CFAR Immunology Core
Volume 2 Issue 1
INSIDE THIS ISSUE The CFAR Immunology Core: Who,
1 Overview What, and Where.
By Jim Riley
2 Reagents for Human T cell
Phenotyping (New Service)
Welcome to the CFAR Immunology Core Newsletter. The goal of this
3 The Human Immunology
Core (New Service) 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
5 “Full Service” research.
I work closely with several technical directors, Don Campbell, Nancy Tustin,
6 The BSL3 Sorting Facility Jean Boyer, Tatiana Golovina, Jay Gardner and Paul Hallberg, who run the
day to day operations of the services listed below. I am also indebted to Mike
7 Primary Cell Services
Betts who has agreed to offer a relatively new service in which reagents to
perform multiparameter T cell phenotyping and functional analysis will be
provided. 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
Jim Riley is also posted on the Penn CFAR website under Immunology Core.
The CFAR Immunology Core provides a wide range of services and reagents to
556 BRB II/III benefit the immunological research of the Penn CFAR community. These
services can be subdivided into self service and full service. Self service
(OFFICE) 215- options include conjugated Abs, tetramers, and primary cells. Here, we
573-6792 provide you with a reagent that undergoes rigorous quality control, enabling
you to perform the experiment that uses the reagent. The full service
options include BLS3 sorting, ELISPOT, NK assays and epitope mapping. With
the full service option, you simply hand off the sample to our highly trained
8590 staff, and they perform the assay for you.
firstname.lastname@example.org Recently, the Cancer Center received funding to establish a Human
.upenn.edu Immunology Core. Carl June is the PI of this Core and Jean Boyer is the
technical director. The goal of this center is to provide immunological
support for the UPENN cancer community. Given the substantial overlap
between the immunological assays and reagents required to analyze cancer
and HIV immune responses, the leadership of both Cores has decided to
combine forces to make more services and reagents available to members of
both communities rather than duplicate services. This arrangement allows us
to greatly expand our full service options and to provide common HIV
tetramers as described below.
Page 2 CFAR Immunology Core
We recently submitted our competitive CFAR renewal application. In addition to the services listed
below, we are proposing an additional service in collaboration with the Xenograft Core that would allow
HIV-1 researchers to perform in vivo studies using immunodeficient NOG mice. 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
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. I am very interested in your feedback. Any comments about this newsletter or the services we
provide would be most welcome.
Reagents for Human T cell Phenotyping
By Michael Betts
Flow cytometric analysis has now moved far beyond the
Jay Gardner standard four-color format familiar to many. It is now
215-746-6526 possible to measure up to 18 different fluorescent
(email@example.com) parameters simultaneously, and the Antibody Conjugation
Core’s goal is to make this possible for researchers at Penn.
Michael Betts Of course, few researchers will need to measure this many
(firstname.lastname@example.org). parameters at once, but even moving from 4 colors to 7 or
8 colors can substantially enhance the information gained
from an experiment.
Antibody Fluorochrome At this time, we are producing antibodies directly
labeled with Quantum Dots or Alexa dyes, the latest set of
CD4 Qdot 585
reagents available for flow cytometry. We currently have
CD4 Qdot 705 available the anti-human conjugates shown in the table.
CD8 Qdot 605 These reagents can be used on either LSRII flow cytometer
CD8 Qdot 655 in the Penn Flow Cytometry Core (John Morgan Building).
CD8 Qdot 705 The cost per test for these conjugates is a nominal charge
CD8 Pacific Orange of $1.50 per test, which is far below the cost to obtain
CD45RO Qdot 705 these reagents as custom conjugates from vendors. An
CD107a Alexa 680 example of staining human peripheral blood lymphocytes
CD57 Qdot 565 with the Qdot 655-anti-CD8 conjugate is shown in the
We will be continuing to add new reagents to our
catalog, including CD3 and CD27 conjugates. Although we
currently do not offer any anti-mouse reagents, we will be
making conjugates in the future.
