University of Rochester
Medical Center
University of Rochester
Medical Center
Mechanisms of T cell mediated
heterosubtypic immune
protection against influenza virus
Dr. David J. Topham
David H. Smith Center for Vaccine Biology & Immunology
Aab Institute for Biomedical Sciences
Department of Microbiology & Immunology
UR Center for Immune Modeling for Biodefense
University of Rochester Medical Center
Rochester, NY
University of Rochester
Medical Center
Influenza Virus
MHC Class I
www.sciencemag.org/cgi/content/full/293/5536/1773
University of Rochester
Medical Center
Mouse Model of Influenza Infection
• Intranasal inoculation with
virus in 30µl PBS
• CD8 CTL appear at day 5 and
antibody at day 7
Virus titer (log10)
• Titers peak by day 5, and is
cleared by day 8-10
Primary influenza infection
5
Virus
4
1x10 7
CTL
8x10 6
6x10 6
3
Ab
2
4x10 6
1
2x10 6
0
0x10 0
0
2
4
6
8
10
12
CD8 T cells in the lung
• Virus replicates exclusively in
the epithelial cells lining the
respiratory tract
C57BL/6 mouse
14
Days after infection
University of Rochester
Medical Center
Protective Immunity Against Influenza
• Antibodies against Neuraminidase (NA) prevent
release of viral particles from the infected cells
–Similar effect with neuramindase inhibitors
•
Matrix 2 (M2)
- Ion channel
Antibodies against Hemagglutinin (HA)
neutralize the virus
– IgA and some IgG in the mucosa
– IgG in the serum
•
Antibodies against internal NP and M1
proteins are not protective
– Antibodies to M2 weakly protective
•
CD8 T cells lyse infected cells
– Recognition of viral peptides in class I MHC
•
CD4 T cells secrete IFN-
– Recognition of viral peptides in class II MHC
Internal proteins:
Cartoon courtesy of R. Webby
NP, PA, PB1, PB2, NS1, NS2, M1
University of Rochester
Medical Center
Why is heterosubtypic immunity relevant?
Reassortment of influenza
Existing human
virus
Emerging avian
virus
Reassortant
virus
University of Rochester
Medical Center
Influenza heterosubtype immunity
• Antibody cross-reactivity
– shared HA, NA epitopes or
proteins (M2)
– Inhibition of infection
– Inhibition of particle release
– Protective against lethal
infection
• T cell cross-reactivity
– Conserved peptide
sequences
– Identical or conserved
internal proteins
• CD8 CTL lyse infected
cells
• CD4 T cells secrete
IFN-
– Reduce virus titers in the
lung
– Sufficient for protection
from lethal infection?
University of Rochester
Medical Center
Mouse model of heterosubtypic
influenza infection
NPPA
Identical NP and PA
internal proteins
(x31)
Day 8
-----
H1N1
(PR8)
(1-3 months)
Day 5
Y Y Y Y Y Y Y Y Y Y Y Y Y
C57/BL6
Secondary
Secondary
viral
clearance
mediated by
NP specific
CD8 CTL
----
YYYY
Primary
H3N2
Primary viral
clearance
mediated by
antibody
and CD8
CTL
University of Rochester
Medical Center
# CD8 NP 1¡
Lung virus 1¡
# CD8 NP 2¡
Lung virus 2¡
Log10 EID50 Lung
Number of CD8 T cells
Accelerated secondary immune response and
viral clearance in heterosubtype immune mice
NP specific
T cells in
airways
200000
150000
2.5 - 4-fold
100000
6
5
Virus titers
in the lung
4
3
2
50000
1
0
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days after infection
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Days after infection
University of Rochester
Medical Center
Optimal immune protection depends
on extralymphoid T cells
• Secondary T cell-mediated immune protection from
influenza wanes with time (Gerhard)
• Central and effector/memory subsets of T cells differ in
their tissue localization (Lanzavecchia)
• CD4 and CD8 memory T cells distribute to extralymphoid
tissues during and after infections (Jenkins, Lefrancois)
• Secondary T cell-mediated immune protection from
influenza is related to the number of virus-specific CD8 T
cells in the tissue (Woodland)
• Intratracheal transfer of virus-specific CD4 or CD8 T cells
into the lung can be protective (Woodland)
University of Rochester
Medical Center
How are memory T cells
retained in non-lymphoid
tissues like the lung?
