated cerebral endothelial cell tight junction protein alterations and
BBB breakdown in the Plasmodium berghei ANKA
(PbA) model of experimental cerebral malaria.
PbA infected C57BL/6 mice showed significant areas of vascular leakage within the central nervous
system six days post infection. Regions of permeability co-localized with disruption of the BBB tight
junction proteins claudin-5 and occludin on microvasculature as shown using confocal microscopy.
Additionally, MRI revealed that PbA infected mice
had vascular leakage specific to the hypothalamus,
thalamus, and brain stem. In contrast, PbA infected perforin deficient mice retained tight junction
integrity and displayed significantly reduced vascular leakage. We propose that perforin released
from CD8 T cells contributes to vascular permeability through BBB tight junction disruption. In
turn, the resulting disruption of homeostasis in vital brain regions likely contributes to the pathology
and morbidity associated with PbA infection.
133
Andrew Shepherd2,ChrisLantz1, Tracy Deem2
1
James Madison University, Harrisonburg, 2Bridgewater
College, Bridgewater
Malaria is a leading cause of death in many developing countries, especially among women and
children. Mechanisms for malaria infection can be
examined with murine models. Previous research
in our lab has shown that IL-3 KO mice survive longer than WT mice possibly due to an overwhelming inflammatory response in WT mice. It was
recently shown that malaria infection leads to increased plasmacytoid dendritic cell (pDC) activation and secretion of IFN-α resulting indirectly in
mast cell release of Flt3L, which activates classical
dendritic cells (cDC) to activate T cells. Since IL-3
is involved in mast cell development and survival,
we hypothesized that IL-3KO mice survive longer
due to differences in dendritic cell activation and
Flt-3L production. Therefore, we used flow cytom-
120
etry to quantify the percentage of pDC and cDC in
Plasmodium berghei-infected KO and WT mice and
used an ELISA to measure splenic and serum levels of Flt3L. Our results show the percentages of
cDC and pDC are the same in both types of infected mice even though Flt3L is elevated in the blood
and spleen of WT mice. Further investigation of DC
co-stimulatory markers will determine if there are
differences in DC and T cell activation that might
account for longer survival in KO mice.
134
1
, Jaci Oliveira2,PedroCarneiro2,Smriti
Sharma3, Olivia Bacellar2,EdgarCarvalho2, Mary
Wilson1, 4, 5
1
Interdisciplinary Graduate Program in Immunology,
University of Iowa 2Fedral University of Bahia (UFBA),
Salvador, Brazil, 3Banaras Hindu University, Varanasi,
4
Veterans Administration Medical Center, Iowa City,
5
Department of Internal Medicine, University of Iowa, Iowa
City
The protozoan parasite Leishmania braziliensis
causes cutaneous leishmaniasis (CL) in individuals
living in endemic regions. Neutrophils (PMN) are
short-lived effector cells, and their phenotype and
potential contribution during the overabundant
inflammatory response of acute leishmaniasis remains unexplored. With investigators from UFBA,
we studied PMN from peripheral blood of patients
with acute CL encountered in a rural leishmania-treatment center in Corte de Pedra, Bahia. Parallel studies were done in a mouse model of leishmaniasis. Surprisingly, both human CL patients and
mouse models of leishmaniasis showed a subset of
neutrophils that express MHCII. MHCII+ PMN were
identified in the site of parasite infection, draining
lymph nodes and circulation in a mouse model of
CL. Further characterization of MHCII+PMN in CL
patients showed increased markers of PMN activation, degranulation, and ROS production (following
stimulation) compared to patient MHCII- PMN. MHCII+ PMN also showed increased surface co-stimulatory molecules (CD80 and CD86). Co-incubation
of PMN and T cells in the presence of Leishmania
antigen resulted in enhanced T cell proliferation in
vitro, suggesting a role for MHCII+ PMN in antigen
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presentation and/or other means of stimulating T
cell responses. These data suggest that Leishmania
infection promotes the emergence of a subset of
PMN with a unique activation and immune profile
in both CL patients and mouse models.
135
Soluble immune complexes promote
Leishmania amazonensis
Marie Bockenstedt,DouglasJones,BrettSponseller
Iowa State University, Ames
The intracellular protozoal parasite Leishmania
can cause cutaneous leishmaniasis, a vector borne
disease that infects approximately 1 million people
each year. C3H mice infected with L. amazonensis
develop chronic cutaneous lesions with large parasite loads. Using an in vitro assay with immune
cells from infected mice we have shown that intracellular killing of L. amazonensis parasites is
dependent upon FcRγ common-chain and NADPH
oxidase-generated superoxide from infected macrophages. We have previously shown that macrophage activation in response to soluble IgG2a immune complexes, IFN-γ and parasite antigen was
effective in killing L. amazonensis. Here we show
that antibody-enhanced intracellular killing is associated with an upregulation of autophagy as
determined by an increase in LC3 II and colocalization of LC3 with parasites within established
parasitophorous vacuoles. These experiments define a mechanism by which antibodies can promote
killing of an intracellular pathogen post-infection.
to Plasmodium berghei
malaria
Erik Ehinger1,ChrisLantz1, Tracy Deem2
1
James Madison University, Harrisonburg, 2Bridgewater
College, Bridgewater
Malaria is a potentially fatal disease caused by
parasites of the genus Plasmodium and spread
by the Anopheles mosquito. In 2012 there were
207 million cases contributing to 627,000 deaths
worldwide (WHO, 2014). In a murine model, IL-3
was shown to decrease survival times compared
to IL-3 KO mice even though IL-3 KO mice were
122
more anemic and had larger spleens (Auclair. et al.,
2013). Differences in survival and spleen size were
not due to differences in hematopoiesis; however,
IFN-g levels were higher in the blood of WT mice
early in infection. Therefore, we hypothesized that
WT mice have decreased survival rates due to an
overwhelming inflammatory response. Investigation of the adaptive immune response by flow
cytometry shows there were no differences in the
percentage of splenic regulatory T cells; however,
WT mice had higher percentages of splenic CD4+
T cells, whereas KO mice had higher percentages
of CD8+ T cells. The percentage of splenic B cells
was not different; however, initial data has shown
higher total antibody levels in KO mice. Further investigation is needed to determine if these antibodies are parasite-specific. Taken together, these data
suggest that IL-3-mediated differences in early innate immune responses may alter adaptive immunity and thus survival rates.
137 Leishmania amazonensis-mediated ERK1/2
Hailie Fowler1,PedroMartinez2,ChristinePetersen1, 2
1
Immunology Program, Carver College of Medicine,
Department of Epidemiology, College of Public Health,
University of Iowa, Iowa City
2
Leishmania amazonensis is a parasitic protozoa
that can result in cutaneous leishmaniasis. Leishmania infects host phagocytic cells and remains
within macrophage parasitophorous vacuoles
during infection. L. amazonensis-mediated ERK
activation occurs secondary to localization of ERK
with MP1 and p14, scaffolding proteins found with
late endosomal parasite-containing vacuoles. Phosphorylated Akt contributes to the NADPH complex
formation within host cells, allowing host cells to
generate reactive oxygen species to combat intracellular infections.
Using murine macrophage cell lines we will determine the role of ERK and Akt in NADPH complex
formation within L. amazonensis infected cells.
Here, we indicate that Leishmania infections resulting in ERK activation, reduced NADPH subunit
co-localization and reduced reactive oxygen spe-
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cies production, in comparison to L. major infections that did not upregulate ERK. This reduction in
NADPH complex formation likely promotes a more
favorable microenvironment for parasite survival
and reproduction, corresponding to the increased
parasitic burden and chronic infection established
by L. amazonensis, suggesting ERK as a potential
drug target for the treatment of chronic Leishmania infections.
138
immunity in IL-3-mediated pathogenesis
Tracy Deem1,ChrisLantz2, Samantha Saylor1
1
Bridgewater College, Bridgewater, 2James Madison University, Harrisonburg
Malaria is a leading cause of death, especially in
children and pregnant women in developing countries. Using a murine model, we examined the role
of Interleukin-3 (IL-3) during malarial infection. We
previously reported Plasmodium berghei NK65-infected IL-3 knock-out (KO) mice survive longer
than WT mice for reasons unrelated to hematopoiesis. In addition, serum levels of Interferon (IFN)-g
are higher in WT mice early in infection. Therefore,
we hypothesized that WT mice die faster than IL-3
KO mice due to a quick and overwhelming inflammatory response mediated by innate immune cells.
We initially looked at innate immune cells known
to secrete IFN-g, namely NK and NKT cells. Flow
cytometry of blood leukocytes and splenocytes
showed no difference in the percentages of NK and
NKT cells, suggesting a currently unidentified cell
type may be the source of the IFN-g. In addition to
these cells, neutrophils and macrophages also play
a role in inflammation. Neutrophil percentages
were higher in the blood and spleen of WT mice.
In addition, WT mice had a higher percentage of
inflammatory macrophages even though KO mice
had a higher total macrophage percentage. Taken together, these data suggest that inflammatory
cells are recruited early and at higher levels in WT
mice.
,
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Janet Fairley3, 5, Mary Wilson1, 2, 3, 4, 5
early
ection
para-
140
relates with disease progression inLeishmania infantum
Robert Schaut1, Ian Lamb1,TaraGrinnage-Pulley1,
PedroMartinez2, Kevin Esch3,ChristinePetersen1
1
University of Iowa, College of Public Health, Iowa City,
Mount Sinai School of Medicine, New York, 3Iowa State
University, College of Veterinary Medicine, Ames
2
Leishmania infantum, a protozoan parasite, is the
causative agent of visceral leishmaniasis (VL). Untreated VL has been shown to produce immune ex-
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haustion and death. Seroconversion to parasite antigen was delayed months to years after infection,
followed by hyperimmunoglobulinemia. Previous
reports have demonstrated atypical or regulatory
IgD expressing B cells arising under chronic inflammatory conditions such as HIV, malaria, lupus
and rheumatoid arthritis and contributing to disease exacerbation. Sources of IL-10 during clinical VL and relative roles for regulatory B cells in
promotion of immune exhaustion are not known.
Here, we describe a novel class of regulatory B cells
which contributed significantly to IL-10 production
during progressive VL. These cells had increased
IgD expression. Gene transcription of STIM-1,
BAFF, FoxP3 and Sema3A, known regulatory transcription factors were all significantly increased in
symptomatic immune cells; whereas transcription
of Th1-inducing STIM-2 was decreased compared
to endemic control animals. As VL progressed B
cell IgD surface expression significantly increased,
whereas total peripheral B cell population size was
not changed. A predominant number of IgD++ B
cells were IL-10-positive via intracellular FACS
regardless of clinical state indicating that IgD++
B cells consistently were an IL-10-producing cell.
This unique class of B cells may be important for VL
progression and immune exhaustion.
141
,CarmelaPratt,CharlesBrown
University of Missouri Department of Veterinary
Pathobiology, Columbia
Leukotriene B4 (LTB4) is a potent lipid chemoattractant produced and released during the innate
immune response to inflammation by neutrophils,
mast cells, and macrophages. When released, LTB4
binds with its high affinity receptor BLT1, which
is located predominately on the surface of leukocytes. Upon binding, LTB4 induces neutrophil and
monocyte chemotaxis to the site of inflammation.
Previous studies in autoimmune arthritis have
shown that inhibition of BLT1 prevents recruitment of neutrophils and macrophages, supporting
that LTB4 and BLT1 act in concert to produce tissue inflammation. Arthritis severity scores demonstrate that the development of Lyme arthritis at the
126
time of peak inflammation on day 21 appeared delayed as compared to C3H WT mice. However, 35
days post-infection, the arthritis severity scores of
BLT1-/- mice were higher than WT mice, though
not statistically significant. Borrelia ear loads were
similar between both strains of mice. These results
suggest that BLT1 is not necessary for the development of Lyme arthritis, and its absence does not
influence the tissue clearance of Borrelia.
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13. Immune response to viruses II
142
state.
Zeb Zacharias, Kevin Legge
Immunology Graduate Program and Department of Pathology, University of Iowa, Iowa City
Respiratory dendritic cells (rDC) have a pivotal role
in the induction of the CD8 T cell response to pulmonary pathogens such as influenza A virus (IAV).
Upon encountering viral antigen, rDC mature and
transiently exhibit enhanced migration to lung
draining lymph nodes (LN) where they interact
with and activate naive IAV-specific CD8 T cells.
Interestingly, the remaining rDC in the lungs are
refractory to further migration stimuli including
secondary pathogens. This inhibition of rDC migration results in a diminished CD8 T cell response to
the secondary pathogen. However, the full extent of
the defect in the CD8 T cell compartment caused
by this lack of DC migration during co-infections
and memory remains unknown. Our studies show
that IAV infections after the induction of the DC refractory state result in reduced IAV-specific CD8 T
cell responses at both effector and memory time
points. Further the central, effector, and tissue resident CD8 T cell memory compartments in the LN
and lungs were differentially reduced. Overall these
findings suggest that pathogen encounters after induction of the rDC refractory state will reduce CD8
T cell responses during the initial coinfection and
may leave the host susceptible to subsequent encounters with the same pathogens.
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Cory Knudson1,LeciaPewe2,DavidMeyerholz3,
KatherineGibson-Corley3,RyanLanglois4, Benjamin
tenOever5, John Harty1, 2, 3,StevenVarga1, 2, 3
1
Interdisciplinary Program in Immunology, 2Department
of Microbiology, 3Department of Pathology, University of
Iowa, Iowa City, 4Department of Microbiology, University
of Minnesota, Minneapolis, 5Department of Microbiology,
Mount Sinai Hospital, New York
CD8 T cells play a critical role in anti-viral immune
responses and mediate viral clearance following
a respiratory syncytial virus (RSV) infection. Here
we questioned if memory CD8 T cells alone could
provide protective immunity against RSV in the absence of CD4 T cell memory and antibodies. High
magnitude CD8 T cell memory responses were generated by immunizing naïve mice with dendritic
cells pulsed with an RSV-derived peptide followed
by a boost with a recombinant L. monocytogenes
engineered to express the same RSV-derived epitope. Memory CD8 T cells significantly reduced
viral titers following RSV challenge at the cost of
increased immunopathology. RSV-specific memory
CD8 T cell responses were associated with significantly increased airway dysfunction, weight loss,
and mortality as compared to mock-immunized
controls. High magnitude CD8 T cell memory responses led to the induction of a proinflammatory cytokine storm in the serum following RSV
challenge. Depletion of the inflammatory cytokine
TNF-α in prime-boosted mice led to a significant
amelioration of weight loss and survival of all subjects. Furthermore, the severe immunopathology
and mortality was observed only in prime-boosted
mice upon RSV challenge and not following infection with a recombinant influenza virus strain that
expresses the same RSV-derived epitope. Our results demonstrate that memory CD8 T cells can mediate protection against RSV infection, but induce
the development of significant immunopathology.
144
,JasonSchenkel,LalitBeura,Clare
Quarnstrom,VaivaVezys
129
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143
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University of Minnesota, Minneapolis
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Certain viruses can outpace and/or impair host
immune responses to establish chronic infections.
During chronic infections CD8 T cells become exhausted and lose function in a hierarchical fashion.
We believe the increasing the number of functional
virus-specific CD8 T cells is critical for clearance
of chronic viral infections. To restore functional
immune responses, we have tested a therapeutic
heterologous-vector vaccination strategy in the
mouse model of chronic infection, LCMV clone-13,
as well as in the non-human primate model of SIV.
In vaccinated mice, the number and function of viral-antigen specific CD8 T cells is increased while
viral burden is decreased. We hypothesize that we
are changing the CD8 repertoire by eliminating the
most exhausted CD8 T cells and allowing the small
percentage of newly formed functional cells to expand. Our method has also shown promise in the
non-human primate model of SIV. We propose that
therapeutic heterologous vaccination is a method
that can augment treatments for chronic infections.
145
Elizabeth Steinert1, 2, 5, Jason Schenkel1, 2, 5, Kathryn
Fraser1, 2, 5, Lalit Beura1, 2, 5, Luke Manlove2, 3, 5, Botond
Igyarto2, 4, 5, David Masopust1, 2, 5
1
Department of Microbiology, 2Center for Immunology,
Department of Laboratory Medicine and Pathology,
4
Department of Dermatology, Minneapolis, 5University of
Minnesota, Minneapolis
3
Memory CD8 T cells protect against intracellular
pathogens by scanning host cell surfaces, thus infection detection rates depend on memory cell
number and distribution. Population analyses rely
on isolation from whole organs and interpretation
is predicated on presumptions of near complete
cell recovery. Paradigmatically, memory is parsed
into central, effector, and resident subsets, ostensibly defined by immunosurveillance patterns, but in
practice identified by phenotypic markers. Because
standard isolation methods ultimately inform models of memory T cell differentiation, protection¬,
and vaccine translation, we tested the validity of
standard methods via parabiosis and quantitative
immunofluorescence microscopy of a mouse mem-
130
ory CD8 T cell population. We found that lymphocyte isolation fails to recover most cells, recovery
is biased against certain subsets and resident cells
greatly outnumber recirculating cells within nonlymphoid tissues. These results revise concepts
of T cell distribution, subsets and inform ongoing
studies of host immunosurveillance.
