CFAR Immunology Core
March, 2013
Volume 5 Issue 1
INSIDE THIS ISSUE
1
2
5
Overview
The Human Immunology
Core-GLP assays
“Full Service”
Immunological Assays
6
The BSL3 Sorting Facility
7
Primary Cell Services
9
Small Animal Model of
HIV-1 Infection
Jim Riley
8th floor TRC
Rm 115
(OFFICE) 215573-6792
(FAX) 215-5738590
rileyj@exchange.
upenn.edu
The CFAR Immunology Core: Who,
What, and Where.
By Jim Riley
Hello, welcome to the CFAR Immunology Core Newsletter. The goal of this
newsletter is to update you on the services we provide and to provide contact
information so you can learn more about how our services can enhance your
research.
I work closely with several technical directors, Florin Tuluc, Nancy Tustin,
Jean Boyer, Lingjie Zheng, Xiaochuan Shan, and Hank Pletcher who run the
day to day operations of the services listed below. We are all dedicated to
make sure that you are satisfied with the breadth and quality of our services
and reagents. If for any reason you are unhappy, please let me know and I
will do my best to get the problem resolved. For technical or scheduling
services, it is best to contact the person in charge of a particular service.
You may wish to keep this newsletter as it has all of our contact info in a
single place. This newsletter is also posted on the Penn CFAR website under
Immunology Core.
The CFAR Immunology Core provides a wide range of services and reagents to
benefit the immunological research of the Penn CFAR community. These
services can be subdivided into self service and full service. Self service
options include primary cells and access to Luminex, Amaxa or other
technology useful for immunological investigations. Here, we provide you
with a reagent/equipment that undergoes rigorous quality control, but you
ultimately perform the experiment that uses the reagent. The full service
options include T and B cell ELISPOT, NK assays and epitope mapping. With
the full service option, you simply hand off the sample to our highly trained
staff, and they perform the assay for you.
Are you proposing a Phase I clinical trial and would like to include
immunological readouts as part of your analysis? The CFAR Immunology Core
will work with you to create custom immune assays done in the spirit of good
laboratory practices (GLP) and will assist in the preparation of regulatory
paperwork.
The CFAR Immunology Core works in concert with the Cancer Center’s Human
Immunology Core and Flow Cytometry Core. Given the substantial overlap
between the immunological assays and reagents required to analyze cancer
and HIV immune responses, the leadership of both Cores have decided to
combine forces to make more services and reagents available to members of
both communities rather than duplicate services.
The CFAR Immunology Core also works with the Xenograft and Stem Core to provide a small animal
model of HIV-1 infection. To date, this has proved to be an effective model to study both vaccine and
therapeutic approaches using human cells. I am very excited to share this model with the CFAR
community as I believe it will accelerate the research of many investigators. We now have a fair amount
of expertise with this in vivo model of HIV-1 and the core now offers several variations of this model that
can be modified to help investigators address a variety of HIV-1 relate d questions. Please contact me if
you feel your research could be assisted by a small animal model of HIV-1 infection.
Lastly, the CFAR Immunology Core is a decentralized core which basically means we offer service
in several buildings throughout Penn and CHOP. In the summaries below we will describe our facilities
and services so that you will know what is available. I am very interested in your feedback. Any
comments about this newsletter or the services we provide would be most welcome.
Full Service GLP Assays offered by Human Immunology Core
By Jean Boyer
Jean Boyer
215-662-2382
The Human Immunology Core has expanded over the past year to aid with trial
design, relative to immune evaluation, and subsequently manages samples from
human subjects in order to evaluate cellular immune responses.