University of Rochester
Medical Center
T cell migration,
adhesion, and
extravasation
Rolling
Blood flow
Basement
Membrane
Tethering
Triggering Strong
Adhesion Diapedesis
Selectins Chemokines VLA-4/VCAM
LFA-1/ICAM-1
VAP-1
?
University of Rochester
Medical Center
VLA-1 and VLA-2 are upregulated on T cells
during influenza infection
CD8
CD4
3
Day 8
0.2
0
1.5
BAL
25
0.5
0.2
VLA-2
VLA-2
Day 0
CD4
32
30
6
11
8
0.1
39
BAL
28
0.1
CD8
2
6
27
3
MLN
3
7
0.8
0.1
MLN
0.4
1
1
0.1
7
8
5
11
SPL
9
3
VLA-1
SPL
2
4
VLA-1
University of Rochester
Medical Center
VLA-1 is expressed on the
majority of influenza-specific
CD8 T cells in the lung
Tetramer
Db/NP
Day 8
2
Db/PA
5 (71)
65
5
5
16 (77)
55
VLA-1
University of Rochester
Medical Center
VLA-1+ CD8+ Db/NP+ T cells accumulate
in the lung after viral clearance
VLA-1+ CD8+ Db/NP+
100%
BAL
80%
MLN
60%
SPL
40%
20%
0%
0
2
4
6
8
10 12 14
Days after infection
University of Rochester
Medical Center
Predominance of VLA-1+ CD8+ T cells among non-lymphoid
organs compared to lymphoid organs
Of the CD8+ cells
Non-lymphoid
Lymphoid
Of the CD8+
DbNP+ cells
Organ
BAL
CD8+
20.9
DbNP+
12.2
VLA-1+
76.4
VLA-1+
86.9
Lung
44.6
11.1
80.7
94.2
Kidney
3.8
33.1
74.4
94.3
Liver
7.5
2.5
86.5
88.0
Salivary
10.6
34.3
88.0
88.0
CLN
23.0
0.4
0.4
31.8
MLN
22.6
1.4
2.1
16.1
MesLN
25.8
0.7
0.9
56.7
Spleen
11.9
3.1
6.0
68.2
PBL
24.6
0.7
4.0
16.9
University of Rochester
Medical Center
Reduced tissue memory in the absence of VLA-1
Day 50 influenza immune mice
Db/NP+ CD8 T cells
WT
KO
100000
3000
10000
50000
80000
2500
8000
40000
6000
30000
4000
20000
2000
60000
1500
40000
1000
20000
500
2000
10000
0
0
0
0
BAL
LUNG
MLN
SPLEEN
Inhibition or absence of VLA-1 results in redistribution to
the lymphoid compartment
University of Rochester
Medical Center
Blocking VLA-1 compromises secondary
heterosubtypic immune protection
Control Ig
CD8 35
Anti-VLA-1
28
55
7
Day 6
Db/NP
Percent survival
100
90
80
70
60
50
40
30
20
10
0
Control
Ig
AntiVLA-1
Nonimmune
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days after infection with PR8
University of Rochester
Medical Center
Percent survival
Increased susceptibility of VLA-1 (–/–) mice to
heterologous secondary infection
100
90
80
70
60
50
40
30
20
10
0
WT
KO
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Days after PR8 infection
3 months after primary
infection
University ofX31
Rochester
Medical Center
CD8 T cell mediated
heterosubtypic protection
• Depends on CD8 T cells in the airways
– Numbers have to be high
– Lymphoid memory cells offer only partial
protection
• Retention of CD8 T cells in the airways
depends on VLA-1
• Protection is not indefinite
– Numbers in the airways wane with time
• Suggests low turnover
University of Rochester
Medical Center
CD4 T cell responses during
influenza infection
University of Rochester
Medical Center
Reverse Genetics insertion of OVA323-339 epitope into
influenza neuramindase
OVA323-339
IQTGNISI-ISQAVHAAHAEINEAGR-ISIVAG
NA
MDCK
NA
University of Rochester
Medical Center
Stimulation of OVA specific
CD4 T cells by influenza:
OVA323-339
Recombinant
influenza
WSN-OVAII
• Assay by Flow cytometry
- Thy1.2+ Tg T cells
- Clonotypic mAb, KJ126
Tg T cells
DO11.10
or
OT-II
Collect: BAL (broncho-alveolar lavage)
MLN (mediastinal lymph node)
SPL (spleen)
Blood
Tissue