Holly Johnson1, 3, 4
1, 2
,FangJin1,
Whitney Manhart1, Istvan Pirko3, Aaron Johnson1, 3
1
Department of Immunology, Mayo Clinic, 2Virology and
Gene Therapy Graduate Program, Mayo Graduate School,
3
Department of Neurology, Mayo Clinic,4Neurobiology
of Disease Graduate Program, Mayo Graduate School,
Rochester
C Numerous neurological disorders are characterized by central nervous system (CNS) vascular permeability. Inflammatory-derived factors leading
to pathology associated with blood-brain barrier
(BBB) disruption remains poorly understood. In
order to address this, we developed an inducible
model of BBB disruption using Theiler’s murine
encephalomyelitis virus (TMEV). This peptide induced fatal syndrome (PIFS) model is initiated by
virus-specific CD8 T cells and is perforin dependent. However, the cellular source of perforin has
remained unidentified. In addition to CD8 T cells,
various innate immune cells also express perforin
and therefore could also contribute to BBB disruption. To investigate this, we isolated the CD8 T cell
as the sole perforin-expressing cell type in the PIFS
model through adoptive transfer techniques. We
determined that C57BL/6 perforin-/- mice reconstituted with perforin competent CD8 T cells and
induced to undergo PIFS exhibited: 1) heightened
CNS vascular permeability, 2) increased astrocyte
activation as measured by GFAP expression, and
3) loss of linear organization of BBB tight junction proteins claudin-5 and occludin in areas of
CNS vascular permeability when compared to
mock-treated controls. These results are consistent with the characteristics associated with PIFS
in perforin competent mice. Therefore, CD8 T cells
are sufficient as a sole perforin-expressing cell type
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to cause BBB disruption in the PIFS model.
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147
April Huseby,FangJin,AaronJohnson
Department of Immunology, Mayo Clinic, Rochester
Theiler’s murine encephalomyelitis virus (TMEV)
infection of C57BL/6 mice has served as a model
of acute neuroinflammation Immune cells have
been shown to be both pathogenic and protective
during the acute stage of TMEV infection. Our lab
has previously investigated the role for CD8 T cells
directly interacting with virally infected neurons
using confocal microscopy. Through this work, we
demonstrated polarized immunological synapse
formation in the CD8 T cell toward the virally infected neuron. IN a subsequent study using confocal microscopy, we observe CD4 T cells are also in
tight apposition to virally infected neurons. This
observation prompts an analysis to define the interaction between the CD4 T cell and the virally
infected neuron. Defining the CD4 subset/s is a
crucial first step to assessing their potential protective effector mechanisms. Future studies will
investigate the role for Kupfer or non-Kupfer type
synapses both statically using confocal microscopy
and dynamically with two-photon laser scanning
microscopy (2PLSM), facilitating the assessment of
CD4 T cell engagement with neurons in vivo.
148
Author’s request:
Perlamn
Do, Stanley
not include
my abstract
the Iowa
online
University ofinIowa,
Cityversion of the
abstract
book
Mice infected
with the
neuroattenuated rJ2.2 strain
of the murine coronavirus, mouse hepatitis virus
(MHV), develop acute encephalitis and acute and
chronic demyelinating diseases. The immuno-
132
ic CD4 T cells derived from the memory pool are
protective. We use Venezuelan equine encephalitis
replicon particles (VRP) expressing the M protein
cytomegalovirus
Maha Almanan1, 2, Jana Raynor1, 2,MeiWang2, 3,
ClaireChougnet1, 2,RhondaCardin2, 3, David
Hildeman1, 2
1
Division of Immunobiology, Cincinnati Children’s Hospital
Medical Center, 2College of Medicine, University of Cincinnati, 3Division of Infectious Diseases, Cincinnati Children’s
Hospital Medical Center
Human cytomegalovirus (HCMV) causes a persistent, lifelong infection. Although the virus persists in a latent state, it also undergoes intermittent
subclinical viral reactivation. Such reactivation
events drive the accumulation of “inflationary” T
cells that suppress viral replication. While T cells
are critical to maintain control of infection, the
factors that prevent T cells from driving complete
viral elimination remain unclear. Here, we investigated the role of regulatory T cells (Treg) in a
mouse model of persistent CMV infection using
FoxP3-diphtheria toxin receptor (DTR) transgenic
mice. FoxP3-DTR mice were infected with murine
CMV (MCMV) and then Treg were depleted via
administration of DT several months later. Treg
depletion resulted in a dramatic increase in the
numbers of MCMV-specific CD4+ and CD8+ T cells
(inflationary and non-inflationary populations)
in the spleen. Functionally, MCMV-specific T cells
from Treg-depleted mice produced significantly
higher levels of IFN-γ and IL-2 upon peptide stimulation compared to Treg sufficient control mice.
Strikingly, Treg depletion led to a significant decrease in splenic viral load. Current experiments
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will examine the effects of Treg depletion on latent
virus using a novel spleen viral reactivation assay.
Combined, these data suggest that Treg promote
persistent MCMV infection, in part, by suppressing
T cell activation.
150 Notch signaling regulates T-cell cytokine
Stephen Gurczynski, Bethany Moore
University of Michigan, Ann Arbor
The Notch signaling pathway has been identified as
an important mediator of T-cell cytokine responses in a variety of immunological maladies such as
graft versus host disease, rheumatoid arthritis,
and systemic lupus erythematosus. Using a genetically altered mouse model wherein CD4+ and
CD8+ T-cells were ablated for all Notch signaling
through expression of a dominant negative version
of the Notch transcriptional regulator Mastermind
Like Ligand (DNMAML) we have further identified
Notch as an important mediator of cytokines during
Murine Gamma Herpesvirus 68 (MHV-68) infection
following both syngeneic and allogeneic bone marrow transplantation (BMT). Wild type syngeneic
bone marrow chimeric mice infected with MHV-68
for 21 days developed pulmonary fibrosis driven,
in part, by increased levels of IL-17. Further, expression of several Notch ligands was markedly
decreased in the lungs following BMT and MHV-68
infection. DNMAML bone marrow chimeric mice
infected for 21 days with MHV-68 developed increased fibrosis in comparison to wild type chimeras, and displayed further elevated levels of IL-17
and IL-6 in the lungs. In contrast, allogeneic transplanted Balb/c mice infected with MHV-68 did not
survive until 21 dpi and succumbed to severe pulmonary pneumonitis and acute lung injury by 1014 dpi due to unchecked viral replication. Together
these data indicate that Notch plays a crucial role
in regulating T-cell cytokine responses during immune reconstitution after BMT.
134
151
Author’s request:
Brian Zamarron,TaleenMergian,Gabriel Do not include my abstract
Martinez-Santibanez,KanakadurgaSinger,Jennifer
in the online version of the
DelProposto,CareyLumeng
abstract book
University of Michigan, Ann Arbor
Obesity induces inflammation in adipose tissue
involving the recruitment and activation of mac-
s showed
en
ht
to
152
immune homeostasis in the adipose
Zuoan Yi
Department of Microbiology, University of Iowa, Iowa City
Chronic inflammation in visceral adipose tissue
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is considered a key element for induction of insulin resistance in obesity. CD40 is required for efficient systemic adaptive immune responses and
is implicated in various inflammatory conditions.
However, its role in modulating immunity in the
microanatomical niches of adipose tissue remains
largely undefined. Here, we show that, in contrast
to its well-documented costimulatory effects, CD40
regulates development of insulin resistance in a
diet-induced obesity (DIO) mouse model by ameliorating local inflammation in adipose tissues.
CD40 deficiency (CD40KO) resulted in greater
body weight gain, more severe inflammation in
epididymal adipose tissue (EAT), and aggravated
insulin resistance in response to DIO. Interestingly, we found that CD40KO CD8(+) T lymphocytes
were major contributors to exacerbated insulin resistance. Specifically, CD8(+) T cells in EAT of DIO
CD40KO mice produced elevated chemokines and
proinflammatory cytokines and were critical for
macrophage recruitment. These results indicate
that CD40 plays distinct roles in different tissues
and, unexpectedly, plays an important role in maintaining immune homeostasis in EAT. Further study
of how CD40 promotes maintenance of healthy metabolism could contribute to better understanding
of and ability to therapeutically manipulate the increasing health problem of obesity and insulin resistance.
153 Endothelial calcium signaling regulates
, Fei Han, William Muller
Northwestern University Feinberg School of Medicine,
Chicago
Leukocyte transendothelial migration (TEM) is a
critical step in the inflammatory response and is
regulated by a multitude of cellular signals. Leukocyte/endothelial PECAM-PECAM interactions,
recruitment of PECAM-rich membrane to surround
the transmigrating leukocyte (targeted recycling,
TR), and a transient increase in endothelial cytosolic Ca2+ ([Ca2+]i) are required for TEM. However, the sequential nature of these events and the
specific Ca2+ channel(s) mediating [Ca2+]i during
TEM are not known. TRPC6, a Ca2+ channel with an
136
established role in inflammation and endothelial
barrier function, co-localized with PECAM at endothelial junctions and surrounded leukocytes during
TEM. Furthermore, polystyrene beads coated with
anti-PECAM Ab recruited local endothelial PECAM
with concomitant TRPC6, suggesting that PECAM
signaling is upstream of TRPC6-mediated [Ca2+]i.
Direct activation of endothelial TRPC6 with a selective agonist was sufficient to restore TEM and TR
in neutrophils blocked with anti-PECAM Ab. Importantly, expression of dominant-negative TRPC6
or knockdown of the channel in endothelial cells
attenuated neutrophil TEM and TR. Whole mount
immunostaining of inflamed tissue revealed a profound defect in neutrophil TEM in chimeric mice
lacking TRPC6 expression in the endothelium. Taken together, our findings demonstrate that TRPC6
is a/the source of endothelial [Ca2+]i at a step
downstream of PECAM interactions in the signaling
pathway that governs leukocyte TEM.
154
Elizabeth Linville, Oliver Oakley, Alex Whitaker
Eastern Kentucky University, Richmond
Ovulation is a naturally occurring process in the
ovary and is preceded by acute inflammation. It
has been hypothesized that estrogen is a key factor
in the inflammatory process and that rising levels
of estradiol produce an anti-inflammatory shield
which is removed during the sharp drop of estradiol after the LH surge prior to ovulation. This experiment examines the role of estradiol and leukocyte function in the ovary during the periovulatory
phase. This relationship is not well understood but
we hypothesize it is an important factor in controlling inflammation and successful ovulation. Immature mice were injected with pregnant mare’s
serum gonadotropin (PMSG) for synchronized
follicular growth, hCG for ovulation induction and
17-beta estradiol. The leukocyte infiltration results
were analyzed at different time points during the
ovulatory cycle using flow cytometry and immunohistochemistry. Results showed that when pretreated with estrogen (17-beta estradiol) the infiltration of leukocytes was delayed or inhibited.
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155
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pre-ovulatory ovary
,ElizabethHorn,OliverOakley
Eastern Kentucky University, Richmond
Ovulation requires the chemotaxis of leukocytes
from the spleen to the ovary. Hormonal signals that
are released during the peri-ovulatory period initiate the release of specific leukocyte populations
that infiltrate the ovary and facilitate the release
of oocytes. We will characterize a specific monocyte population that infiltrates the ovary during a
precise period of time, as well as M1 and M2-like
macrophage populations. Before ovulation occurs,
a Ly-6 monocyte is likely promoting inflammation
and follicular rupture. The Ly-6 monocyte infiltration will start to diminish as they differentiate into
M1 macrophages to further promote inflammation.
These studies will utilize a super-ovulation protocol to initiate ovulation in immature balb/c mice.
Spleens and ovaries will be collected post hCG injection and analyzed by multi-color flow cytometry. Immunohistochemistry will be used to show
the location of the leukocyte populations in the
ovaries. Results from splenectomized and reconstituted mice will demonstrate reduced and corrected
ovulation rates, respectively. Antagonists for CCR2
on the Ly-6 monocyte should also show a reduced
ovulation rate from restricted chemotactic abilities.
Studies should establish that without these subsets
of monocytes and macrophages, the rate of ovulation is reduced. In this regard, in the ovary, the Ly-6
may function by inducing ovulatory inflammation
while the M1 macrophage helps continue the inflammatory process.
Nicholas Goplen, Mark Daniels, Emma Teixeiro
University of Missouri School of Medicine Department of
Molecular Microbiology & Immunology, Columbia
Effector CD8 T-cells provide protection from intracellular pathogens & serve an important role
in cancer surveillance. It has become evident that
pro-inflammatory cytokines can activate effector
CD8 T-cells in the absence of a pathogenic antigen.
How this occurs, the biological relevance and there-
138
fore the impact on disease outcome are unclear.
Strikingly, we have found that upon stimulation
with many cytokines, T-cell Receptor (TCR) or antigen-dependent signaling is induced in effector CD8
T-cells when antigen is not present. For at least one
inflammatory cytokine essential to CD8 responses,
IL-12, its signaling through the TCR enhances multiple effector functions. We have been characterizing biochemical mechanisms by which IL-12 uses
TCR signaling machinery to regulate CD8 T cell
responses. Our data shows that CD8 and self-peptide MHC interactions together with activity of src
kinases are key in this process. These findings may
have unique implications for vaccine design, autoimmune & cancer therapies.
157
Shannon Rapovy1, 2, Stephanie Schmidt1, Rebecca
Bricker1, Joseph Qualls1, 2
1
Cincinnati Children’s Hospital Medical Center, 2University
of Cincinnati
L-Arginine is necessary for T cell function. Diminished L-arginine triggers T cell hypo-responsiveness and the immune system exploits this phenomenon to down-regulate T cell function. Despite this,
T cells may overcome suppression by synthesizing
L-arginine via intracellular L-citrulline metabolism. Host defense during Mycobacterium tuberculosis infection is inhibited in mice lacking L-citrulline metabolism in hematopoietic cells, yet the role
of L-citrulline metabolism for anti-mycobacterial
T cell responses remains unknown. We hypothesize that L-citrulline metabolism is necessary
for antigen-specific T cell function. We first investigated the effects of L-citrulline availability on
antigen-specific T cell function in vitro. Our data
show that antigen-specific T cells utilize L-citrulline for proliferation as efficiently as L-arginine
and overcome arginase-mediated suppression.
Furthermore, mycobacterial antigen-specific T
cells displayed enhanced proliferation in the presence of L-arginine and L-citrulline, compared to
either amino acid alone, in response to heat-killed
M. bovis BCG. Taken together, we demonstrate that
L-citrulline enhances T cell proliferation in vitro,
suggesting an unappreciated role for L-citrulline
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metabolism in T cells in vivo. The contribution of
L-citrulline metabolism for anti-mycobacterial T
cell function in vivo will be investigated in future
experiments utilizing novel mouse models in which
T cells are deficient in L-citrulline metabolism.
158
Sreya Bagchi1,HongZhang2,Chyung-RuWang1
1
Department of Microbiology-Immunology, Feinberg School
of Medicine, Northwestern University, Chicago,
2
Department of Surgery, Thomas E. Starzl Transplantation
Institute, University of Pittsburgh Medical Center
Psoriasis, a chronic inflammatory skin disease, is
associated with hyperlipidemia. While conventional T cells usually exacerbate psoriasis, the role of
self-lipid reactive CD1-restricted T cells remains
elusive. CD1 molecules, which present lipid antigens to T cells, are divided in to two groups. Group 1
includes CD1a, CD1b and CD1c while CD1d belongs
to group 2. Humans express both group 1 and group
2 CD1 molecules whereas mice only have CD1d.
Thus, due to the lack of a suitable animal model,
the role of autoreactive group 1 CD1-restricted T
cells in hyperlipidemia-associated inflammatory
diseases is unknown. To overcome this challenge,
our lab generated mice that expressed human
CD1b and a CD1b-autoreactive T cell receptor (hCD1Tg/HJ1Tg). hCD1Tg/HJ1Tg mice were crossed
to the ApoE-/- background (hCD1Tg/HJ1Tg/ApoE/) to examine the role of CD1b-restricted T cells
in hyperlipidemia. Interestingly, hCD1Tg/HJ1Tg/
ApoE-/- mice developed severe psoriasis-like skin
inflammation characterized by T cell and neutrophil infiltration and a Th17-biased cytokine milieu.
They also showed significantly increased CD1b
expression and polar lipid accumulation in the
skin as compared to wildtype counterparts in the
ApoE sufficient background. This suggested that
the presence of excessive lipids in the skin resulted
in the recruitment and activation of the CD1b-autoreacitve T cells, resulting in the development of
skin disease.
healing
140
AIC 2014 • Chicago, IL
, John Kubasiak2, Frederick
Kohlhapp ,VidyaratnaFleetwood2,JevgenijsLusciks1,
AndrewZloza1, 3
1
Departments of Immunology/Microbiology, 2General
Surgery, 3Internal Medicine, Rush University Medical
Center, Chicago
Background: Engagement of NKG2D on NK cells
and CD8+ T cells results in improved control of infection and tumor growth. However, the effect of
such engagement in the context of wound healing
is currently not well understood. Therefore, we
sought to determine whether engagement of NKG2D utilizing stimulation antibody delivery could
be used to improve wound healing. Methods: A
3-mm punch biopsy was used to create six wounds
on the naired dorsal surface of C57BL/6 mice (510 per group). On days 0 and 1, NKG2D stimulation
antibody (clone A10, 100 µg/mouse) or control
IgG was administered to mice via intraperitoneal
injection. On postoperative day 0 and every day
thereafter, images of dorsal wounds were obtained
and wound healing (i.e., wound size) was analyzed
using ImageJ software. Results: NKG2D stimulation resulted in the hastening of wound healing by
approximately 2 days (P<0.05) of the 7 days total
needed for healing in control treatment mice. Depletion of both NK and CD8+ T cells resulted in decreased effects of NKG2D engagement on wound
healing. Conclusions: These findings provide evidence that NKG2D engagement may be utilized to
improve wound healing. Ultimately, we aim to understand the mechanism/s by which augmented
NKG2D engagement on NK and CD8+ T cells results
in improved would healing.