The induction of a cellular immune response is an intricate web of interactions
between antigen, cells, cytokines and antibodies. A CD8 cytotoxic lymphocyte
response is important for clearing viral infections and tumors. CD8 cells
recognize antigens that are processed intracellularly and presented to them via
the MHC class I pathway on activated dendritic cells. Extracellular proteins can
be endocytosed by professional antigen presenting cells and presented to CD4
lymphocytes through the MHC class II pathway. CD4 T cells not only play a role in
S E R V I C E S O F F E R E D stemming viral infection and tumor growth directly, but are crucial in the
maintenance of virus-specific CD8 effector cells and the development of antibody
BY THE HIC
responses.
boyerj@mail.med.
upenn.edu
1
Epitope Mapping
2
T lymphocyte proliferation
3
4
5
T cell Subset
characterization
Luminex
Sample Processing and
Sample Storage
Modern methods for analysis of effector and memory T cell function rely upon
measurements of proliferation, cytokine profiles, cell surface markers and
tetramer binding to antigen specific T cell receptors. These methods are
sensitive enough to rapidly assess rare events characteristic of cognate memory
T cell responses. The Immunology Core has available immunological techniques
to assess the cellular immune response of human subjects. The assays
performed include: tetramer staining, intracellular staining for cytokines,
ELIspot and proliferation assays. These are complimentary and can provide
researchers with a comprehensive view of the immune responses to the various
treatments and conditions under consideration.
Epitope Mapping
The sensitivity of the ELIspot assay and the ease of synthesizing peptides has led
to a very accurate and sensitive method of mapping out the dominant CD8 and
CD4 epitopes. Overlapping peptides encompassing an entire protein allows an
investigator to look at a broad immune response. The cellular responses can be
mapped to dominant and subdominant epitopes by mixing the peptides in a
matrix format. Peptides are mixed as pools. Each peptide is present in two
pools. The intersection of reactive pools defines reactive epitopes. Defining the
dominant CD8 epitopes is necessary for tetramer design. In addition, this
information can be utilized by the Principle Investigators to define targets for
cancer therapy or engineering vaccines.
T lymphocyte proliferation.
Mounting evidence suggests that T cell proliferative capacity in response to antigen stimulation is an
important parameter to assess. Indeed, proliferative capacity is important with regard to assessing
the immunological function and capabilities of the antigen specific lymphocytes. Traditionally,
proliferation was assessed by adding tritiated thymidine to cells that had been stimulated with
antigen in vitro. As the lymphocytes continued to divide thymidine was incorporated into the DNA.
The cells were then harvested and higher levels of radioactivity indicated higher levels of
proliferation. However, newer methods have been developed that are more informative and can
provide an extended amount of data on the types of cells proliferating concurrently with the number
of cell divisions. This new method utilizes Carboxy-fluorescein diacetate, succinimidyl ester (CFSE).
CFSE consists of a fluorescein molecule containing a succinimidyl ester functional group and two
acetate moieties. CFSE diffuses freely into cells and intracellular esterases cleave the acetate
groups converting it to a fluorescent, membrane impermeant dye. The dye is not transferred to
adjacent cells. CFSE is retained by the cell in the cytoplasm and does not adversely affect cellular
function. During each round of cell division, the relative intensity of the dye is decreased by half.
T cell proliferative capacity may be central to assessing the strength of vaccine response or
immunotherapeutic treatments. Further importance lies in the techniques themselves that utilize
flow cytometry. The assessment of proliferation responses based CFSE allows for the utilization of
flow cytometry and is advantageous over methods measuring incorporation of radioactive thymidine
since CFSE allow the simultaneous detection of specific T cells, e.g., by cell surface or tetramer
staining.
Luminex.
Luminex is also offered as a full service. Luminex is a powerful tool to analyse in a multiplex format
a number of molecules with a limited sample size. We work with reagents from a number of
companies that best fit your needs. These include: cytokines, transcription factors, RNA and
phosphoproteins. We work with human, primate, mouse, and rat samples.
T cell Subset Analysis.
We can also perform flow cytometry. Flow cytometry can be a costly endeavour. The core can save
you money. We have developed a number of flow cytometry panels that are currently ready for use
(see partial list below) or we can custom design a panel. We will help you find the panel that best
asks the question you are investigating.