University of Rochester
Medical Center
IFN- SFC/106
Induction of OVA-specific CD4 and NP-specific CD8 T cells by WSN-OVAII
OVA323-339
Number of Lymphocytes
WSN-OVAII
NP366-374
WSN-HEL
BAL
LUNG
University of Rochester
Medical Center
Induction of VLA-1 and VLA-2 on B6 wild-type
CD4+ T cells 8 days after WSN-OVAII infection
A
CD8
Airways
Lung Tissue
46
18
35
CD4
VLA-2
B
30
65
24
1
54
15
1
VLA-1
University of Rochester
Medical Center
Percent VLA-1+ CD4
T cells
Late accumulation of VLA-1+ CD4 T cells in the lung
and airways during acute influenza infection
50
40
B
Airways
J
Lung
B
J
B
J
30
J
20
J
B
10
JB
B
J
B
6
7
0
4
5
8
9 10 11 12
Days after WSN-OVAII
infection
University of Rochester
Medical Center
Memory and recall response in WSN-OVAII immune mice
WSN-OVAII
Day 30
WSN-OVAII
OVA323-339
*Undetectable
in the airways
Recall with
OVA323-339 + LPS
*
* b.d.= below detection
University of Rochester
Medical Center
Antigen is required for proliferation of OT-II CD4 T
cells in a flu infection
WSN-HEL
WSN-OVAII
Lung
MLN
SPL
Day 5
CFSE
University of Rochester
Medical Center
Influenza primed OT-II airway memory CD4 T cells
Day 30 WSN-OVAII Airways
10
4
10
4
24 .6
10
10
2
1
10
10
0
10 0
10
FL 2-H
10
3
VLA-2
FL 4-H
Thy 1.1
0.33
10
10
10 1
102
FL 3-H
25 .5
25
103
104
26
3
2
1
0
1111 .4
38 .5
10 0
CD4
10 1
102
FL 1-H
103
104
VLA-1
Does low VLA-1 expression account for
the low number of cells in the airways?
University of Rochester
Medical Center
Number
Distribution of OVA-specific OT-II CD4 T cells 30
days after WSN-OVAII influenza infection
20000
18000
16000
14000
12000
10000
8000
6000
4000
2000
0
300000
Airways
MLN
250000
Spleen
Lung
200000
150000
100000
50000
0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Day
Day
60000
50000
Few cells remain
in the airways or
lung tissue
OT-II cells
40000
Day 30
30000
20000
10000
0
Airway
Lung
MLN
Spleen
University of Rochester
Medical Center
OVA specific response is intermediate between two
endogenous CD4 T cell epitopes during WSN-OVAII infection
1000
SFC/106 cells
IFN-g
IL2
4000
800
OVA323-339
600
NA161-175
400
HA91-105
250
200
150
100
50
0
OVA 323-339
3000
NA161-175
2000
HA91-105
800
600
400
200
Not done
BAL
Lung
MLN
Spleen
0
BAL
Lung
MLN
Spleen
Day 9 WSN-OVAII
University of Rochester
Medical Center
CD4 T cells in flu infection
• Novel system for single cell analysis of CD4 T
cells in flu infection
• Analysis suggests that very low numbers of
memory CD4 T cells are retained in the lung
or airways
– Low proportion of VLA-1+ CD4 T cells
– Are there enough cells in the lung to provide
heterosubtypic protection?
University of Rochester
Medical Center
CD4 mediated protection
against influenza
Pros:
Cons:
• Priming of CD4 T cells or
adoptive transfer of primed
cells accelerates virus
clearance from lung (Braciale, Swain,
• In vivo primed influenza
specific CD4 T cells are not
cytolytic
Woodland)
• Intratracheal transfer of
virus-specific CD4 T cells
can reduce titers (Woodland)
– Protection mediated by IFN-g
and help for B cells or CD8
• Low number of endogenous
CD4 T cells in the airways or
lung tissue
– Suggests endogenous
protection is derived from
lymphoid memory cells
University of Rochester
Medical Center
Antigen-specific and non
specific recruitment of T cells
to the lung during influenza
infection
Activation or antigen?