Cara Hrusch,JoshuaCasaos,CatherineBonham,
KellyBlaine,DanaBryazka,MohammadJaffery,
StephenieTakahashi,ImreNoth,AnneSperling
University of Chicago
Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease causing irreversible lung scarring
and loss of pulmonary function. Microarray analysis has shown that patients with IPF have reduced
141
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Autumn Immunology Conference 2014
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ICOS signaling in peripheral blood mononuclear
cells. However, the role of ICOS in the pathogenesis of IPF is unclear. We now show that ICOS expression decreases on circulating CD4 T cells in
patients as they progress. In a mouse model of
bleomycin-induced pulmonary fibrosis, ICOS was
increased on lung CD4+ T cells by 3 days post-challenge and remained elevated over 21 days at the
peak of collagen deposition. ICOS-deficient mice
had increased weight loss and mortality after bleomycin challenge compared to wild-type mice, suggesting a protective role for ICOS after lung injury.
ICOS did not play a role in collagen formation, as
both wild-type and surviving ICOS-deficient mice
had similar amounts of collagen in the lung at 21
days post-challenge. Overall, these results indicate
that ICOS expression on T cells does not directly
drive collagen deposition but does play a key role
in recovery after lung injury.
142
15. Innate immunity II
Félix Casiano-Rivera,Chun-YuTung,Hua-Chen
Chang,MarciFinizio
Department of Biology, Indiana University-Purdue
University Indianapolis
Lunasin is a seed peptide containing 43 amino acids, originally isolated from soybeans. Recently, a
novel function of lunasin was discovered, as it acts
as an immune modulating agent regulating gene
expression of various innate immune cells. Lunasin
strongly activated the expression of genes encoding
for type I interferon and inflammatory cytokines.
Nonetheless, the molecular mechanisms of lunasin’s gene regulation are relatively unknown. Our
current hypothesis states that lunasin is able to induce activation of various transcription factors, resulting in gene expression in immune cells. Human
dendritic cells (DCs) or monocytes were purified
from peripheral blood mononuclear cells (PBMCs)
in order to determine the activation of transcription factors. Phosphorylation of STAT1 and NF-ĸB
(p65) were evident in cells treated with lunasin
using flow cytometry and Western blotting. The results will ultimately lead to the signaling pathways
involved in gene expression regulated by lunasin
in innate immune cells. By defining the signaling
pathway of lunasin, we can have a better understanding of its application in immune modulation.
iNKT cell development
Aditya Rao,TiffanyCarr,MihalisVerykokakis,Barbara
Kee
Department of Pathology, University of Chicago
Invariant natural killer T (iNKT) cells are innate-like T cells that are activated by glycolipid
antigens, rapidly producing cytokines that impact
anti-microbial immune responses, asthma, and autoimmunity. Intrathymic expansion of iNKT cells
and acquisition of multiple effector fates during
thymic development prior to foreign antigen stimulation are distinct features of the iNKT lineage. In-
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terestingly, the number of Th2-type effector iNKT
cells (NKT2) varies across different mouse strains,
and is correlated with systemic alterations of the
function of other immune cells. The molecular
mechanisms that regulate these important developmental processes are poorly understood. Here,
we show that the transcription factor Lymphoid
Enhancer Factor-1 (LEF1) is required for the intrathymic expansion of iNKT cells and NKT2 differentiation. To test whether LEF1 is sufficient to
induce some aspects of iNKT cell development, we
will overexpress LEF1 in iNKT cells. To that end,
we generated a retroviral vector that can inducibly
express LEF1 upon Cre recombination. Using this
system, we will transduce hematopoietic stem cells
(HSC) from CD4-driven Cre transgenic mice and
we will investigate iNKT cell development from
HSC-reconstituted mice overexpressing LEF1 in
CD4+CD8+ cells.
Sara E. Jones,MalloryL.Paynich,KatherineL.Knight
Loyola University Chicago, Microbiology and Immunology
Department, Maywood
Beneficial microbes modulate host immune responses and can prevent disease during pathogen
infection; currently, molecular mechanisms involved in protection are poorly characterized. Previously, we showed that a single i.p. injection of purified exopolysaccharides (EPS) from probiotic B.
subtilis protected wt, but not TLR2 KO or TLR4 KO,
mice from the enteric pathogen C. rodentium. Here,
we identify host cells involved in protection. Using
flow cytometry, we show that F4/80+CD11b+ peritoneal macrophages bind EPS. To test if these cells
are important for protection, we transferred macrophage-rich peritoneal cells from an EPS-treated
wt mouse to naïve wt mice and found that these
cells prevented pathogen-induced disease. We repeated these adoptive transfer studies using TLR4
KO and TLR2 KO mice, and as expected, cells from
an EPS-treated TLR4 KO mouse did not protect recipient mice of any genotype. We were surprised to
find that peritoneal cells from an EPS-treated TLR2
KO mouse conveyed protection but that TLR2 KO
recipients were not protected. These data suggest
that two cells are needed for EPS-mediated pro-
144
tection; one cell is a TLR4-utilizing peritoneal cell
(hypothesized macrophage) and the other cell acts
downstream and uses TLR2.
resistance to an enteric pathogen
Kevin Muite,YangXin-Fu
Committee on Immunology, University of Chicago
Protection against gut pathogens is regulated by
different mechanisms. Lymphotoxin (LT), a member of the Tumor Necrosis Factor (TNF) super
family has been shown to play a critical role in the
control of the mouse extracellular enteric pathogen
C. rodentium. Recently, it has been shown that LT
binding onto the lymphotoxin-β receptor (LTβR)
plays a role in regulating the gut flora, specifically,
LTβR-/- mice harbor an outgrowth of segmented
filamentous bacteria (SFB), which has also been
shown to protect against C. rodentium colonization.
Here we examine the role of a LT regulated microbiota and colonization resistance to C. rodentium.
Surprisingly, despite an outgrowth of SFB, LTβR/mice still had higher levels of C. rodentium fecal
CFU compared to that of LTβR+/- hosts as early as
1 day post-infection. LTβR+/+ germ-free (GF) mice
reconstituted with LTβR-/- cecal contents (LTβR-/-→
GF LTβR+/+) also showed an early outgrowth of C.
rodentium compared to that of LTβR+/-→ GF LTβR+/+
controls, indicating an important role for a LT regulated microbiota in colonization resistance. Interestingly, GF LTβR-/- were more susceptible to a C.
rodentium challenge compared to that of LTβR-/- in
specific pathogen free (SPF) housing. This suggests
a model in which a physiological defect in LT-/- animals results in an altered microbiota that is incapable of resisting C. rodentium colonization.
Author’s request:
Do not include my abstract
Meghan Painter1, Eric Poeschla2,MosesRodriguez3
in the online version of the
1
Mayo Graduate
Schoolbook
of Medicine, Mayo Clinic,
abstract
2
Rochester, Division of Infectious Diseases, University of
Colorado Anschutz Medical Campus, Aurora,3Department
of
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malian genomes lack RdRPs, and RNA viruses sequester them tightly within specialized membra-
immunity
Katherine Eichinger,LoretoEgana,JacobOrend,
Kerry Empey
University of Pittsburgh, School of Pharmacy, Pittsburgh
Severe RSV disproportionately affects infants due
to biased T helper 2 (Th2) responses and impaired
innate immunity which enable prolonged viral
replication. This leads to necrotic airway epithelial cells and infiltrating immune cells occluding the
narrow lumen of the infant airway. Although T cells
are credited with facilitating viral clearance, their
infiltration occurs late in infection after significant
pathology has already occurred. The present study
examined the role the primary Th1 cytokine, IFNγ,
plays in activating innate mediators of viral clearance; alveolar macrophages (AM) and natural killer
(NK) cells. Intra-nasal IFNγ improved viral clear-
146
ance in a dose-dependent manner prior to T cell infiltration in infant BALB/c mice. AMs displayed early dose-dependent activation that coincided with
improvements in viral clearance. IFNγ treatment
of macrophages promoted a classic M1 phenotype
capable of suppressing viral replication. In infants,
only high-dose IFNγ elicited improvements in NK
cell recruitment and activation with increases in
the cytotoxic NK cell phenotype occurring early in
infection. These data support IFNγ targeting of AMs
and NK cells as early initiators of viral clearance.
Topical leukotriene B4 improves methicillin-resistant Staphylococcus aureus skin
Stephanie Brandt,SoujuanWang,StacyBlank,
NathanDelafield,SebastianCarrasco,C.Henrique
Serezani
Indiana University, Indianapolis
Methicillin resistant Staphylococcus aureus (MRSA)
commonly cause skin infections. Leukotriene B4
(LTB4) is produced by the 5-lipoxygenase (5LO)
metabolism of arachidonic acid; an important component of host defense. We hypothesize that 5LO-/and mice lacking LTB4 receptor (BLT1-/-) are more
prone to MRSA skin infection than wild type (WT)
mice. 5LO-/- and BLT1-/- mice infected subcutaneously with MRSA had increased abscess and higher bacterial burden than WT mice. Macrophages
(MFs) were the main source of MRSA-induced LTB4
production in vitro and in vivo. MRSA ingestion was
similar in MFs from WT and BLT1-/-. 5LO-/- MFs produced less antimicrobial molecules, nitric oxide
and defensins, than WT MFs. 5LO-/- mice produced
similar TNFα levels but less IL1β than WT, suggesting LTB4 is needed for IL1β.Topical treatment of
WT and 5LO-/- mice with LTB4-containing ointment
decreases abscess formation and improves bacterial clearance. These findings suggest that LTB4 is vital for skin host defense mechanisms and suggests
that topical LTB4 treatment is a likely candidate for
an immunotherapeutic agent to control MRSA infections.
ing cells in cervix
,NellLurain,JeffreyMartinson,
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PariaMirmonsef,AlanLanday,GregSpear
SUNDAY ABSTRACTS
Rush University Medical center, Chicago, IL
IL22 and IL17 have important antimicrobial and
epithelial repair functions. However their role in
the female lower genital tract has not been well defined. We determined the frequency and phenotype
of cells producing these cytokines at this mucosal
site, because we postulated that they contribute to
resistance to sexually transmitted infections.
Cervical tissue was obtained from benign hysterectomies. Cells were obtained by collagenase digestion, stimulated with PMA/ionomycin/Monensin
for 4 hrs and stained for flow cytometry. On average in endocervical tissue, the CD3+CD45+ cells
were 71% (CD4+ ~50%) and CD3- CD45+ cells
were 24%. 20% of CD45+ cells were Lin- (negative for CD3, CD14, CD16, CD19, CD20 and CD56).
Unstimulated CD4+ T-cells were < 0.05% IL22+,
IL17+ and IL22+IL17+, while after stimulation the
percentages were 1.9%, 5.5% and 1.6% respectively. Unstimulated CD3- cells in cervical tissue were
2.3% IL22+, 3% IL17+ and 4.6% of IL22+IL17+,
and the percentages after stimulation were 1.6%,
6% and 1.9% positive respectively.
Our data show that in cervical tissue, there are
CD3+ and CD3- cells capable of making IL22 and
IL17. This is important because this is the first
study to show what types of cells in the genital
tract are making these cytokines, and suggests that
these cells could induce antimicrobial peptide production by cervical epithelial cells in response to
sexually transmitted pathogens.
Human T-cell lymphotropic virus type 2 Tax
in peripheral blood mononuclear cells
Christy S. Barrios1 , 2,LauraCastillo1, 2,
MarkA.Beilke1, 2
1
Department of Medicine, Medical College of Wisconsin,
Milwaukee, 2Research Service, Clement J. Zablocki VA Medical Center, Milwaukee, WI
HTLV-2 is a benign retrovirus linked to high lymphocyte and platelet counts but unrelated to disease; while HTLV-1 cause neurological and lymphoproliferative diseases, drives the spontaneous
proliferation and T cell transformation of infected
148
cells. These pathogenic discrepancies are attributed to the transcriptional regulatory protein Tax,
essential for virus replication. Tax1 modulates host
genes leading to cell proliferation and immortalization and inhibits or induces apoptosis. Tax2 role on
cell proliferation and apoptosis is unclear.
We hypothesized that Tax2 modulates mononuclear cell number in peripheral compartments. The
ability of Tax2 to regulate the maintenance of the
cell pool was studied in PBMCs persistently treated
with Tax, and also pre-treated with a NF-kB inhibitor (PDTC) before Tax.
Higher levels of viable cells were determined in
Tax1-treated after 3 and 6 days of culture, while
higher viable cells were observed after 6 days
when treated daily with Tax2 compared to controls
(p<0.05). High proliferative responses were seen in
both Tax1- and Tax2-treated cells, but Tax1 induced
more cell proliferation than Tax2 (day 6, p<0.001).
PDTC pre-treated cells before Tax addition reduced
proliferation (p<0.05). The persistent presence of
Tax induced higher percent of cells under apoptosis (day 6, p<0.0001).
Results suggest that HTLV-2 Tax regulates the cell
pool number maintenance in the peripheral compartment of infected individuals mainly through
NF-kB pathway.
170
Kathryn Pothoven, Atsushi Kato, Bruce Tan, Paul
Bryce, Robert Schleimer
Northwestern University
Epithelial barrier dysfunction is thought to play a
role in the pathogenesis of mucosal disease, including allergic asthma (AA), chronic rhinosinusitis
(CRS), and eosinophilic esophagitis (EoE). We cultured nasal epithelial cells from CRS patients and
controls at air liquid interface. No differences in barrier function, as measured by electrical resistance,
could be seen in cells from patients and controls,
suggesting barrier dysfunction in CRS is epithelial
cell-extrinsic. We have previously shown that oncostatin M (OSM) is elevated in CRS, and wanted
to determine if OSM is elevated in other mucosal
diseases. OSM mRNA was increased in esophageal
biopsies of EoE patients vs. controls,(3 fold p<.01)
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and OSM protein was elevated in bronchoalveolar
lavage(BAL) of AA patients following segmental allergen challenge(SAC) vs. saline challenge (22.8 ±
6.3 vs. 0.56 ± 0.56, p<.0001). To determine whether
OSM may mediate barrier dysfunction in vivo, we
measured levels of OSM protein in tissue from CRS
patients and controls, and correlated them with
levels of α2-macroglobulin, a marker of epithelial
leak, in matched nasal secretions (r=.51, p<.01). In
AA, OSM levels in BAL from SAC correlated with albumin, a marker of epithelial leak (r=.81, p<.001),
suggesting that OSM may mediate epithelial dysfunction in mucosal disease.
171
Ruth Eckel, Joyce Doan
Bethel University, St.Paul
Resveratrol, a naturally occurring polyphenol, possesses well-documented anti-inflammatory properties. Previous studies from our group and others
demonstrate that resveratrol significantly attenuates classical macrophage activation by IFNγ and
LPS. Based on these data we hypothesized that a
decline in classical macrophage activities would
be associated with an augmentation of alternative
macrophage functions. The present study aimed to
investigate the ability of resveratrol either alone or
in combination with IL-4 and/or LPS to induce or
potentiate markers of alternative macrophage activation. Specifically, RAW 264.7 cells or murine bone
marrow-derived macrophages (BMM) were stimulated individually or in tandem with IL-4 and LPS,
in the absence or presence of a 2-hour resveratrol
pretreatment. Surprisingly, resveratrol pretreatment was found to attenuate arginase production
when IL-4 and LPS were added alone or in tandem (p<0.001). However, in spite of the seemingly
broad downregulatory capabilities of resveratrol
in RAW 264.7 cells and BMM, these observations
did not extend to analysis of cytokine production
in RAW 264.7 cells by ELISA where IL-6 production
was significantly diminished by resveratrol pretreatment (p<0.05) but IL-10 and TNFγ production
were not.
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172
Renée de Pooter1,MargaretA.Goodell2, Mikael
Sigvardsson3
1
Dept. of Pathology, University of Chicago, 2Baylor College
of Medicine, Houston, 3Linköping University, Sweden
Hematopoietic stem cells (HSCs) generate lymphocytes via increasingly lineage-restricted progeny. The most differentiated progenitor to retain
robust potential for both myeloid and lymphoid
fates is the lymphoid-primed multipotent progenitor (LMPP). Thus, the LMPP lies at the bifurcation point between the myeloid and lymphoid
lineages. Recently basic helix-loop-helix (bHLH)
transcription factors have been implicated in regulating myeloid versus lymphoid fate choices. The
ubiquitous Class I bHLH factor E2A is required for
efficient LMPP generation and early hematopoiesis
is fundamentally altered in E2A-/- mice: the expression of lymphoid-associated genes (lymphoid
priming) is impaired as early as the HSCs and the
number of lymphoid progenitors downstream of
the LMPPs is significantly reduced. In contrast to
E2A, expression of the Class II bHLH factor TAL-1
declines sharply with lymphoid bias and ectopic
TAL-1 is incompatible with lymphopoiesis. Using a
conditional deletion model, we show that TAL-1 restrains lymphoid priming and preserves alternate
potential in LMPPs. TAL-1 functions in opposition
to the highly homologous Class II bHLH factor LYL1, and the ratio of these factors regulates lymphoid
output from the LMPP. Our experiments lend insight into an important developmental process and
have broader implications for how stage-specific
factors modulate broadly-expressed transcriptional regulators.
173
progenitors
Siva Gandhapudi1,ChibingTan1, Julie Marino1,
Ashlee Taylor1, Jonathan Wren2,KentTeague1
1
University of Oklahoma, Tulsa, 2Oklahoma Medical Re-
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search Foundation, Oklahoma City
SUNDAY ABSTRACTS
Although IL-18 has not previously been shown to
promote T lymphopoiesis, results obtained via a
novel data mining algorithm (GAMMA), led us to
explore a predicted role for this cytokine in T cell
development. IL-18 is a member of the IL-1 cytokine family that has been extensively characterized
as a mediator of inflammatory immune responses.