Human T cell subset phenotyping: CD3, CD4, CD8, CD27, CCR7 and CD45RO
Cytotoxic/Degranulation: CD3, CD4, CD8, CD107a, Perforin, GranzymeB, IFNg
Immunological Activation Phenotyping: CD3, CD4, CD25, CD38, PD-1, HLA-DR
Activation Panel with Memory Support: CD3, CD4, CD8, CD25, CD27, CD38, CD45RO, CD127,
HLA-DR
Regulatory T-cell Panel: CD3, CD4, CD25, CD27, CD45RO, CD127, CTLA-4, CD279 (PD-1) and FoxP3
Poly-functional Cytokine Panel: CD3, CD4, CD8, CD107a, IL-2, IFNg, TNFa
Human B cell Panel: CD19, CD20, IgM, IgD, CD10, CD27, CD38, CD5, CD24
Tscm Phenotype: CD3+ CD4+ CD8+ CCR7+ CD45RA+ CD45RO- CD62L+ CD27+ CD28+ CD127+ CD95+
CD122+
T follicular Helper Cells: CD278, CD279, BCL6, CXCR5
Sample Processing and Sample Storage
In addition to immunological evaluation, the Immunology Core has available sample preparation and
sample storage. Lymphocytes can be isolated from peripheral blood as well as mucosal biopsy samples.
Clinical specimens can be banked for long term liquid nitrogen storage.
Data Storage:
Raw data, reports, and disks are in the care of department management. Computers are password
protected. Only the Human Immunology Core personnel have access to raw data, reports, and disks.
Final reports and subject information are filed in binders in a locked room within the department.
“Full Service” Immunological Assays
By Florin Tuluc and Nancy Tustin
Are you a clinician who doesn’t have a lab but wants to perform immunological
assays? Do you need to perform an assay once or twice to finish up a paper but
CHOP, Abramson Research don’t want to devote the time and reagents to have your lab learn? If so, the
Center, 12th Floor
CFAR Immunology Core has a group of highly trained scientists that will consult
with you on the assay to be performed, take your samples, perform the
experiments and help you interpret the results. This facility is located at The
Children’s Hospital of Philadelphia and it performs a wide array of standard
Florin Tuluc
immunological assays. Note that the Functional Natural Killer assay (FNKA) and
(267)-426-5350
be used for
tuluc@email.chop.edu the single platform CD4 counts are CLIA certified and can therefore
7
patient management. The reported parameters are LU20/10 PBMC, LU20/107
NK and % PBMC NK. This assay needs to be scheduled with the laboratory at
least 24 hours in advance.
Nancy Tustin
In addition, this facility offers a number of assays in which the core will perform
215-590-2043
complete assays or only certain steps of the assays or procedures, as required by
tustin@email.chop.edu each investigator. Please contact Nancy Tustin for cell based assays and Florin
Full Service Assays
Flow Cytometry
- CD4 counts
-Cell surface
phenotyping
-Intracellular flow
-CBAs
-Intracellular calcium
Cell Assays
-Functional Natural
Killer Assay (FNKA)
Tuluc for FACS based assays for more details and pricing. These “full service”
assays include:


Cell based Assays and Services
 Functional Natural Killer Cell Assay
 Inducible cytokines
 ELISA
 Other assays and services are available upon request (separation
of PBMC from whole blood, separation and cryopreservation of
plasma, etc.)
Flow based Assays and Service
 Flow cytometric, single platform CD4 counts
 Multicolor immunophenotyping: leukocyte subsets (T cells, B cells,
NK cells, monocytes), T cell subsets (naïve, memory), other
phenotypes upon request.