University of Rochester
Medical Center
The OT-I and WSN-OVAI
model
• OT-I TCR transgenic mouse
– Thy1.2+ CD8+ T cells specific for MHC Class I Kb
restricted ovalbumin peptide (siinfekl)
• WSN-OVAI
– Influenza virus with ovalbumin peptide engineered
into the stalk of the viral neuraminidase protein
(Dr. Maria Castrucci, Istituto Superiore de Sanita, Rome, Italy)
University of Rochester
Medical Center
Experimental design:
Naïve
Thy1.2+
CD8+
OT-I T cells
Thy1.1+ mouse
48 h
WSN-OVAI
WSN-PEP2
7-10 days
Collect: BAL (broncheo-alveolar lavage)
MLN (mediastinal lymph node)
SPL (spleen)
Assay by flow cytometry for
• Thy1.2+ Tg T cells
• Kb/siinfekl (OVA257-264)
• Intracellular IFN-g secretion
University of Rochester
Medical Center
Pre- and post-transfer phenotype of the OT-I T cells
A
CD62L
Vß5
70.34
4.13
97.10
3.26
CD8
Day 0 Pre–
transfer
donor cells
B
29.64
V2
C
0.26
18.16
CD44
D
0.38
Day 12 after
transfer
no infection
Thy1.2
University of Rochester
Medical Center
+ CD8+
100000
d8 W
80000
0
35000
30000
25000
20000
15000
10000
0
300000
BAL
250000
200000
150000
100000
50000
5000
0
5 x 107
1 x 107
Number of OT-1 Tg T cells
Number of Thy1.2
Antigen-specific recruitment60000
of
40000
50000
OT-I Tg T cells to the airways
20000
0
7
WSN-OVA
II
2 x 106 Control
1 x 10
Number of OT-1 Tg T c ells trans ferred
University of Rochester
Medical Center
Recruitment of Memory T cells
Thy1.2+
CD8+
Tg T cells
1 x 107
Thy1.1+ mouse
48 h
Prime T cells with Vacc-OVA
6 weeks
WSN-OVAI
OR
WSN-PEPII
days 5 or 11
Collect & Assay: BAL (broncheo-alveolar lavage)
MLN (mediastinal lymph node)
SPL (spleen)
University of Rochester
Medical Center
Visualization of the 1° NP and 2° OVA
responses
d5 BAL
d11 BAL
Peptide stimulus
IFN-
Flu-NP264-272
OVA257-264
Thy1.2
University of Rochester
Medical Center
Visualization of the 1° NP and 2° OVA
responses
d5 BAL
d11 BAL
Peptide stimulus
IFN-
1
°
Flu-NP264-272
2°
OVA257-264
Thy1.2
University of Rochester
Medical Center
Antigen-independent recruitment of CD8 T cells to the
airways occurs early in the infection
University of Rochester
Medical Center
Antigen-independent recruitment of CD8 T cells to the
airways occurs early in the infection
University of Rochester
Medical Center
Antigen-independent recruitment of CD8 T cells to the
airways occurs early in the infection
University of Rochester
Medical Center
Without antigen, CD4 cells do not expand or
traffic to the influenza infected lung
Naïve CD44lo CD62hi OT-II CD4 T cells sorted prior to transfer and infection
Frequency and number of OT-II cells day 9 post-treatment*
BAL
Lung
MLN
Spleen
Number
3286
Frequency
9.95E-04
Number
846
Frequency
4.71E-03
Number
29438
Frequency
1.11E-03
Number
111000
Totals
144569
Control WSN
Allantoic fluid
Uninfected
7.80E-05
4.67E-04
7.65E-04
77
4
14
1.52E-04
1.55E-04
9.80E-05
79
28
16
1.68E-04
1.27E-04
1.92E-04
1008
91
5
1.91E-04
2.29E-04
2.00E-04
19100
22900
20000
20264
23023
20034
Number CD4+ Thy1.1+ OT-II cells
WSN-OVAII
Frequency
2.12E-03
*Frequency calculated from number of CD4+Thy1.2+ cells in lymphocyte gate
160000
140000
120000
4500
4000
Total
Airways +
Lung
3500
(MLN, spleen airways, lung tissue)
100000
3000
2500
80000
2000
60000
1500
40000
1000
20000
500
0
0
WSN-OVAII WSN-OVAI Allantoic
fluid
PBS
WSN-OVAII WSN-OVAI
Allantoic
fluid
PBS
University of Rochester
Medical Center
Primed Th1 CD4 T cells are traffic to the lung independently of
antigen or infection
Day 6
• In vitro primed OT-II Th1 cells
(peptide + IL12 + anti-IL4) were
rested for 1 week by transfer into
normal recipients. Donor cells were
reisolated and 3 x 106 were injected
into naive recipients that were then
infected with flu and administered
BrdU. Response is measured on day
6 after infection.