To assess a potential role for IL-18 in T cell development, we sort-purified mouse early thymic progenitors (ETP) and DN2 thymocytes and cultured
these populations on OP9-DL4 stromal layers in
the presence or absence of IL-18 and/or IL-7. After one week of culture, IL-18 promoted proliferation and accelerated differentiation of ETPs to the
DN3 stage, similar in efficiency to IL-7. Interestingly, IL-18 acted in synergy with IL-7 and greatly
enhanced the proliferation of thymus derived progenitor cells. This synergistic effect correlated with
increased surface expression of c-Kit and IL-7 receptors on the IL-18-treated cells. In summary, we
successfully validated the GAMMA prediction that
IL-18 affects T lymphopoiesis and demonstrated
that IL-18 can positively T cell development, presumably via interaction with the c-Kit and IL-7 signaling axis.
174
Puspa Thapa1, MeiboChen1,DougMcWilliams1,VirginiaShapiro1,DerekSant’Angelo3,ScottHiebert2
1
Dept. of Immunology, Mayo Clinic, Rochester, MN,2 Dept.
of Biochemistry, Vanderbilt University, Nashville, TN,3 Dept.
of Pediatrics, Robert Wood Johnson Medical School/ Rutgers, New Brunswick, NJ
After positive selection into iNKT lineage at the
DP stage, iNKT cells mature and differentiate into
their functional subsets known as NKT1, NKT2 and
NKT17. Previously, we demonstrated that the transcriptional repressor NKAP is required for positive
selection of DP thymocytes into the iNKT cell lineage. To study the role of NKAP in iNKT cell development and differentiation, we generated PLZF-cre
NKAP cKO mice with NKAP deletion occurring after entry into the iNKT lineage at stage 0, bypassing the previous block in iNKT development at the
152
DP stage. In these mice, there was a significant decrease in the absolute number, which was not due
to decreased proliferation or increased apoptosis.
In the PLZF-cre NKAP cKO mice, there are very
few T-bet expressing NKT1 cells, almost no ROR-γt
expressing NKT17 cells and decreased number of
Gata3 expressing NKT2 cells present. Concurrently with defect in NKT1 and NKT17 lineages, there
is lower production of IFN-gamma and a defect in
production of IL17. Interestingly, in PLZF-cre NKAP
cKO mice, the early stage 0-1 iNKTs have lower
PLZF and T-bet expression, which could account for
the early block in development and decreased differentiation. Deletion of NKAP associated protein
Hdac3 using PLZF-cre also shows an iNKT cell developmental defect, with decreased expression of
Tbet and its regulated gene CXCR3. Thus, NKAP together with Hdac3 regulate iNKT cell development
and differentiation.
175
Kun-Po Li1, 2, Eron Roy1, Harris Perlman3, David
Hildeman1, 2
1
Cincinnati Children’s Hospital Medical Center, 2University
of Cincinnati, 3Northwestern University, Chicago
Regulation of apoptosis during thymocyte development is critical to eliminate self-reactive T cells
whilst maintaining a repertoire of protective T
cells. Although Bim promotes thymocyte apoptosis,
its role is controversial due to the minor effect of
Bim on restoring endogenous superantigen-reactive T cells. We further investigated the role of Bim
on thymocyte selection. Bim-/- mice had a striking
accumulation of DN4 thymocytes that expressed
high levels of surface TCR. DN4 cells from Bim/mice failed to generate DP cells in OP9 culture.
Further, dLckCre-driven deletion of Bim resulted in
a normal DN4 compartment, while CD4cre-driven
deletion of Bim resulted in aberrant TCR+DN4 cells.
Combined, these data suggest that the TCR+DN4
cells failed negative selection, and then downregulated both CD4 and CD8. Further, we found that
DN4 cells may sequentially develop into peripheral
CD8αα T cells, as increased intestinal intraepithelial and splenic CD8αα T cells in Bim-/-, but not dLckCre+Bimf/f mice. In conclusion, these data further
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delineate the role of Bim on thymocyte fate; and establish a new model in which the effects of Bim on
thymocyte selection are uncoupled from the effects
of Bim on peripheral T cell survival.
Rachael Philips,MeiboChen,DougMcWilliams,Paul
Belmonte,MeganConstans,VirginiaShapiro
Department of Immunology, Mayo Clinic, Rochester MN
Proper gene regulation is critical during T cell development in order to generate functional T cells.
Development involves both the activation and repression of genes as cells progress through each
developmental checkpoint. Histone acetylation/
deacetylation is one mechanism of regulating gene
expression in which acetylation by histone acetyl
transferases (HATs) allows for gene expression
and deacetylation by histone deacetylase (HDAC)
enzymes promote gene silencing, and their role
in T cell development is still under investigation.
In this study, our lab focused on HDAC3. We examined the role HDAC3 plays during T cell development by utilizing a CD2-Cre-mediated loss of
HDAC3 (CD2icre HDAC3-cKO), which deletes early
in thymocyte development. When HDAC3 is absent,
there is a developmental block during positive selection, leading to very few CD4 and CD8 SP thymocytes as well as T cells in the periphery. During
positive selection, HDAC3-deficient thymocytes fail
to up-regulate Bcl-2 and show increased apoptosis. Mechanistically, thymocytes fail to up-regulate
the transcription factor c-Rel that regulates Bcl-2
expression after positive selection. This correlates
with a failure to down-regulate RORγt, which is required for c-Rel expression. These results suggest
that HDAC3 is required for the down-regulation of
RORγt during positive selection. This work is supported by NIH RO1 to V.S.S as well as internal funds
from the Mayo Clinic.
177
, Fan-Chi Hsu, Paul Belmonte, Megan
Constans,VirginiaShapiro
Department of Immunology, Mayo Clinic, Rochester
T cell development depends on coordinated gene
154
expression changes to pass each developmental
checkpoint. Previously, the loss of transcription
factor Runx1 in CD4 single positive (SP) thymocytes and peripheral T cells led to decreased expression of IL-7Ra, a key player in T cell homeostasis and survival. Thus, low IL-7Ra was thought to
be the cause of low CD4 T cell production. However,
we show that constitutive expression of an IL-7Rα
transgene in CD4-cre Runx1 conditional knockout
(cKO) mice does not restore thymic or peripheral
CD4 cellularity. We also see similar expression of
Bcl-2 and Bcl-xL in WT and Runx1-deficient CD4 T
cells, indicating that a Runx1-mediated IL-7Ra-independent mechanism must be responsible. As IL7Ra expression rises during T cell maturation, we
asked if Runx1 is needed for maturation. During
thymic maturation, post-positive selection CD4 SP
thymocytes migrate from cortex to medulla, changing from CCR7lowCD24high to CCR7highCD24high and
finally CCR7highCD24low before thymic egress. We
saw a block at the CCR7lowCD24high stage in CD4-cre
Runx1 cKO CD4 SP thymocytes. Additionally, other
maturation markers including CD55 and Qa2 were
also downregulated. Thus, Runx1 is required for
thymic maturation of CD4 SP thymocytes.
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couples TCR signal strength to thymic Treg
1
,ShawnMahmud1, Luke Manlove1,
HeatherSchmitz1,YanXing1,YanyanWang2, Jason
Schenkel1, Jonathan Boomer4, Jonathan Green4,
HideoYagita3,HongboChi2,KristinHogquist1,
Michael Farrar1
1
Center for Immunology, University of Minnesota,
Minneapolis, 2St Jude Childrens Research Hospital,
Memphis, 3Juntendo University School of Medicine,
Tokyo,4Washington University, St. Louis
Regulatory T cells express several TNF receptor
superfamily (TNFRSF) members, but their role in
thymic Treg development is undefined. We demonstrate that Treg progenitors express high levels
of the TNFRSF members GITR, OX40, and TNFR2.
Expression of these receptors requires TAK1 and
CD28 and correlates directly with TCR signal
strength. Neutralizing TNFSF ligands markedly reduced Treg development. Conversely, TNFRSF ago-
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nists enhanced Treg differentiation by augmenting
IL2R/STAT5 responsiveness. Importantly, GITR-L
costimulation elicited a dose-dependent enrichment of lower-affinity cells within the Treg repertoire. In vivo, GITR- or OX40-deficiency modestly
attenuated Treg development; in contrast, combined inhibition of GITR, OX40 and TNFR2 abrogated Treg development. Thus TNFRSF expression on
Treg progenitors translates strong TCR signals into
molecular parameters that specifically promote
Treg differentiation and shape the Treg repertoire.
Pak2 links TCR signaling strength to the
Author’s request:
regulatory
cells my abstract
Do not Tinclude
in the online version of the
abstract book
evelopment of regulatory T (Treg) cells is
of peripheral imm
oleritthe
156
Treg lineage while helping to facilitate peripheral
immune tolerance.
180 Immature recent thymic emigrants are
eliminated by complement
Fan-Chi Hsu,MeiboChen,DouglasMcWilliams,LaurenSeaburg,SarahTangen,VirginiaShapiro
Department of Immunology, Mayo Clinic, Rochester, MN
Recent thymic emigrants (RTEs) must undergo
phenotypic and functional maturation to become
long-lived mature naïve T cells. In CD4-cre NKAP
conditional knockout mice, NKAP-deficient RTEs
fail to complete T cell maturation. Here, we demonstrate that NKAP-deficient immature RTEs do not
undergo apoptosis, but are eliminated by complement. C3, C4 and C1q are bound to NKAP-deficient
peripheral T cells, demonstrating activation of the
classical arm of the complement pathway. As thymocytes mature and exit to the periphery, they
increase sialic acid incorporation into cell surface
glycans. This is essential to peripheral lymphocyte
survival, as stripping sialic acid with neuraminidase leads to the binding of natural IgM and complement fixation. NKAP-deficient T cells have a defect in sialylation on cell surface glycans, leading to
IgM recruitment. We demonstrate that the defect
in sialylation is due to aberrant α2,8-linked sialylation, and the expression of three genes (ST8sia1,
ST8sia4 and ST8sia6) that mediate α2,8 sialylation
are downregulated in NKAP-defcient RTEs. The
maturation of peripheral NKAP-deficient T cells
is partially rescued in a C3-deficient environment.
Thus, sialylation during T cell maturation is critical
to protect immature RTEs from complement in the
periphery.
181
Jinyong Choi,KyleL.O’Hagan,OlgaPryshchep,
HyewonPhee
Northwestern University, Feinberg School of Medicine,
Chicago
As an effector molecule for the Rho family GTPases
Rac and Cdc42 that are key regulators for actin cytoskeleton, Pak2 regulates diverse functions of T
cells. However, the roles of Pak2 in the maintenance
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and differentiation of regulatory T cells are not
clearly determined. Our recent finding showed that
T cell-specific deletion of Pak2 resulted in dramatic
decrease in the number and frequency of natural
Tregs (nTregs) in thymus, suggesting that Pak2 is
required for the development of nTregs in thymus.
Next, we sought to determine the role of Pak2 in peripheral Tregs. Although we found that the number
of peripheral Tregs was reduced in the absence of
Pak2, the reduced number of CD4 T cells in the periphery of T-cell specific Pak2-deficient mice made
it difficult to interpret the result. To overcome this
difficulty, we recently generated tamoxifen-inducible Pak2 knockout mice, in which Pak2 is deleted inducibly by the treatment of tamoxifen. When
Pak2 was deleted in these mice, the frequency of
peripheral Tregs was markedly decreased, suggesting Pak2 is required for the maintenance of peripheral Tregs. Furthermore, inducible deletion of Pak2
greatly inhibited differentiation of inducible Tregs
(iTregs) from naïve CD4 T cells, as was evidenced
by failure to induce Foxp3, CTLA4 and CD25. Together, we show that Pak2 maintains the Treg pools
in the periphery by providing survival signals to
the peripheral Tregs as well as controlling differentiation of CD4 T cells to become iTregs.
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17. T cell signaling
182
Brendan Reed, Alejandro Ferrer, Mike Bell, Diana Gil
Pages,AdamSchrum
Mayo Clinic, Rochester, Minnesota
Decades of work has helped to characterize the
interaction between the T cell antigen receptor
(TCR) complex and peptide-loaded MHC molecules (pMHC). In the “Goldilocks” model of thymic
development, conventional alpha-beta thymocytes undergo positive selection when the molecular summation of TCR/pMHC contacts results in a
moderate, “just right” signal, but negative selection
when the binding interaction is strong. The threshold between these two responses largely defines
the repertoire of antigenic T cell reactivity potential in the periphery. Previous studies have relied
on murine models to define the T cell response
hierarchy to panels of altered-peptide ligands,
and the central tolerance T cell threshold. Here,
we present our design for an experimental system
to test the degree of evolutionary conservation of
hierarchical TCR response patterns and central
tolerance thresholds between murine and human
T cells. Preliminary results allude to a highly conserved reactivity hierarchy between mouse and human, when T cells of either species are presented
with panels of altered-peptide ligands. The extent
to which both species display identical, similar, or
different central tolerance T cell thresholds is currently being investigated.
183
Sydney Blevins1, 2, Brian Baker1, 2
1
Department of Chemistry and Biochemistry, 2Harper
Cancer Research Institute, University of Notre Dame, Notre
Dame
The Adaptive Immune System is the body’s way
of identifying and destroying foreign pathogens.
This process involves a recognition event utilizing
two major proteins: a T cell receptor (TCR) and a
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Major Histocompatibility Complex (MHC) protein
presenting a peptide. The complete mechanism by
which a TCR recognizes an antigen (peptide- MHC)
and proceeds to initiate the signaling cascade to
destroy infected cells is still not well understood.
Much of our work aims to better understand the
properties that might influence how a T cell receptor recognizes a peptide-MHC. One area of interest
is the role of conserved TCR residues and how this
might influence TCR behavior. Preliminary bioinformatics work has shown that over 80% of TCRs
have a conserved histidine residue in the CDR1β
loop. Structural observation revealed that the histidine is contacting a surprisingly conserved serine or threonine residue in the CDR3β loop. To
explore potential roles this interaction might have,
two complementary approaches will be used to examine the effects of this interaction within T cell
receptor CDR loops. Surface plasmon resonance
will be used for the assessment of non-additivity
between remote CDR loops and fluorescence anisotropy will be used to detect changes in CDR loop
flexibility.
184
increases T cell responses to poorly
,CarlosMolinaMendiola,Michael
Hansen,LarryPease,AdamSchrum,GilPagesDiana
Mayo Clinic, Department of Immunology, Rochester
T cell receptor (TCR) stimulation induces a conformational change in the CD3 complex (CD3Dc)
that exposes a cryptic proline-rich motif in the
cytosolic tail of CD3e. CD3Dc is a molecular event
necessary but not sufficient for productive TCR/
CD3 triggering. CD3Dc fails to occur when mature
T cells engage weak antigens. Here, a monovalent
Fab fragment specific for the CD3eg dimer of the
CD3 (Mono-7D6-Fab) that exogenously provides
CD3Dc was engineered to increase T cell sensitivity to poor antigenic stimulation. When bound
to T cells, Mono-7D6-Fab (i) induces CD3Dc in
the absence of further downstream signaling, (ii)
does not interfere with TCR/Antigen interactions
and (iii) does not crosslink TCR/CD3 complexes.
The addition of Mono-7D6-Fab to T cells encoun-
160
tering weak antigens significantly increased their
functional responses when tested in vitro in different TCR transgenic mouse models. In vivo, Mono-7D6-Fab administered to healthy wild-type
mice was inert. However, Mono-7D6-Fab displayed
anti-tumor effects against a poorly immunogenic
lung-metastatic melanoma model that are dependent on melanoma-specific CD4 and CD8 T cells.
Thus, Mono-7D6-Fab decreases the threshold of
TCR/CD3 triggering, increasing T cell sensitivity to
poorly immunogenic antigens in the context of disease. We propose that Mono-7D6-Fab represents
a novel treatment that allows T cells to overcome
poor TCR/antigenic stimulation by targeting the
non-polymorphic CD3.
185
1
,NicoleChapman2, Jon Houtman1
1
University of Iowa, Iowa City, 2St. Jude Children’s Research
Hospital, Memphis
CD4 T cells are constantly exposed to inflammatory
cytokines that alter their function. One example is
IL-12, which enhances IFN-γ production when given concurrently with TCR induction. However, how
prior exposure to IL-12 alters subsequent TCR-mediated functions in CD4 T cells is unexplored. To address this knowledge gap, human CD4 T cells were
exposed to IL-12, washed and stimulated with anti-TCR antibodies. We found that IL-12 treatment
increased TCR-induced IFN-γ, TNF-α, IL-13 and
IL-4 production in comparison to untreated cells.
This suggests that prior exposure to IL-12 primes
the TCR-induced release of a range of cytokines.
The potentiation of TCR-mediated cytokine release
required at least 45 minutes of IL-12 treatment and
only lasted for 6-12 hours after cessation of IL-12
treatment. Interestingly, we found that IL-12 exposure lead to an increase in the phosphorylation of
AKT and LCK following TCR stimulation without
altering other TCR signaling molecules. Our data
suggests that IL-12 mediates its priming effects
on cytokine release by increasing the ability of the
TCR to activate select signaling molecules. A novel
function of IL-12 beyond its well-studied costimulatory effects provides insight into potential methods to enhance or suppress CD4 T cell activity that
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could be harnessed as therapeutics.
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TRAF3 restrains CSK to enhance T cell
receptor (TCR) signaling
1
, Zuoan Yi2, Gail Bishop1, 2, 3, 4
1
Graduate Program in Immunology, Iowa City, 2Dept of Microbiology, Iowa City, 3Internal Medicine, Iowa City, 4VAMC,
Iowa City
TCR signaling is critical for T cell activation, so further elucidation of this process is necessary both to
understanding T cell biology, and to manipulate T
cell activation in various clinical settings. Extensive
research on TCR signaling has revealed many details, but additional information on the molecular
mechanisms continues to be revealed. Our group
previously produced a T cell-conditional (CD4-Cre)
TRAF3-/- mouse, which demonstrated that TRAF3
plays an important positive role in TCR signaling
and T cell functions. After TCR engagement, TRAF3
associates with the TCR complex, and TRAF3-/cells display a marked reduction in the early phosphorylation of Lck and CD3-zeta activation sites.