 Cell sorting (BSL-1 or BSL-2 containment)
 Cytometric Bead Array (CBA) for TH1/TH2 cytokines and
customized CBAs
 Single cell intracellular cytokine measurements
 Cell cycle and apoptosis assays
The BSL3 Cell Sorting Facility
By Hank Pletcher
Biohazardous Cell Sorting
By Hank Pletcher
Hank Pletcher
215-573-7048
pletcher@upenn.
edu
Paul Hallberg
215-573-8099
hallberg@upenn.
edu
Viable cell sorting of potentially infectious samples has evolved in recent years at Penn.
The BSL3 Cell Sorting Facility was originally created in BSL3 space in the ARC at CHOP with
a BD FACSVantage DiVa, to meet the needs of the CFAR community. No longer in operation,
that facility has been replaced by a more modern BD FACSAria II SORP, now installed in a
biosafety cabinet, in the Biomedical Research Building II/III.
Traditional electrostatic droplet generating cell sorters, whether cuvette based (FACSAria)
or jet-in-air (FACSVantage), generate aerosols at significant frequencies and velocities, and
have the potential to expose the operator to infectious pathogens. To address that issue,
guidelines have been developed, and continue to evolve, with the cytometry community
and the ISAC Biosafety Committee leading the way, along with the recently adopted NIH
Policy for Biosafety of Cell Sorters.
To meet the needs of some of the more demanding multi-color cell sorting applications, a
four-laser, fourteen-color BD FACSAria II SORP was installed in a custom designed Baker
BioPROtect IV biosafety cabinet, in 578 BRB II/III. With a dedicated operator, and
controlled access and anteroom, this facility replaces the original FACSVantage (now
retired), and provides enhanced fluorescence sensitivity when cell sorting, along with
improved access to end-users.
Administered as a part of the ACC Flow Cytometry & Cell Sorting Shared Resource, access
to biohazardous cell sorting is the same as any other cell sorting application in the PSOM at
Penn. Once an active project is created in Path BioResource, a sample description form
must be completed, a risk assessment performed, and a cell sorter is assigned. As it
currently stands, we expect to meet the demands of all potentially infectious cell sorting,
as well as meet the existing and developing biosafety guidelines.
We’re available for consultation and help with experimental design as well. Simply give us
a call if you’re interested in viable cell sorting.
Primary Human Cells
By Jim Riley
The experimental systems to study HIV in primary cells have improved over the last several years and
many journals now require results to be at least confirmed using primary human cells. But where you
do get primary cells from? Is it worth your time to get your own non exempt or expedited IRB? How
will you recruit donors? And where will the donor donate? This is how our facility benefits you. We
maintain the IRB approval; we recruit the donors; and we isolate the cell types that you are interested
in. Moreover, by providing this service to the entire Penn community our prices are below what you
could do yourself and you don’t have to waste your time preparing the cells. Below is a list of
Frequently Asked Questions from this service. If you have a question that we should include on the
list, please contact me.
Do I need IRB approval to receive cells from your facility?
For Penn faculty, the answer is No. The Immunology Core
maintains the IRB-approved protocol and the individual core users
receipt of these cells is considered secondary use of de-identified
human specimens which is not considered human use by both NIH
and our IRB. For grant submissions, we recommend that you claim
your use of the cells we provide you is NOT human subjects
research since you have no contact with the human and no way to identify who the
donor is/was.
For Wistar and CHOP investigators, please consult your local IRBs. We are happy to
provide whatever paperwork they require.
Who are the donors?
Donors are members of the community, recruited through word of mouth. They are
generally between 20-30 yrs of age, of both genders, and representative of our diverse
community. We generally schedule donors 1.5 to 2 months ahead (please our donor
calendar for specifics), and we will do our best to schedule a requested donor. HIV
infected individuals can be scheduled. Any other patient populations will need their
protocol and thus cannot be recruited by this protocol. Also, each lab receiving cells
from HIV infected will have to acknowledge by email that they know that they are
receiving HIV infected blood products.
What days of the week are primary cells available?