University of Rochester
Medical Center
CD4 T cells in flu infection
• Antigen is required to expand CD4 T cells
• Primed (but not naïve) CD4 T cells are nonspecifically recruited to the lung during
infection but do not proliferate
• If the starting number in the airways is low,
then protection may depend on the expansion
and recruitment of CD4 memory cells from
the lymphoid organs
University of Rochester
Medical Center
Summary: Activation status
versus antigen specificity
• Recruitment of naïve (unprimed) T cells to the
lung is negligible
• Antigen is required to expand memory T cells
• Trafficking of memory (primed) T cells is
significant
• The trafficking of either CD4 or CD8 T cells to
the lung is dependent upon the activation
status, not antigen specificity
University of Rochester
Medical Center
Revised model of T cell
mediated immunity in the lung
•
Primary infection sets up populations of lymphoid
and extralymphoid memory T cells
•
•
Extralymphoid memory CD8 T cells retained in the tissue
by VLA-1
Relatively few extralymphoid CD4 memory T cells in the
lung
•
•
•
•
•
Low VLA-1
Could be different with altered priming
Airway and lung tissue memory T cells turn over
at a low rate, but are not maintained indefinitely
Expansion of CD4 and CD8 T cells depends on
antigen
Recruitment to the infected lung depends on
activation, not antigen
University of Rochester
Medical Center
Model of secondary immune
responses in the lung
•
Secondary protection is optimal when sufficient numbers of
memory T cells are in the tissue
•
•
•
Short term (1-6 months) protection
May explain observed long term loss of T cell mediated
heterosubtypic protection in humans
Secondary heterotypic immunity relies on at least three
distinct populations of memory T cells
•
•
•
Resident tissue memory T cells
Circulating memory T cells in the blood
Lymphoid memory T cells in the spleen, lymph nodes, and BM
University of Rochester
Medical Center
Number of flu-specific T cells
Model of primary and secondary T
cell responses in the lung
1°
infection
Lymphoid
Primary
0 1
2 3 4
5 6 7
Circulating
memory
8
9 10 11 12 13 14 15 16
2°
Lymphoid
infection
memory
1
2
3
Diagrams modeled after:
Tissue
memory
0 1
2 3 4
5 6 7
8 9 10 11 12 13 14 15 16
Days after infection
Woodland, D. L., and I. Scott. 2005. T
cell memory in the lung airways. Proc
Am Thorac Soc 2:126-31.
University of Rochester
Medical Center
Thanks!
Snezhana
Dimitrova,
Technician
Jane Baer,
Technician
Steve Ray,
Graduate
Student
Noelle Polakos,
Graduate
Student
Tim Chapman,
Graduate
Student
Sarah Austin,
Graduate
Student
Martin Richter,
Postdoctoral
Fellow
Steve Ray, Martin Richter
Pivotal role of VLA-1 in CD8 T cells retention and survival (RO1-AG021970)
Tim Chapman
OT-II WSN-OVAII model of CD4 T cells in the Lung (N01-AI50020, Rochester Center for Immune Modeling for Biodefense)
Sarah Austin
Role of VLA-2 on Natural Killer cell migration within virus-infected tissues
Snezhana Dimitrova
Inhibition of influenza by RNAi (Alnylam)
Jane Baer
Human immune responses to smallpox, malaria, and influenza vaccines (NO1-AI-25460, Rochester VTEU)
Noelle Polakos
Influenza hepatitis and the fate of virus-specific T cells in the liver
Alnylam, Inc.
Dr. Tony de Fougerolles
Sidney Kimmel Cancer Institute, San Diego, CA
Dr. Linda Bradley
Biogen, Inc.
Humphrie Gardner
Istituto Superiore de Sanita, Rome, Italy
Dr. Maria Castrucci
Trudeau Institute, Saranac
Lake, NY
David Woodland
University of Rochester
Medical Center