This role for TRAF3 in proximal TCR signaling
events led us to hypothesize that TRAF3 regulates
TCR signaling through CSK, a T cell membrane-localized protein that binds to and inhibits Src family
kinases, such as Lck. Through immunoprecipitation, TRAF3 was shown to interact with CSK upon
TCR stimulation. In the absence of TRAF3, elevated
amounts of CSK remained in the plasma membrane
of stimulated T cells. Ongoing experiments are
testing the prediction that TRAF3 sequesters CSK
in the cytoplasm to restrain its ability to inhibit
TCR-associated Lck activation.
187 Glycerol monolaurate (GML) inhibits the
Michael Zhang, Jon Houtman
University of Iowa, Iowa City
Glycerol monolaurate (GML) is an antimicrobial
that is used to prevent menstrual toxic shock syndrome. The bactericidal and anti-toxin effects of
GML have been well characterized. However GML
has also been found to modulate immune cell biology. Peterson et al. observed that GML suppresses
162
cell proliferation and inositol triphosphate levels
in TSST-1 stimulated PBMCs. Thus, GML may inhibit T cell activation. Nevertheless, GML’s specific
mechanism to prevent TCR-mediated signaling is
unknown. In this study, we address this knowledge
gap by examining GML’s effects on primary human
effector T cells. We observed that GML suppresses
T cell effector functions through interference of T
cell signaling cluster formation. Specifically, GML
treated cells have lowered membrane localization
of phosphorylated PLC-γ and LAT. We also found
that GML treatment markedly reduces TCR-induced
calcium influx and phosphorylation of SLP-76. GML
induced T cell signaling changes have consequences on effector T cell physiology. GML treatment decreases IL-2 and IFN-γ production as well as cell
adhesion in a dose dependent manner. Altogether,
GML hinders effector T cell functions by inhibiting
membrane localization of PLC-γ and LAT. GML’s immunomodulatory properties have applications in
limiting the T cell mediated cytokine storm in TSS
as well as being a potential treatment option for
other T cell mediated inflammatory conditions.
188
150
nucleases to inhibit human C-C chemokine
receptor 7 gene expression to enhance
,ColinKnight,ColinBill,Charlotte
Vines
Text
University of Texas at El Paso
An adaptive immune response leads to elevated
antibody production following exposure to antigen. C-C chemokine receptor 7 (CCR7) is an important modulator of antibody production during
adaptive immunity. In these studies homologous
deletion of the CCR7 locus in mice resulted in elevated production of antibodies. To determine if the
same CCR7-signaling pathways regulate antibody
production in human cells during the adaptive
immune response, we have constructed transcription activator-like effector nucleases (TALENs)
that recognize specific sequences of the human
CCR7 gene. TALENs induce double strand breaks
in the CCR7 locus in cells transfected with these
nucleases, which results in loss of CCR7 protein
expression. We hypothesize that the disruption of
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AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SUNDAY ABSTRACTS
the CCR7 gene and subsequent reduction in CCR7
signaling will lead to enhanced antibody production during an adaptive immune response in
vitro, which has the potential to enhance individual immune responses to vaccination. As a proof
of principle we have inhibited CCR7 in human
immune cells and found that signaling through
CCR7 is lost. We expect that this technology will be
useful for improving antibody production during
vaccination.
Author’s request:
Do not include my abstract
maintenance
in the online version of the
abstract book
Karin Knudson, Mark Daniels, Emma Teixeiro
The development of memory CD8+ T cells during
infection is required for long-term immunity. How
nd ren. The
design
cleases to inhibit C-C Chemokine Receptor 7
gene expression in experimental autoimmune
Colin Knight,ColinBill,CharlotteVines,Brenda
Hinojoza
University of Texas at El Paso
164
Although it is known that C-C Chemokine Receptor
7 (CCR7) is required to induce experimental autoimmune encephalomyelitis (EAE), an animal model
of brain inflammation used to model human multiple sclerosis, the role of CCR7 in the etiology of
EAE remains unclear. Since EAE manifestations depend upon the migration of T-cells into the central
nervous system (CNS) and T-cell migration is CCR7
dependent, we hypothesize that if we can block
T-cells from expressing CCR7 in mice suffering
from EAE, we can prevent T cell migration into the
CNS and the subsequent autoimmune response. To
this end we have designed a panel of Transcription
Activator-like Effector Nucleases (TALENs) to cut
specific regions of the CCR7 gene in mouse T-cells
to knockdown CCR7 gene expression. Indeed, our
preliminary data showed that targeted reduction of
CCR7 in a human T-cell line led to a decreased cell
migration using a transwell migration assay. We
are currently assessing the therapies that have the
greatest reduction of CCR7-mediated migration for
use in our mouse model of EAE.
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1
,JoohoChung1, Ann Friedman1,
ToddV.Brennan2,ChristianW.Siebel3, Ivan Maillard1
1
University of Michigan, Ann Arbor, 2Duke Universiy,
Durham, 3Genentech Inc, South San Francisco
Blocking Notch signaling after allogeneic bone
marrow transplantation prevents acute graft-versus-host disease (GVHD) in mice. Notch inhibition
within a short 2-day window after transplantation
provides maximal benefits. We describe new tools
to study alloantigen-specific T-cells within this window, and provide insights into early events associated with Notch blockade. In a MHC-mismatched
model, transgenic CD4+ 4C T-cells recognizing
I-Ad induced lethal GVHD in BALB/c recipients. 4C
T-cells were sensitive to Notch blockade, showing
marked decrease in inflammatory cytokine production, despite preserved activation, proliferation and
expansion. In the B10.D2->BALB/c MHC-matched
model of GVHD, donor-derived CD4+Vβ3+ T-cells,
responding to a Mtv6-encoded BALB/c superantigen, demonstrated massive expansion in lymphoid
tissues and liver. Notch blockade inhibited cytokine
production, but preserved Vβ3+ T-cell activation
and proliferation. Our findings document the early
onset of dissociated effects of Notch inhibition on
proliferation and cytokine production in alloantigen-specific T-cells, allowing further focused evaluation of Notch’s role in the alloantigen-specific
T-cell response.
Stromal cells in secondary lymphoid organs
drive Notch-mediated T cell pathogenicity in
J Chung1,CEbens1,VRadojcic1, A Friedman1,
CSiebel2, I Maillard1
1
University of Michigan, Ann Arbor, 2Genentech, San
Francisco
Notch signaling is a critical regulator of T cell effector functions during acute graft-versus-host disease (aGVHD). Pan-Notch inhibition in donor-de-
166
rived T cells or systemic blockade of Delta-like1
(Dll1) and Delta-like4 (Dll4) Notch ligands results
in near-complete protection from aGVHD. Here, we
sought to: 1) determine the kinetic requirements
for Notch during aGVHD; 2) identify the cellular
compartment that delivers Dll1 and Dll4 ligands to
donor T cells. In the B6 anti-BALB/c model, a single
dose of anti-Dll1/4 abolished T cell cytokine production, increased Tregs, and conferred long-term
protection from aGVHD. Mx1-Cre mediated genetic
inactivation of Dll1 and Dll4 in donor or host hematopoietic tissues had no effect on aGVHD. In contrast, Ccl19-Cremediated Dll1 and Dll4 inactivation
impaired donor T cell cytokine production, leading to long-term protection from aGVHD. Lineage
tracing revealed Ccl19-Cre activity within CD45lymph node and spleen stromal cells, but not in
professional hematopoietic antigen-presenting
cells. These data suggest that a specialized subset
of non-hematopoietic stromal cells delivers an early pulse of Notch signaling to alloreactive T cells
during aGVHD.
disease
Brad Griesenauer,AbdulraoufRamadan,JiluZhang,
SophiePaczesny
Indiana University, Indianapolis
Graft-versus-host disease (GVHD) hinders the efficacy of allogeneic bone marrow transplantation
(BMT) as a curative treatment, as GVHD develops
in half of patients receiving BMT. Mortality of those
who develop GVHD hovers near 50%. MyD88 signaling, which is activated through either Toll-like
receptors or IL1 superfamily receptors, in host
antigen presenting cells has no effect on GVHD (Li
H, 2011). The role of MyD88 signaling in donor
T cells has yet to be elucidated. We hypothesized
that knocking out MyD88 in donor T cells will have
a protective effect in GVHD by reducing Th1 cell
differentiation through prevention of IL1 receptor signaling. Using the B6 -> Balb/c model, mice
receiving MyD88 KO T cells survived longer (71%
vs 13%, P < 0.001, n=15), reduced GVHD severity
(3.8 vs 5.5 P < 0.001, n=15), had fewer Th1 cells in
classical organ targets of GVHD (fewer infiltrating
cells and fewer IFNγ secreting cells), and secreted
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less IFNγ (0.89 vs 2.66 ng/mL, P = 0.009) and IL1β
(0.51 vs 1.05 ng/mL, P = 0.02) in plasma at early
time points. This data suggests that MyD88 signaling in the donor T cells plays an important role in
the development of GVHD.
Author’s request:
Yuk Man (Kevin)
Do notLei,LuqiuChen,CarolineBartman,
include my abstract
YingWang,LekhaNair,AndrewStefka,CathyNagler,
in the online version of the
abstract book
LucianaMolinero,AnitaChong,Maria-LuisaAlegre
University of Chicago
The kinetics of transplant rejection are determined
both by genetic and environmental factors. One po-
ial
to
for
Pawan Gupta1,QiangWu1,SarahWagner1, David
Wilkes2,RebeccaShilling1
1
University of Illinois, Chicago, 2Indiana University,
Indianapolis
Obliterative bronchiolitis (OB) is immune mediat-
168
ed fibrous obliteration of airways and limits long
term lung transplant survival. Previous studies in
a mouse model of minor MHC mismatch orthotopic
left lung transplant (C57Bl/10,H-2b→C57Bl/6,H2b) suggested a role for IL-17A in OB development.
We have investigated the cellular sources of IL-17A
and their contribution to OB development in this
model. We found that the increased IL-17A+ cells
infiltration in allografts coincided with OB development at day 21 post-transplant. IL-17A+ cells were
primarily CD4+ T cells (Th17) and gδ T cells. CD4+
T cell depletion led to decreased infiltration of IL17A+ cells and IL-17A+gδ T cells and abrogated OB.
Unexpectedly, allografts from Th17 cells deficient
mice developed OB. The frequency of IL-17A+ cells
remained intact in these allografts because of IL17A+gδ T cells infiltration suggesting a role for gδ
T cells in allograft injury. However, allografts from
TCRδ-/- mice developed acute rejection and fibrosis similar to control allografts. Our data suggest
that CD4+ T cells are required for OB and promote
IL-17A responses from innate immune cells, whereas recipient gδ T cells are not necessary for OB development. Supported by NIH (R01HL109310).
IL-17C and IL-17E may play a role in the
Collin Langer,PawanGupta,QiangWu,SarahWagner,RebeccaShilling
University of Illinois at Chicago, Chicago
Obliterative bronchiolitis (OB) is the immune mediated fibrotic obliteration of small airways, which
can limit the survival of lung transplant recipients.
Previous work in a mouse model of minor MHC
mismatch orthotopic left lung transplant found
that the administration of IL-17RA-Fc abrogated
OB. Our lab has found that IL-17A/F producing
CD4+ T cells (Th17) cells are not necessary for OB
development. These data suggest that other IL-17
family members that can also bind to IL-17RA may
be involved in the development of OB. IL-17C and
IL-17E can bind IL-17RA and are made predominantly by epithelium. We investigated if damage
to the epithelium resulting from the transplantation procedure was associated with an increase
in IL-17C and IL-17E in the lungs after transplant.
Using real time PCR to evaluate expression in lung
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allografts and native lungs we found that lung allografts expressed less IL-17C on average relative
to native lungs and more IL-17E. Expression was
also evaluated in spleen tissue, but no noticeable
expression was observed for IL-17C or IL-17E.
This is consistent with these cytokines not being
expressed by lymphoid tissues. IL-17C and IL-17E
are expressed in the lungs after transplant and may
be contributing to the development of OB.
Michelle L Miller1, Melvin D Daniels1,Tongmin
Wang1,JianjunChen1,JamesYoung1,JingXu1,Ying
Wang1,DengpingYin1, Aliya Husain1, James Moon2,
AnitaS.Chong1,Maria-LuisaAlegre1
1
University of Chicago, 2Massachusetts General Hospital,
Boston
Non-immunosuppressed recipients of a primary allograft are known to develop memory T cells
that can trigger accelerated rejection of a second
donor-matched transplant. In contrast, our data
show that mice tolerant to a cardiac allograft after
costimulation blockade, but later rejected these
grafts following infection with Listeria monocytogenes (Lm), spontaneously accepted second donor
heart grafts after the infection cleared. Using fluorescent MHC:peptide multimers to track endogenous T cells specific for model antigens expressed
in the graft or the bacteria, as well as graft-reactive
TCR-transgenic T cells, we found that Lm infection
transiently increased alloreactive and Lm-specific T cells in the primary allografts, reducing Treg
percentages. However, Tregs reaccumulated in high
percentages in the second tolerated grafts. Moreover, the restored tolerance in the second grafts
was dependent on CD25+ regulatory cells. Thus,
dominant regulation can restore a tolerant state
following infection-mediated rejection. These data
may have implications for relapsing/remitting autoimmune diseases or tumor recurrence as tolerance to self- or tumor antigens may dominate when
inflammation subsides.
Pregnancy primed maternal regulatory
microchimeric cells.
170
AIC 2014 • Chicago, IL
1
Cincinnati Children’s Hospital, Cincinnati, 2University of
Cincinnati, Cincinnati
During pregnancy, expansion of immune suppressive maternal regulatory CD4+ T cells (Tregs) is essential to accommodate foreign paternal antigens
expressed by the fetus. In turn, blunted accumulation of maternal Tregs occurs in many human pregnancy complications including preeclampsia and
spontaneous abortion. The importance of Tregs
is recapitulated in animals where even partial depletion of maternal Tregs cells during pregnancy
disrupts fetal tolerance and triggers fetal wastage.
By using a mating strategy that transforms defined model antigens into surrogate fetal antigen
we have shown a selective expansion of maternal
Tregs with fetal specificity occurs during pregnancy. Interestingly, the postnatal persistence of maternal Tregs with specificity to fetal antigen, along
with their rapid expansion during secondary pregnancy was also observed and provided increased
resiliency against pregnancy complications induced by disruptions in fetal tolerance. These results highlight protective memory features for
maternal Tregs with fetal specificity in mice which
parallel human partner-specific protective benefits
against complications in subsequent pregnancies.
Here we show that postpartum retention of memory maternal Tregs requires ongoing stimulation by
microchimeric fetal cells retained in mothers after
pregnancy.
1, 2
,RobertGower1,JesseZhang1, Fallon
1, 2
Noto , Lonnie Shea1, 2
1
Northwestern University, Department of Biological and
Chemical Engineering, Evanston, 2University of Michigan,
Department of Biomedical Engineering, Ann Arbor
Allogeneic islet transplantation represents a promising experimental therapeutic option to restore
endogenous insulin production in Type-1 diabetes
patients. However, the immune response remains
a significant hurdle for long-term islet allograft
survival. We have previously demonstrated that
171
SUNDAY ABSTRACTS
Jeremy Kinder1, 2,SingSingWay1
Autumn Immunology Conference 2014
SUNDAY ABSTRACTS
islets transplanted on porous poly-lactide-co-glycolide (PLG) scaffolds within the epididymal fat
pad can restore long-term euglycemia in a streptozotocin-induced diabetic mouse model. Here, we
explore the use of the scaffolds to modulate the immune response within the local microenvironment
by engineering them to release two protein factors:
TGF-β1 and IL-35. Diabetic C57BL/6 recipients
receiving BALB/c islets transplanted on TGF-β1
releasing scaffolds showed extended euglycemia
and islet survival. Flow cytometry and histology
revealed TGF-β1 loaded scaffolds had significant
decreases in leukocyte infiltration of the islet graft,
most prominently amongst innate immune cell lineages like macrophages and natural killer cells. We
also found preliminary evidence that IL-35 release
from the scaffold preferentially recruits regulatory T cells to the implant site, suggesting IL-35’s
potential for promotion of long-term allograft tolerance. Future studies will examine using factor
release to polarize infiltrating antigen-presenting
cells towards a tolerogenic phenotype and induce
differentiation of Tregs within the scaffold microenvironment.
200
Author’s request:
Do not include my abstract
in the online version of the
abstract book
,WilliamBurlingham
Patient-specific induced pluripotent stem cells
ve
text of the patient-specific immune environment.
Humanized mouse models offer a tractable
-
172
AIC 2014 • Chicago, IL
SUNDAY ABSTRACTS
vo
201
The Cleveland Clinic, Cleveland
Antibody-mediated lymphoablation is used as an
induction therapy in transplant patients. We investigated the requirements for T cell recovery
following lymphoablation in a mouse model of
heart transplantation. We previously reported that
memory CD4 T cells are less susceptible to depletion by murine antithymocyte globulin (mATG)
than CD8 T cells. Additional CD4 T cell depletion
impaired CD8 T cell recovery after mATG treatment
in B6 recipients of BALB/c heart allografts and in
non-transplanted B6 mice. Treatment with blocking anti-CD154 mAb plus mATG inhibited CD8 T
cell recovery, while agonistic anti-CD40 mAb restored memory CD8 T cell expansion after mATG
treatment of CD4 T cell depleted recipients. To test
whether B cells are required for CD8 T cell recovery, we used B6.huCD20tg mice. Treatment with
anti-human CD20 mAb depleted >90% of peripheral B cells and markedly inhibited CD8 T cell expansion in huCD20tg recipients compared to B cell-sufficient mice. Next, B cells were isolated from mATG
treated recipients with or without CD4 depletion.