Purified cells are generally ready by 3pm on Tuesday, Thursday and every other
Wednesday. Follow order status on Twitter via @ImmuneCellsRUs
How do I place an order
1. Go to Request Primary Cells
2. Choose an account to order
3. Pick a date you want cells
4. Indicate how many cells you want. We also ask you to put the minimum
number of cells. When there is a lot of orders or just a low yield from the
donor, we try our best to ration the cells so if you also tell the minimal number
of cells you need for a meaningful experiment, it will increase your chance of
getting cells in times of scarcity.
5. Click Submit Immunology.
When is the last call for cells?
After 9 am the day the cells are being prepared, no more orders will be accepted.
Exactly how are the cells purified?
Apheresis material is placed in a Ficoll gradient to purify PBMCs.
All other cell types are purified using RossetteSep kits from Stem Cell Technologies. Ab
used to perform negative selection can be found on this website
http://www.stemcell.com/en/Products/Popular-Product-Lines/RosetteSep.aspx
Do you know the HLA type of the donor?
HLA types of the donors are available generally 3 to 4 weeks after their initial
donation. Since many of donors contribute often, there is a good chance we will know
the HLA type prior to donation. This information as well as the gender of the donor is
kept on the Donor calendar.
Can I request that my cells be placed in specific media?
For PBMC and monocytes, cells are put in Aim V with no serum at 5 million per ml
rotating at 4oC in cold room. All other cells are in RPMI 10% FCS at 5 million per ml at
37°C in HIC incubator. Any other specific media will have to be provided by the
requestor. We recommend that you count your cells prior to use.
Small Animal Model of HIV-1 Infection
By Gwenn Danet-Desnoyers- Technical Director of the The Stem
and Xenograft facility
https://somapps.med.upenn.edu/pbr/portal/scxc/
SERVICES OFFERED
1
Immune-deficient (NSG) mice
2
Human Immune system mice:
CD34-transplanted and BLT
3
4
5
Full range of in vivo services
for HIV studies
Dedicated BSL-2+ animal
spaces in Smillow and Hill
Dedicated in vivo optical
imaging in Smillow
Gwenn DanetDesnoyers
215-746-0181
gdanet@upenn.edu
Xiaochuan Shan
215-573-8581
shanx@upenn.edu
Tony Secreto
215-573-3705
asecreto@upenn.edu
The use of mouse xenograft models for HIV research represents a valuable
and economical alternative to simian models. Our facility has a great deal of
experience with human immune reconstitution using hematopoietic stem cell
transplantation in immune-deficient mice. In recent years, we have
established an improved model of human immune reconstitution consisting of
fetal liver-derived CD34+ cells co-transplanted with autologous thymus. This
model, commonly designated as BLT mice, improves human T-cell
developement, function and peripheral tissue distribution allowing efficient
mucosal HIV infection.
We are committed to making these human immune system models available
to the HIV research community at Penn. We work closely with individual
groups to satisfy your needs within the constrains of human CD34+ cell and
fetal tissue procurement. We routinely transplant immune-deficient mice (up
to 40 mice per fetal tissue donor) in order to reduce the lead-time needed to
provide you with fully reconstituted animals. Please contact Xiaochuan Shan
for questions on BLT mice availability.
In addition, our facility offers dedicated BSL-2+ space in the animal barrier in
the Smillow 6 (TRC) and Hill Pavilion facilities. Our staff can provide a full
range of in vivo services to support your experiments:

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




Human immune reconstitution analysis by flow
Injections (iv, ip, sc, intra-femoral, HIV inoculation)
Retro-orbital bleeding
Mouse health monitoring and body scoring
In vivo imaging on dedicated Xenogen Spectrum (Smillow only)
Tissue harvesting
Custom flow cytometric analysis of tissues

Consultation on feasibility and experimental design
Please contact Gwenn Danet-Desnoyers or Xiaochuan Shan for model
applications, feasibility, and experimental design; or Tony Secreto for
experiment setup and implementation.