In the absence of CD4 T cells, B cells expressed
lower levels of MHC class I, CD80, CD86, and TNFa
suggesting that both cognate interactions and cytokines promote CD8 T cell expansion. Our results
show that CD4 T cell help mediated through B cells
and CD40/CD154 interactions drives CD8 T cell
reconstitution following antibody-mediated depletion.
173
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202
SUNDAY ABSTRACTS
CCR7 levels in T cells as a strategy to prevent
in the CNS
,OlgaSoto,ColinBill,CharlotteVines
The University of Texas at El Paso
T cells that migrate past the blood brain barrier into
the central nervous system (CNS) view myelin as
foreign and induce inflammation and degradation
of myelin sheaths in multiple sclerosis patients. We
and others have shown that C-C chemokine receptor 7 (CCR7) is required for T cells to enter the CNS.
We hypothesize that blocking expression of CCR7
will inhibit the migration of T cells into the brain
and prevent myelin sheath destruction in multiple
sclerosis patients. We have developed a panel of
Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR) associated with Cas9 RNA
guided DNA nucleases targeted to different exon
regions of CCR7 to specifically disrupt the CCR7 locus in human T cells. We have shown by immunofluorescence microscopy that individual CRISPR/
Cas9 transfected into human cells are targeted to
the nucleus and that these cells express the fulllength CRISPR/Cas9 nuclease by Western blots. We
are currently exploring the efficiency of CRISPR/
Cas9 cutting of the CCR7 locus and optimizing the
combinations of CRISPR/Cas9 targeting to CCR7 to
maximize the level of CCR7 gene editing prior to assessing the effects on T cell migration into the CNS.
174
203
1
, Erica Huelsmann1, Jason
Schenkel2, Janet Zayas1, Frederick Kohlhapp1,Andrew
Zloza1, 3
1
Department of Immunology/Microbiology, Rush University Medical Center, 2Center for Immunology, Department
of Microbiology, University of Minnesota, Minneapolis,
3
Department of Internal Medicine, Rush University Medical
Center, Chicago
Background: Emerging epidemiologic studies describe an increased prevalence of tumors in patients with non-oncogenic viral disease However, there is a lack of basic understanding by what
mechanism infections result in increased unrelated
cancer formation (i.e., in tissues not associated with
the infection). Methods: We examined the effect of
influenza infection in a mouse model of genetically
driven BrafV600E-induced, Pten-deficient metastatic melanoma, which mimics key pathophysiological aspects of the human disease. Mice were
challenged with influenza (A/PR/8 on day 0), and/
or tamoxifen (Days 0, 1, and 2). Mice were then observed for melanoma formation and photographed
weekly. Results: Influenza infection hastened the
formation of melanoma in a tamoxifen-inducible
genetically driven mouse model at day 48 to the
same level as tamoxifen administration (up to 80%
of treated mice) compared to <20% of untreated
mice (P<0.01). All mice (100%) of mice receiving
both tamoxifen and influenza infection demonstrated tumors at day 48. Conclusions: These studies suggest a previously unrecognized role for acute
viral infection in the emergence of cancer and may
answer the long-standing questions of why certain
individuals are more susceptible to tumor formation or less responsive to cancer therapies.
204
growth in a lung melanoma mouse model
Raquel Redondo1, Joseph Broucek2, Hubert
Dolubizno1, Janet Zayas1, Frederick Kohlhapp1, An-
175
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Autumn Immunology Conference 2014
drewZloza1, 3
SUNDAY ABSTRACTS
1
Departments of Immunology/Microbiology, 2General
Surgery, 3Department of Internal Medicine, Rush University
Medical Center, Chicago
Background: We have discovered that influenza infection results in decreased anti-melanoma
CD8+ T cell responses. This is in part due to the
migration of anti-tumor CD8+ T cells to the site
of the infection (lungs) from the site of the tumor
(intradermal space) and their subsequent activation/exhaustion. Based on this finding, we sought
to determine whether such recruitment of anti-tumor CD8+ T cells by viral infection at the site of a
tumor in combination with blocked of exhaustion
(using a PD-1 blocking antibody) could be used to
decreased tumor growth. Methods: C57BL/6 mice
were challenged with B16-F10 melanoma (via retroorbital injection with 150,000 cells) on day 0.
Some mice were treated via intranasal influenza
(A/PR/8, 8,000 EID50 in 40 µl via intranasal injection on day -3 and anti-PD-1 (250 µg/mouse) via
intraperitonal injection on day 0. Mouse weights
and overall survey of health were recorded every
other day until animals were sacrificed. The primary outcome included lung tumor burden on day
14. Results: Combination immunotherapy with influenza and anti-PD1 significantly decreased (by
>80%, P<0.01) the number of tumor foci identified
on mouse lungs when compared to influenza alone,
anti-PD1 alone, or placebo (PBS/IgG). Conclusions:
These findings provide evidence that combination
therapy with viral infection and PD-1 blockade may
be utilized to decrease tumor growth. Ultimately,
we aim to understand the mechanisms involved in
these findings.
205 In vitro
Grace Blitzer,WeiqingJing,JamesWeber,Laura
McOlash, Jill Gershan, Bryon Johnson
Medical College of Wisconsin, Milwaukee
High-risk hematological malignancies, including
myeloma, are difficult to treat with current therapies. Adoptive transfer of tumor-specific T-cells
could be an effective treatment, but identification of
the cancer-specific T-cells is difficult and the appropriate growth factors for expansion of the T-cells
176
are unclear. Programmed cell death protein-1 (PD1) appears to be a viable marker for the tumor-antigen experienced T-cells. PD-1+ T-cells are known
to be dysfunctional, preventing effective immune
targeting of tumors. Whether normal function of
PD-1+ T-cells can be restored by manipulating the
cells ex vivo is unknown, but data suggests that
proliferation of the PD-1+ T-cells may allow them
to recover their anti-tumor activity. A combination
of IL-15 and IL-21 facilitated the expansion of normal murine T-cells more than 15-fold with maintenance of cell function. PD-1+ T-cells from myeloma-bearing mice also expanded well in the IL-15/
IL-21 conditions and maintained their tumor cell
specificity in IFN- ELISPOT assays; tumor reactivity was detected in expanded PD-1+ T-cells but not
in PD-1- cells. These data suggest that although
PD-1+ T-cells are dysfunctional after exposure to
PD-L1-expressing tumor cells, they can be revived
to target the tumor once again. Further studies will
assess the mechanisms involved in recovery of PD1+ T-cell function after in vitro expansion.
Lance Hellman
University of Notre Dame Department of Chemistry and
Biochemistry, Notre Dame and Harper Cancer Research
Institute, South Bend
Recognition of malignantly transformed or virally
infected cells by T-cells is mediated through the
T-cell receptor (TCR). Malignant melanoma is one
such malignancy that is immunosensitive. One of
the melanoma antigens presented by the MHC is
the MART-127-35 (AAGIGILTV) nonameric peptide,
which is recognized by the TCRs DMF4 and DMF5.
Clinical trials involving adoptive cell therapy (ACT)
of melanoma patients showed cancer regression
of 13% and 30% for clonally expanded T-cells genetically engineered to express DMF4 and DMF5.
Our work involves using structure-guided computational design to enhance the affinity of DMF5 towards the MART-127-35 peptide, with the eventual
goal of assessing the impact of enhanced affinity on
anti-tumor immunity in mouse models of melanoma. Thus far, we have generated five higher affinity mutants of DMF5; αD26Y, αD26W, βL98W, and
two double-mutants αD26Y/βL98W and αD26W/
177
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βL98W, and are optimizing a retroviral expression
system to generate gene-modified human T cells.
207
melanoma using b lymphocytes
1
,ClaireM.Buchta2,LauraStunz1, 4,
GailA.Bishop1, 2, 3, 4
Dept. of Microbiology, Immunology and Molecular
1
Department of Microbiology, 2Department of Internal
Medicine, 3Graduate Program of Immunology, University of
Iowa, Iowa City, 4VAMC, Iowa City
Immunotherapy has provided a promising avenue
in the fight against cancer, and cellular vaccines
have emerged as a strong candidate for further
development. Such vaccines employ antigen presenting cells (APCs), typically dendritic Cells (DCs),
to deliver tumor associated antigens (TAAs) to a
patient. Subsequent presentation of these TAAs
induces adaptive immune responses against the
tumor. Though DCs possess advantages in cellular
vaccines, their difficulty of isolation from humans,
low numbers in peripheral blood, and inability to
expand in vitro are distinct drawbacks. Cellular
vaccines harnessing activated B cells have advantages in addressing these challenges. Easily isolated in large numbers from the peripheral blood, B
cells which have been activated through optimized
stimuli are effective APCs. In addition, they induce
strong antigen-specific CD8 T cell responses. Our
model seeks to optimize B cell vaccines against murine melanoma. Preliminary data indicate that our
B cell vaccines, using two optimized stimulating
conditions and several melanoma antigens, effectively extend survival and reduce melanoma tumor
growth in mice. For some antigens, tumor growth
is eradicated. Current directions seek to enhance
delivery of B cell in vitro stimuli through the use of
nanoparticle delivery.
208
Adam Scheid1,VirginiaVanKeulen1, Sara Felts1, Yuji
Zhang2, Svetomir Markovic1, 3, 4, Larry Pease1
1
Department of Immunology, Mayo Clinic College of
Medicine, 2Division of Biomedical Statistics and Informatics, Department of Health Sciences, Mayo Clinic College of
178
Medicine, 3Division of Hematology, 4Department of Medical
Oncology, Mayo Clinic, Rochester, MN
The nature of tumor-associated systemic immune
modulation is controversial. In order to investigate
this controversy we used RNAseq to compare and
contrast peripheral blood T cell transcriptomes of
healthy blood donors to those of stage IV melanoma patients. Using this approach we have found
that consistent profiles of CD4+ and CD8+ T cell
gene expression differentiate healthy individuals
from one another and differentiate melanoma patients from one another as well. Interestingly, these
expression profiles also group healthy individuals
differently than they do melanoma patients, suggesting that gene expression differs between peripheral blood T cells of healthy individuals and
cancer patients. Some of the gene transcripts that
are differentially expressed between these groups
are known to be involved in immune functions,
while others are long intergenic non-coding (linc)
RNAs. Differences in expression of transcripts like
these could result in alterations of peripheral T cell
functions in cancer patients relative to healthy individuals. Further validation and investigation of
our findings could provide new insights into cancer
immunology and lay foundations for more effective
clinical cancer immunotherapies.
tumorigenesis
Ann Janowski1, 2,SuzanneCassel1, 2, 3,Fayyaz
Sutterwala1, 2, 3
1
Interdisciplinary Graduate Program in Immunology,
Inflammation Program, 3Department of Internal Medicine,
Iowa City
2
Inflammation plays a critical role in tumorigenesis
and can contribute to oncogenic mutations, tumor
promotion and angiogenesis. Tumor promoting
inflammation is driven by many factors including
the presence of the pro-inflammatory cytokine IL1β. One major source of IL-1β secretion is through
the activation of inflammasomes. Various inflammasomes have been implicated in cancer including
the NLRC4 inflammasome, which has been shown
to play a protective role in the development of
colorectal cancer. Additionally, NLRC4 expression
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is downregulated in human lung and breast cancer tissue compared to normal healthy tissue. We
sought to future determine the role of the NLRC4
inflammasome in tumor progression by utilizing
subcutaneous models of B16 melanoma and Lewis lung carcinoma. Surprisingly, we found that NLRC4-deficient mice, but not ASC- or caspase-1-deficient, mice developed larger tumors than wild type
mice in a model of B16 melanoma. An increased
tumor size was also observed in NLRC4-deficient
mice during a subcutaneous model of Lewis Lung
Carcinoma. Bone marrow chimera experiments
suggest that an absence of NLRC4 in a radioresistant cell leads to increased tumor size. Taken together these data demonstrate that expression of
NLRC4, and not caspase-1 or ASC, in radioresistant
cells is important for protection in models of subcutaneous tumors.
210
immunity in melanoma
Ayelet Sivan,LeticiaCorrales,ThomasGajewski
University of Chicago
T cell infiltration of solid tumors is associated with
favorable patient outcomes, yet the underlying
mechanisms are not well understood. To examine
the role of microbial composition in spontaneous
immunity to melanoma, we compared B16 melanoma growth in genetically identical C57BL/6
mice derived from 2 different facilities, Jackson
labs (JAX) and Taconic farms (TAC). We found that
B16.SIY tumors exhibited an increased growth rate
in mice derived from TAC, accompanied by reduced
induction and infiltration of tumor-specific CD8+ T
cells. Strikingly, these differences were ablated in
animals cohoused prior to tumor inoculation, consistent with a microbiota-derived effect. Transfer of
JAX fecal material into TAC mice prior to tumor inoculation was sufficient to improve anti-tumor responses. Adoptive transfer of SIY-specific 2C TCR Tg
T cells into tumor-bearing mice revealed reduced
IFN-γ induction in TAC mice, likely pointing to an
effect via antigen-presenting cells. Administration
of JAX feces in combination with αPD-L1 to TAC recipients with established tumors improved tumor
control in comparison to αPD-L1 treatment alone.
Our data provide evidence implicating the microbi-
180
ota in shaping anti-tumor immunity. Further study
may provide the foundation for manipulation of
commensal microbes as a cancer therapeutic.
211 Melanoma intrinsic b-catenin signaling
prevents T cell-dependent immunity
1
, Riyue Bao2,ThomasGajewski1, 3
1
Department of Pathology, 2Center for Research
Informatics, 3Department of Medicine, University of
Chicago
A subset of melanoma patients has evidence for
spontaneous T cell infiltration into tumor sites,
which is associated with clinical benefit. However,
the molecular mechanisms explaining absence of
a T cell response are not yet defined. Analyses of
human melanoma metastases have revealed that
T cell signature low tumors show alterations in
the Wnt/β-catenin signaling pathway. We utilized
an inducible mouse model driven by inducible
BrafV600E and PTEN-deletion, with or without active b-catenin (CAT-STA) to test if tumor- intrinsic
active β-catenin can block anti-tumor immunity.
While Braf/PTEN melanomas showed presence of
a T cell infiltrate, T cells were nearly completely
eliminated in tumors expressing active β-catenin. Adoptive transfer experiments revealed defective T cell priming when tumors expressed active
β-catenin. Analysis of the antigen-presenting cell
compartment revealed a selective decrease in the
CD103+ DC subset within the tumor microenvironment, which was associated with β-catenin-depended inhibition of expression of the chemokine
CCL4 within tumor cells. Therefore, our data have
identified the first defined molecular pathway in
tumor cells that results in defective spontaneous
anti-tumor T cell responses.
212
responses
Janet Zayas1, Frederick Kohlhapp1, Joseph Broucek2,
JevgenijsLusciks1,AndrewZloza1, 3,HowardKaufman4
1
Departments of Immunology/Microbiology, 2General
Surgery, and 3Internal Medicine, Rush University Medical
Center, Chicago, 4Rutgers Cancer Institute of New Jersey,
New Brunswick
181
SUNDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
SUNDAY ABSTRACTS
Background: Ipilimumab (CTLA-4 blocking antibody) and IL-2 (an activating cytokine) are each
approved for the treatment of metastatic melanoma. However, whether a more potent regimen can
be achieved by combining these two therapies has
not been sufficiently investigated. Therefore, we
investigated the ability of this combination to enhance therapeutic responses. Methods: C57BL/6
mice were challenged via intradermal injection
with B16-F10 melanoma (120,000 cells) on day
0. Anti-CTLA-4 (100 µg in 100 µl or 100 µg IgG
control) was administered via intraperitoneal injection on days 3, 6, and 9. IL-2 (100,000 units in
100 µl or 100µl PBS) was administered every 12
h on days 4-8. The tumor area was measured every 2-3 days until mice were sacrificed for flow cytometry analysis of the tumor microenvironment.
Results: Tumor growth was significantly reduced
with combination anti-CTLA-4 and IL-2 treatment
(average tumor area: 2mm2 on day 14) compared
to anti-CTLA-4 only, IL-2 only, and placebo treatment (14, 29, and 68 mm2, respectively, on day 14)
(P<0.01 for all comparisons). Depletion of NK cells
resulted in the abrogation of these combination immunotherapy effects. Conclusions: These findings
propose a previously unrecognized mechanism for
combination anti-CTLA-4 and IL-2 immunotherapy
and highlight the potential synergistic action of anti-CTLA-4 combined with IL-2 for the treatment of
melanoma.
213
and senescence in responder T cells
Jian Ye,FangWang,GuangyongPeng
St. Louis University, St. Louis
Regulatory T (Treg) cells have broad suppressive
activity on host immunity, but the molecular processes and functions of suppressed responder T
cells remains largely unknown. We discovered a
novel suppressive mechanism whereby human
Treg cells induce senescence in naïve/effector T
cells that then exhibit potent suppressive activity
and amplify immune suppression. Treg-induced
senescent T cells were distinctlydifferent from the
exhausted or anergic T cells based on the transcriptome analyses and phenotypic profiles. We futher
identified that the nuclear kinase ataxia-telangiec-
182
tasia mutated protein (ATM) induced DNA damage
wascritical and the main cause for the induction
of responder T cell senescence and dysfunction
mediated by human Treg cells. In addition MAPK
ERK1/2 and p38 were involved in the regulation
of senescence process in responder T cells. Importantly, we futher revealed that human Treg-induced senescence and suppresson function could
be blocked by specific ATM signaling /or ERK1/2
AND P38 signling inhibiton in vitro and in vivo in
animal models. Our studies identify a novel molecular mechanism responsible for human Treg cell
suppression, and provide new insights relevant for
the development of strategies capable of preventing and/or reversing Treg-induced immune suppression in various clinical settings. Supported by
the Melanoma Research Alliance, American Cancer
Society and the National Institutes of Health.
183
SUNDAY ABSTRACTS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
Notes:
184
AIC 2014 • Chicago, IL
Abdala-Valencia, H - 6
Abernathy, L - 52, 53
Abuaita, B - 67
Agashe, V - 15
Alegre, M - 123, 194, 197
Alhakeem, S - 29
Almanan, M - 149
Amarsaikhan, N - 40
Amjadi, M - 94
Anderson, E - 118
Arkee, T - 25
Arnoys, E - 102
Ashbaugh, A - 119
Ashley, S - 120
Aust, S - 19
Ayasoufi, K - 201
Bacellar, O - 134
Badovinac, V - 74, 75, 76
Bagchi, S - 158
Baker, B - 183
Banuelos, J - 78
Bao, R - 211
Barrett, N - 4
Barrios, C - 169
Bartman, C - 123, 194
Bassuk, D - 202
Beilke, M - 169
Bell, M - 182
Belmonte, P - 176, 177
Benck, C - 119
Benet, Z - 11
Berdnikovs, S - 7
Berges, B - 121
Berrien-Elliott, M - 83
Beura, L - 144
Beura, L - 145
Bill, C - 111, 188, 190,
202
Bishop, G - 25, 186, 207
Blaine, K - 160
Blank, S - 167
Blevins, S - 183
Blitzer, G - 205
Bobeck, E - 71
Bockenstedt, M - 135
Bohlson, S - 107
Bohn, P - 35
Boles, B - 67
Bondada, S - 29
Bongard, J - 101
Bonham, C - 160
Boomer, J - 178
Bordner, A - 81
Boyle-Vavra, S - 123
Brandt, S - 32, 167
Brennan, T - 191
Bricker, R - 157
Brockmeier, S - 47, 48
Brooks, A - 99
Broucek, J - 204, 212
Brown, C - 31, 34, 108,
125, 141
Brown, M - 33, 200
Bruns, H - 99
Bryazka, D - 160
Bryce, P - 2, 3, 170
Buchta, C - 207
Burkholder, K - 67
Burlingham, W - 15, 200
Burrage, A - 41
Butsch Kovacic, M - 116
Byrd, J - 29
Cain-Veal, A - 10
Campbell, E - 41
Campbell, J - 6
Cao, Y - 17
Cardin, R - 149
Carneiro, P - 134
Carolan, J - 60, 62
Carr, T - 162
Carrasco, S - 32, 167
Carvalho, E - 134
Casaos, J - 160
Casiano-Rivera, F - 161
185
Autumn Immunology Conference 2014
Cassel, S - 209
Castillo, L - 169
Catalano, M - 110
Cervantes, J - 111
Chang, H - 115, 161
Chapman, N - 185
Chatterjea, D - 119
Chen, C - 118
Chen, J - 197
Chen, L - 123, 194
Chen, M - 174, 176, 180
Chen, S - 84, 85
Chen, X - 88
Chhiba, K - 2, 3
Chi, H - 178
Cho, C - 73
Cho, W - 37, 38, 39
Choi, B - 69
Choi, J - 179, 181
Chong, A - 123, 194, 197
Chougnet, C - 149
Chung, J - 39, 191, 192
Clark, M - 22
Cohen, I - 79
Colin, B - 112
Constans, M - 176, 177
Contreras, A - 73
Cook-Mills, J - 6
Corrales, L - 66, 210
Cory, T - 63
Crabtree, J - 45
Curry, M - 99
Curtis, J - 65
D’Orazio, S - 128, 129
Daniels, M - 156, 189,
197
Dash, B - 177
Daum, R - 123
David, J - 53
David, M - 123
Davis, M - 38
Davis, R - 134
de Pooter, R - 172
Deem, T - 133, 136, 138
del Barrio, L - 124
Delafield, N - 167
DelProposto, J - 151
Diana, G - 184
Doan, J - 97, 171
Dolence, J - 23
Dolubizno, H - 18, 159,
204
Domingo-Gonzalez, R 65, 68
Dominguez, D - 84, 85
Dorsam, G - 5
Dow, C - 35
Duncker, P - 56
Eastman, A - 37, 38, 39,
60, 62
Ebens, C - 192
Eberle, K - 44
Eckel, R - 171
Egana, L - 166
Ehinger, E - 136
Eichinger, K - 166
Empey, K - 166
Epping, L - 16
Esch, K - 140
Evans, H - 36
Fairley, J - 19, 139
Fan, J - 84, 85
Farrar, M - 178
Feau, S - 59
Felts, S - 208
Feola, D - 54, 63, 64
Ferrer, A - 182
Fife, B - 14
Finizio, M - 161
Finnegan, A - 17
Fleetwood, V - 18, 159
Fleming, E - 126
Folmsbee, S - 8
Fosco, D - 88
186
AIC 2014 • Chicago, IL
Fountain, M - 52, 53
Fowler, H - 137
Fraser, K - 145
Frazier, W - 58
Friedman, A - 191, 192
Friedman, N - 79
Gachuki, B - 29
Gajewski, T - 66, 72, 86,
210, 211
Gal, H - 79
Galvis, E - 57
Gan, E - 15
Gandhapudi, S - 173
Ganusov, V - 49
Garnett-Benson, C - 87
Garvy, B - 36
Gates, K - 110
Gauger, P - 48
Gershan, J - 205
Ghosh, S - 5, 106
Gibbons Johnson, R 105
Gibson-Corley, K - 143
Gil Pages, D - 182
Giles, D - 12
Gilpin, T - 35
Gomer, R - 57
Gonser, E - 107
Gonzalez, R - 54
Goodell, M - 172
Goodman, J - 30
Goplen, N - 156
Gottardi, C - 8
Gower, R - 199
Green, J - 178
Greenlee-Wacker, M - 94,
122
Griesenauer, B - 193
Grifka-Walk, H - 12
Grigorova, I - 11, 24
Grinnage-Pulley, T - 140
Gross, B - 89
Guan, W - 45
Gubbels Bupp, M - 91,
96, 98
Gullicksrud, J - 77
Gumperz, J - 71
Gupta, P - 195, 196
Gurczynski, S - 150
Gwin, K - 23
Hair, B - 121
Han, F - 153
Hansen, M - 81, 184
Hanson, B - 25, 207
Harty, J - 16, 74, 77, 143
Hatch, J - 121
Hawksworth, D - 50
Haydar, D - 64
Haynes, L - 15
Hedin, K - 92
Heffernan, J - 14
Hellman, L - 206
Hemann, E - 75
Hennies, C - 59
Hiebert, S - 174
Hildeman, D - 149, 175
Hillman, G - 52, 53
Hinojoza, B - 188, 190
Hofer, E - 105
Hoffmann, M - 184
Hogquist, K - 70, 178
Holbreich, M - 114
Hoogstra, D - 52
Horn, E - 155
Horton, B - 86
Hoselton, S - 5, 106
Hostetter, J - 130
Houtman, J - 185, 187
Hrsuch, C - 4, 114, 160
Hsu, F - 23, 177, 180
Huang, S - 65
Huber, A - 12, 56
Huelsmann, E - 203
Huggins, M - 132
187
Autumn Immunology Conference 2014
Hughes, H - 47, 48
Husain, A - 197
Huseby, A - 147
Hussain, S - 26
Igartua, K - 114
Igyarto, B - 145
Iijima, K - 118
Incrocci, R - 26
Ippolito, J - 33
Itani, F - 16
Jacobsen, J - 21
Jaffery, M - 160
Janowski, A - 209
Janssen, E - 59
Jensen, K - 121
Jergens, A - 130
Jin, F - 82, 132, 146, 147
Jing, W - 205
Johnson, A - 82, 132,
146, 147
Johnson, B - 205
Johnson, E - 49
Johnson, H - 10, 146
Johnston, L - 2, 3
Jones, D - 135
Jones, G - 128
Jones, S - 127, 163
Kaplan, M - 68
Karandikar, N - 16
Karki, S - 22
Karl, J - 35
Kato, A - 170
Kaufman, H - 212
Kee, B - 21, 162
Keleher, L - 34
Keller, M - 15
Kennedy, D - 20
Kephart, G - 118
Kern, R - 9
Khan, S - 75
Kherallah, Y - 11
Kim, M - 74
Kinder, J - 198
Kita, H - 118
Klarquist, J - 59
Kline, D - 88
Kline, J - 88
Klutse, L - 97
Knight, C - 188, 190
Knight, K - 20, 127, 163
Knudson, C - 143
Knudson, K - 189
Kobayashi, T - 118
Koblinski, J - 55
Kohlhapp, F - 18, 159,
203, 204, 212
Kovacs, E - 33
Kryczek, I - 60
Kubasiak, J - 159
Kumari, A - 87
Kunkel, S - 60
Kurtz, J - 131
Kusick, E - 106
Lacey, C - 34
Lamb, I - 140
Landay, A - 28, 168
Langer, C - 196
Langlois, R - 143
Lantz, C - 133, 136, 138
Laouar, Y - 61, 65, 69
Larkin, J - 10
Larsen, B - 103
Lasky, C - 31, 108, 125
LaVallee, K - 141
Lee, Y - 70
Legge, K - 75, 142
Lehn, M - 59
Lei, Y - 194
Lemke, L - 95
Lewkowich, I - 113, 116,
117
Li, H - 40
Li, K - 175
Lin, J - 100
188
AIC 2014 • Chicago, IL
Linville, E - 154
Liu, J - 199
Loffredo, L - 7
Lonardo, F - 52
Long, A - 84
Longnecker, R - 42
Loomis-King, H - 43
Looyenga, B - 102
Louters, L - 102
Loving, C - 46, 47, 48
Lu, N - 78
Lumeng, C - 151
Lurain, N - 168
Lusciks, J - 18, 159, 203,
204, 212
Madi, A - 79
Mahmud, S - 178
Maillard, I - 39, 191, 192
Makinde, H - 168
Malachoswki, A - 37, 60,
62
Mambetsariev, N - 25
Manhart, W - 146
Manlove, L - 145, 178
Manoj, S - 50
Marino, J - 173
Markovic, S - 208
Martin, M - 76
Martinez, P - 137, 140
Martinez-Colon, G - 68,
151
Martinov, T - 14, 119
Martinson, J - 168
Marvin, S - 41
Masopust, D - 145
McAlees, J - 117
McGill, J - 44
McKenna, K - 29
McLachlan, J - 131
McOlash, L - 205
McWilliams, D - 174,
176, 180
Medina, K - 23
Mergian, T - 151
Messingham, K - 19, 139
Meyerholz, D - 143
Meyers, J - 73
Miller, M - 197
Minnerath, J - 90, 101,
103
Mirmonsef, P - 168
Molenaar, M - 92, 105
Molina Mendiola, C - 184
Molinero, L - 194
Moon, J - 197
Moore, B - 43, 65, 68,
120, 150
Morman, R - 27
Mothe, B - 35
Muite, K - 164
Muller, W - 42, 55, 153
Murphy, B - 63
Murphy, S - 98
Muthusamy, N - 29
Myers-Morales, T - 128,
129
Nagler, C - 194
Nair, L - 194
Nauseef, W - 94, 122
Neal, L - 38, 39
Nelson, C - 144
Nguyen, T - 109
Nipper, A - 28
Norian, L - 75, 89
Noth, I - 160
Noto, F - 199
O’Conner, S - 107
O’Hagan, K - 179, 181
O’Riordan, M - 67
O`Dea, E - 40
Oakley, O - 154
Oakley, O - 155
Ober, C - 114
Olalekan, S - 17
189
Autumn Immunology Conference 2014
Oliveira, J - 134
Olson, R - 31
Olson, Z - 46, 48
Olszewski, M - 37, 38,
39, 62
Orend, J - 166
Owen, D - 178
Paczesny, S - 193
Painter, M - 165
Parks, C - 81
Parney, I - 82
Pauken, K - 14
Pavelko, K - 82
Paynich, M - 127, 163
Pease, L - 81, 184, 208
Peng, G - 213
Perez, D - 46, 47
Perlamn, S - 148
Perlman, H - 175
Person, T - 80
Peters, A - 9
Petersen, C - 137, 140
Peterson, E - 45
Pewe, L - 143
Phee, H - 179
Phee, H - 181
Philips, R - 176
Phillips, G - 130
Pincus, N - 42
Pirko, I - 146
Pitts, M - 129
Poeschla, E - 165
Pohl, N - 30
Poston, S - 27
Potchen, N - 60, 62
Pothoven, K - 170
Powers, S - 22
Pratt, C - 108, 125, 141
Proctor, A - 130
Pryshchep, O - 179, 181
Qin, L - 84, 85
Qiu, Y - 37, 38
Qualls, J - 157
Quan, J - 95
Quarnstrom, C - 144
Qui, Y - 39
Radojcic, V - 191, 192
Ramadan, A - 193
Ramirez, L - 33
Rao, A - 162
Rapovy, S - 157
Raynor, J - 149
Re, F - 124
Reboulet, R - 59
Redondo, R - 204
Reed, B - 182
Renner, D - 82
Reynolds, J - 124
Richer, M - 74
Richgels, P - 113
Rienstra, M - 104
Rocholl, H - 106
Rodriguez, M - 165
Roy, E - 175
Roychoudhury, R - 30
Sacco, R - 44
Sahli, N - 14
Sahoo, M - 124
Salem, A - 89
Sandbulte, M - 46
Sant’Angelo, D - 174
Saylor, S - 138
Schaut, R - 140
Scheid, A - 208
Schenkel, J - 144, 145,
178, 203
Schleimer, R - 9, 170
Schmidt, S - 157
Schmitz, H - 178
Schrum, A - 182, 184
Schuh, J - 5, 106
Schuiteman, S - 102
Scorza, B - 139
Scott, S - 91
190
AIC 2014 • Chicago, IL
Seaburg, L - 180
Segal, B - 12, 56
Sen, S - 73
Serezani, C - 167
Serezani, H - 32
Shapiro, V - 23, 174, 176,
177, 180
Sharma, S - 134
Shea, L - 199
Shen, A - 109
Shepherd, A - 133
Shifrut, E - 79
Shilling, R - 195, 196
Siebel, C - 191, 192
Siewe, B - 28
Sigvardsson, M - 172
Simon, S - 87
Singer, K - 151
Sinha, S - 16
Sinniah, R - 110
Sivan, A - 210
Sivaprasad, U - 116
Skyberg, J - 34, 125
Smith, C - 68
Smith, L - 93
Soto, O - 112, 202
Souza, C - 46
Soveg, F - 6
Spanier, J - 14
Spear, G - 168
Sperling, A - 4, 114, 160
Sponseller, B - 135
Spranger, S - 86, 211
Spranger, S - 211
Srinand, P - 73
Stangel, A - 105
Steffan, B - 5, 106
Stefka, A - 194
Stein, M - 114
Steinert, E - 145
Stevens, W - 9
Stoffers, S - 116
Stoolman, J - 56
Stunz, L - 25, 207
Sukka-Ganesh, B - 10
Sullivan, J - 15
Sutterwala, F - 209
Swanson-Mungerson,
M - 26
Swier, L - 83
Takahashi, S - 160
Tan, B - 170
Tan, C - 173
Tangen, S - 180
Taparowsky, E - 27
Tatar, A - 73
Taylor, A - 173
Teague, K - 173
Teague, R - 83
Teixeiro, E - 156, 189
Templeton, S - 40
tenOever, B - 143
Thapa, P - 174
Thompson, E - 13
Thorne, P - 114
Thorp, E - 58
Tien, S - 45
Tjota, M - 4
Torres Ocampo, A - 37
Troxell, B - 32
Tuna, H - 63
Tung, C - 115, 161
Turner, J - 24
Vacaflores, A - 185
Van Keulen, V - 208
van Lier, A - 116
Varga, S - 143
Vela Ramirez, J - 30
Vercelli, D - 114
Verykokakis, M - 162
Vezys, V - 13, 144
Vincent, A - 46, 48
Vines, C - 111, 112, 188,
190, 202
191
Autumn Immunology Conference 2014
Von Mutius, E - 114
Wacker, M - 139
Wagner, D - 30
Wagner, S - 195, 196
Wai, L - 25
Wallis, A - 186
Wang, C - 158
Wang, F - 213
Wang, H - 70
Wang, M - 149
Wang, S - 167
Wang, T - 197
Wang, Y - 178, 194, 197
Wannemuehler, M - 30,
130
Way, S - 198
Weber, E - 153
Weber, J - 205
Weber, T - 105
Wertz, J - 100
Weston, T - 96
Wheeler, L - 148
Whitaker, A - 154, 155
White, M - 57
White, T - 121
Wienclaw, T - 121
Wiethoff, C - 41
Wilcox, D - 42
Wilke, C - 43, 120
Wilkes, D - 195
Willenbring, R - 146
Williams, J - 72, 86
Wilson, M - 134, 139
Winford, E - 108
Winger, R - 55
Wiseman, R - 35
Wodrich, H - 41
Won, T - 61
Wongrakpanich, A - 89
Woo, S - 66
Woodard, J - 21
Wren, J - 173
Wu, Q - 195, 196
Wu, R - 11
Wymore Brand, M - 130
Xin-Fu, Y - 164
Xing, E - 37, 62
Xing, Y - 178
Xu, J - 197
Xu, X - 51, 71
Xue, H - 77
Yagita, H - 178
Yang, F - 32
Yang, X - 115
Yang, Y - 32
Yanik, G - 65
Ye, J - 213
Yeah, X - 58
Yi, Z - 152, 186
Yin, D - 197
Young, J - 197
Yunker, C - 52, 53
Zacharias, Z - 142
Zamarron, B - 151
Zawacki, A - 90
Zayas, J - 18, 203, 204,
212
Zhang, B - 84, 85
Zhang, H - 158
Zhang, J - 193, 199
Zhang, M - 85, 187
Zhang, S - 58
Zhang, X - 116
Zhang, Y - 208
Zheng, Y - 72
Zhou, B - 115
Zhou, X - 43
Ziemann, R - 50
Zloza, A - 212, 159, 203,
204, 18
Zook, E - 1
192
Thomas Mitchell
Conference chair
SandraBurnett
Secretary
StevenVarga
Workshop coordinator
UniversityofLouisville
BrighamYoungUniversity
UniversityofIowa
Subba Bondada
Liaison to sponsors
UniversityofKentucky
TKentTeague
Liaison to sponsors
UniversityofOklahoma
Dave Bruns
Treasurer
PricewaterhouseCoopers,LLP
JohnHackett
Conference cohost
AbbottDiagnostics
CarlWaltenbaugh
Conference cohost
NorthwesternUniversity
Michael Zimmer
Conference cohost
PurdueUniversity,Calumet
RafaelFernandez-Botran
Diversity
UniversityofLouisville
Heather Bruns
Undergraduate program
BallStateUniversity
FotiniGounari
Liaison to academia
UniversityofChicago
VirginiaShapiro
Awards coordinator
MayoClinic,Rochester,
Minnesota
E.CharlesSnow
Chief executive officer
DavidLubaroff
Advisor
Maria-LuisaAlegre
Councilor
UniversityofKentucky
UniversityofIowa UniversityofChicago
193
AIC COUNCIL MEMBERS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
AIC COUNCIL MEMBERS
Gretchen Pinkerton
Administrative assistant
AIC General Council Members
Paul Bryce NorthwesternUniversity
Richard DiPaolo St.LouisUniversity
Dave Feola UniversityofKentucky
BrianFife UniversityofMinnesota
Marian Kohut IowaStateUniversity
IanLewkowich CincinnatiChildren’s
Hospital
DeminWang BloodCenterofWisconsin
VladimirBadovinac UniversityofIowa
Hua-chenChang IndianaUniversity-Purdue
UniversityIndianapolis
WilbertDerbigny IndianaUniversitySchool
ofMedicine,Indianapolis
Kay Medina MayoClinic,Rochester,
Minnesota
Lyse Norian UniversityofIowa
RyanTeague SaintLouisUniversity
SchoolofMedicine
CharlotteVines UniversityofTexas,ElPaso
SuzanneBohlson DesMoinesUniversity
CharlieBrown UniversityofMissouri
Ivan Maillard UniversityofMichigan
RebeccaShilling UniversityofIllinois,
Chicago
Keith Hamel UniversityofChicago
Postdoctoral
representative
AldoVacafloresSalinas UniversityofIowa
Graduate student
representative
194
AIC 2014 • Chicago, IL
2014 AIC Academic Sponsors
We appreciate the support of our academic
sponsors.
DesMoinesUniversity Departmentof IllinoisStateUniversity
IndianaUniversity
SchoolofMedicine
IowaStateUniversity
LoyolaUniversity
MedicalCenter
MayoClinicCollege
ofMedicine
NorthwesternUniversity
PurdueUniversity
Rosalind Franklin
University
SaintLouisUniversity
UniversityofChicago
UniversityofCincinnati
UniversityofIowa
UniversityofIowa
UniversityofKentucky
UniversityofLouisville
UniversityofMichigan
UniversityofMichigan
UniversityofMinnesota
UniversityofWyoming
WashingtonUniversity
Microbiologyand Immunology
SchoolofBiological
Sciences
Departmentof
Microbiologyand Immunology
ImmunologyGraduate
Program
Departmentof
Microbiologyand Immunology
Departmentof
Immunology
Medicine,Divisionof
AllergyandImmunology
Comparative
Pathobiology
Departmentof
Microbiologyand Immunology
Departmentof
MolecularMicrobiology
andImmunology
Committeeon
Immunology
CincinnatiChildren’s
Hospital
Departmentof
Microbiology
Interdisciplinary
GraduateProgramin
Immunology
Departmentof
Microbiology,Immunology
andMolecularGenetics
Departmentof
Microbiologyand Immunology
GraduatePrograminImmunology
Departmentof
Microbiologyand Immunology
CenterforImmunology
CollegeofArtsand
Sciences
Departmentof
Pathologyand
Immunology
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Autumn Immunology Conference 2014
AIC 2014 Exhibitors
AIC 2014 EXHIBITORS
eBioscience,
anAffymetrix
Company
eBioscience, an Affymetrix company,
develops and manufactures over 12,000
antibodies, proteins, immunoassays
and multiplex assays at ISO-certified
facilities worldwide. Focused on accelerating immunologic discoveries with
reagents to measure gene and protein
expression in single cells, we provide
innovative solutions to researchers and
clinicians looking to answer questions
driving today’s life science communities.
American
Associationof
Immunologists
The AAI is an association of professionally trained scientists from all over
the world dedicated to advancing the
knowledge of immunology, fostering
interchange of ideas and information
among investigators, and addressing
the potential integration of immunologic principles into clinical practice.
Ancell
Since 1992, Ancell Corporation has
been providing the immunology research community with high quality antibodies, conjugates and recombinant
proteins.
Bangs
Laboratories,
Inc.
Bangs Laboratories, Inc. is a manufacturer of uniform polymer, silica, and
magnetic microsphere products for diagnostic, research, and flow cytometry
applications.
BioLegend
World-Class Antibodies, Proteins, Assays and Research Solutions. Complete
Brilliant Violet™ Antibody Conjugates
for the Violet Laser: BV510™ , BV711™
, BV785™. Personalized Multicolor Flow
Cytometry Panel Design. New LEGENDScreen™ Human Cell Screening (PE)
Kits. Request Bulk Cytokines & Chemokines for Bioassay. Ultra-LEAF™ (Low
Endotoxin, Azide-Free) Antibodies.
New ELISA Kits: IL-35, Active TGF-β1.
Bio-techne
Bio-Techne combines the best-in-class
products and services from R&D Systems, Novus Biologicals and Tocris,
making us the best strategic partners
for immunology researchers. Come see
how Bio-Techne is building innovation
opportunities to propel your research
to the next level. Learn about our vast
product range and how we manufacture over 90% of our bioactive proteins, application-qualified antibodies,
Quantikine® ELISAs, Luminex Assays,
and small molecule inhibitors and activators.
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CellSignaling
Technology
Founded by research scientists in
1999, Cell Signaling Technology (CST)
is a private, family-owned company
with over 400 employees worldwide.
Active in the field of applied systems
biology research, particularly as it relates to cancer, CST understands that
antibodies with high levels of specificity and lot-to-lot consistency are
critical for successful results. It’s why
we produce all of our antibodies in
house, and perform painstaking validations for multiple applications. And
the same CST scientists who produce
our antibodies also pro vide technical
support for customers, helping them
design experiments, troubleshoot, and
achieve reliable results. We do this because that’s what we’d want if we were
in the lab. Because, actually, we are.
Cellular
Technology
Limited(C.T.L.)
Cellular Technology Limited (CTL),
headquartered in Cleveland, OH, is a
global biotechnology company with
locations and distributors worldwide.
CTL provides the tools for specializing
in cellular immune assays: PBMC Library:Cryopreserved, HLA-typed donors with established T cell reactivity.
All-around ELISPOT: ImmunoSpot®
Readers, ELISPOT Kits, Standardization
Packs, Hands-on Training, Consultation,
and Contract Research. Serum-free Cell
Culture Media for standardized freezing, thawing, and testing of PBMC. BioSpot® Analyzers for serum antibody
neutralization tests: Viral Plaque, PRNT,
Viral ICA, Serum Neutralization Assays,
Serum Bactericidal Assays.
Cytek
Cytek Development provides the flow
cytometry tools scientists need at a
price they can afford. Our upgradable
product lines allow clients to upgrade
their equipment at a fraction of the cost
of buying new; and our comprehensive
service plans keep your cytometer running at peak performance.
Cytobank
Cytobank is a web-based platform to
manage, analyze, and share your flow
and mass cytometry experiments. Using
a web browser, users can log in to the
Cytobank website, create new experiments, upload a set of FCS files associated with that experiment, and generate
novel visualizations like SPADE and
viSNE plots . Each experiment can contain context rich attachments such as
PDFs, PowerPoints, and Excel spreadsheets.
197
AIC 2014 EXHIBITORS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
AIC 2014 EXHIBITORS
Users have an email-like interface to
organize and search their experiments
and can share their experiments with
other users on Cytobank.
De Novo
Software
FCS Express 4 is one of the leading flow
cytometry data analysis software packages. Powerful analysis, visualization
capabilities and sophisticated presentation features make it the tool of choice
for thousands of researchers needing
quick results. FCS Express 4 Image cytometry provides the same support and
flexibility for Image Cytometry data.
EMD Millipore
EMD Millipore provides comprehensive
solutions to life science researchers.
From flow cytometers to antibodies, reagents and chemicals-- EMD Millipore’s
portfolio of products is growing. We are
much more than a water system and
filtration company and we invite you to
visit our website to see for yourself.
Essential
Pharmaceuticals,LLC
Essential Pharmaceuticals offers quality cell culture media products for the
next generation of research. Now cell
culture can be conducted with all the
benefits and none of the negative consequences of serum use. Cell-Ess® serum
replacement is chemically defined and
fully synthetic. Save time. Be in control.
Be certain.
FLOWJO,LLC
FlowJo is a full suite of flow cytometry
data analysis tools. From simple gating, to complex clustering algorithms,
FlowJo has it all. With over 20 years in
the industry, FlowJo has everything you
need for your data analysis. FlowJo’s
revolutionary analysis paradigm allows
you to easily scale from analyzing two
colors to 40, so once you learn FlowJo,
you’ll never need anything else. In 2013,
FlowJo was cited by over 90% of the
highest impact journal articles using
3rd party flow cytometry data analysis
software!
Hycult
Since 1994, Hycult Biotech designs, develops, produces and markets antibodies, antibody based products and more
specifically immunoassays for innate
immunity and directly related fields,
with an emphasis on complement, neutrophil proteins, TLR, scavenger receptors and acute phase proteins.
Immudex
Based in Copenhagen, Denmark with
North American operations based in
Fairfax, Virginia, Immudex manufactures
MHC Dextramers, the leading MHC mul
198
timer reagent for the detection of antigen-specific T cells. Under Cancer Immunotherapy Consortium (CIC) and the
European Cancer Immunotherapy Consortium (CIMT), Immudex also provides
MHC Multimer and Elispot proficiency
panel services worldwide. Immudex’s
MHC Dextramer® products are utilized
for the quantification or sorting of antigen-specific T cells in life science research, in vitro diagnostics, as well as
the development of immunotherapeutics and vaccines. The primary focus is
research-use-only products for the immune monitoring of immunotherapy
development, and monitoring of CMV
cellular immunity in transplant and other immune-deficient patients.
InvivoGen
InvivoGen is the leading supplier of
research reagents for studying innate
and adaptive immunity. We offer a wide
range of engineered cell lines and the
largest choice of agonists for the myriad PRRs involved in the innate immune
system. In addition to standard research
grade agonists we supply many VacciGrade™ ligands for use as vaccine adjuvants. InvivoGen also provides a custom
ligand-screening service to determine if
a given sample will act as an agonist or
antagonist against a given PRR.
Leinco
Technologies,
Inc
Leinco Technologies is specialty manufacturer of antibodies, recombinant
proteins, second step reagents, and
specialty buffers/substrates for use
in Flow Cytometry, ELISA, WB, ImmunohistoChemistry. in vivo functional
studies, immunofluorescence and other
life science applications. We are also a
premier provider of custom research
and protein/antibody manufacturing
services.
Life
Technologies
Life Technologies™ products harness
the power of science to transform lives.
As a member of the Thermo Fisher
Scientific family of brands, our instruments, everyday tools, and services offer high-quality, innovative life science
solutions for every lab and application.
Go to lifetechnologies.com to learn
more.
Miltenyi Biotec
Miltenyi Biotec provides MACS© Technology, the gold standard in cell separation for research and clinical scale, as
well as sample preparation, cell analysis and molecular biology products
and services to the biomedical research
community.
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AIC 2014 EXHIBITORS
AIC 2014 • Chicago, IL
Autumn Immunology Conference 2014
Supporting Life Science Research since
1988, PeproTech is the trusted source
for the developing and manufacturing
of high quality cytokine products for
the life-science and cell therapy market. Over the past 26 years the company
has grown into a global enterprise with
state-of-the-art manufacturing facilities
in the US, and offices around the world.
With over 2,000 products PeproTech
has developed and refined innovated
protocols to ensure quality, reliability and consistency. Our mission is to
provide the highest quality products
and premium support that address the
needs and demands of today’s scientists
and researchers.
PerkinElmer
Biological complexity raises questions
requiring translational research from
the well, to the cell, to the animal and
back again. PerkinElmer enables you
to approach your target from multiple
perspectives: locate, detect and quantitate your biology of interest; analyze
and understand it in wider physiological contexts.
Sarstedt
Sarstedt develops, manufactures, and
markets equipment and consumables
for medicine and research. Products
for the research laboratory include cell
culture labware; consumables for PCR,
molecular biology, and cryopreservation; and benchtop instruments. Sarstedt’s specialty cell culture products are
designed for superior cell growth, ease
of use and better value. Included are lumox® film-based plates and dishes for
low autofluorescence and effective gas
exchange; flexiPERM® silicone inserts
for subdivision or parallel analysis; and
the miniPERM® benchtop bioreactor
for high density cell product yield.
Seahorse
Seahorse Bioscience metabolic analyzers
and XF stress test kits are the industry
standard for measuring cell metabolism,
in real-time, in a microplate. XF Extracellular Flux Analyzers simultaneously
measure the two major energy pathways
of the cell, mitochondrial respiration and
glycolysis, providing a full bioenergetic
profile. Now it’s easy to determine the
ATP and biosynthetic demands of immune cell proliferation, differentiation
and effector function, generating new
insights into mitochondrial dysfunction
and opening the door to a new understanding of immunology, cancer, aging,
and metabolic, cardiovascular, and neurodegenerative diseases.
AIC 2014 EXHIBITORS
PeproTech
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AIC 2014 • Chicago, IL
Shenandoah
Biotechnology,
Inc
Shenandoah Biotechnology specializes
in manufacturing recombinant proteins
for research, including cytokines, chemokines and growth factors. We are one of
the top (3) manufacturers of cytokines
and growth factors in the United States.
Our scientists are experts in developing
and producing biologically active and
highly pure proteins.
Sigma-Aldrich
Sigma-Aldrich is a leading Life Science
and High Technology company whose
biochemical, organic chemical products,
kits and services are used in scientific research, including genomic and proteomic
research, biotechnology, pharmaceutical
development, the diagnosis of disease
and as key components in pharmaceutical, diagnostics and high technology
manufacturing. Sigma-Aldrich customers include more than 1.3 million scientists and technologists in life science
companies, university and government
institutions, hospitals and industry.
Spherotech
Spherotech manufactures a variety of
microparticles for flow cytometers and
assay development. These particles are
used for calibration, alignment, multiplexing, compensation, absolute counting and drop delay determination. Specifically, the calibration, alignment, and
drop delay particles are used extensively
for QC and long term performance tracking. In addition, Spherotech has particles
for confocal fluorescence microscopy.
STEMCELL
Technologies
“Ready. Sep. Go. Cell separation in as little as 15 minutes. STEMCELL Technologies enables the fast and easy isolation of
highly purified T cells, B cells, NK cells,
dendritic cells, monocytes, granulocytes
and other cell types. Isolate fully functional cells with our column-free immunomagnetic (EasySep™ and RoboSep™)
or immunodensity (RosetteSep™) systems, and then move immediately to
your downstream application of choice.”
St Jude
Children’s
Research
Hospital
Since opening 50 years ago, St. Jude
research has played a pivotal role in
pushing overall U.S. pediatric cancer
survival rates from 20 to 80 percent.
Our strength comes from an unparalleled integration of research and clinical care. The breadth of research at St.
Jude spans fundamental basic sciences,
translational research, clinical trials
and long-term follow-up for our patients. These interwoven research efforts include unparalleled integration of
research and clinical care.
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Autumn Immunology Conference 2014
TheAutumnImmunologyConferenceappreciates
theFriendsofAICfortheirgeneroussupport:
MarisaAlegre
JohnHackett
Gail Bishop
Barbara Kee
Subba Bondada
Marian Kohut
Paul Bryce
DavidLubaroff
LeticiaCorrales
Tom Mitchell
BonnieDittel
Lyse Norian
Kerry Empey
AnneSperling
Robert Fairchild
T.KentTeague
Beth Garvy
CharlotteVines
FotiniGounari
CarlWaltenbaugh
Grant Support
Funding for this conference was made possible, in
part, by grant R13-074121 from the National Institute of Allergy and Infectious Diseases. The views
expressed in written conference materials or publicationsandbyspeakersandmoderatorsdonotnecessarilyreflecttheofficialpoliciesoftheDepartment
ofHealthandHumanServices;nordoesmentionof
tradenames,commercialpractices,ororganizations
implyendorsementbytheU.S.Government.
AutumnImmunology,Inc.isincorporatedasatax-exempt Scientific Organization under section 501(c)(3)
oftheInternalRevenueCode.
©williamburnettphotography.com
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2014 AIC Sponsors
Weappreciatethesupportofoursponsors.
®
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2014 AIC SPONSORS
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Exhibit Hall 7th Floor Salon I/II
EMD
Millipore
Cytek
Hycult
Cellular
Technology
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Miltenyi
Bang's
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©williamburnettphotography.com
44th Annual Autumn
November 20-23, 2015
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