CFAR Immunology Core Page 3
The Human Immunology Core
By Jean Boyer
The Immunology Core has expanded over the past year to aid with trial design
Jean Boyer relative to immune evaluation, and will subsequently manage samples from
human subjects in order to evaluate cellular immune responses.
The induction of a cellular immune response is an intricate web of
email@example.com. interactions between antigen, cells, cytokines and antibodies. A CD8
upenn.edu 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 stemming viral infection and tumor growth
SERVICES OFFERED directly, but are crucial in the maintenance of virus-specific CD8 effector cells
BY THE HIC and the development of antibody responses.
Modern methods for analysis of effector and memory T cell function rely upon
1 Epitope Mapping
measurements of proliferation, cytokine profiles, cell surface markers and
2 Sample Processing and tetramer binding to antigen specific T cell receptors. These methods are
Sample Storage sensitive enough to rapidly assess rare events characteristic of cognate
memory T cell responses. The Immunology Core has available immunological
3 T lymphocyte proliferation techniques to assess the cellular immune response of human subjects. The
assays performed include: tetramer staining, intracellular staining for
4 Tetramers cytokines, ELISpot and proliferation assays. These are complimentary and can
provide researchers with a comprehensive view of the immune responses to
5 Data and Specimen
Storage the various treatments and conditions under consideration.
The sensitivity of the ELISpot assay and the ease of synthesizing peptides has
lead 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 Principal Investigators to define targets for
cancer therapy or engineering vaccines.
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.
Page 4 CFAR Immunology Core
Human Immunology Core from page 3
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.
The assessment of antigen-specific T cell immunity for both research and clinical
experiments has been revolutionized by the recent development of MHC/peptide
tetramer technology. Tetramers are assembled following the production of
recombinant MHC class I or class II molecules, which fold together such that specific
peptide epitopes fit into the groove of MHC. The term “tetramer” is applied to the
final product, which includes four such peptide/MHC complexes brought together in a
biotin-avidin system and bound to a fluorochrome. As a high-avidity synthetic complex
that presents a desired peptide antigen in the context of MHC, tetramers bind to the T
cell antigen receptor and permit the quantification and isolation of antigen-specific T
lymphocytes found in patient blood or tissue using flow cytometry. The technique is
highly sensitive and can be combined with cell surface staining for multiparametric
functional analysis and isolation of antigen-specific patient cells.
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.
CFAR Immunology Core Page 5
“Full Service” Immunological Assays
By Nancy Tustin and Don Campbell
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 don’t want to devote the time and reagents to
have your lab learn? If so, the 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
immunological assays. Note that the Functional Natural Killer assay is
CLIA certified and can therefore be used for patient management. The
reported parameters are LU20/107 PBMC, LU20/107 NK and % PBMC
NK. This assay needs to be scheduled with the laboratory at least 24
hours in advance.
In addition, this facility offers a number of assays in which the core
will do as much work as the investigator desires. Please contact Nancy
Tustin for cell based assays and Don Campbell for FACS based assays
for more details and pricing. These “full service” assays include:
• Cell based Assays
Nancy Tustin • Lymphoproliferative Assays to a wide array of mitogens
215-590-2043 and recall antigens
Tustin@email.chop.edu • Natural Killer Cell Assays
• Inducible Cytokines
Don Campbell • Flow based Assays and Service
(215) 590-2353 • Flow cytometric, single platform CD4 Counts, Multi-
firstname.lastname@example.org Color Immunophenotyping markers for naïve and
memory T cells, as well as cytotoxic T cells.
• Non-Infectious Cell Sorting
• Cytometric Bead Array for TH1/TH2 Cytokines
• Single cell intracellular cytokine measurements
• Apoptosis assay based on propidium iodide and Annexin
Page 6 CFAR Immunology Core
The BSL3 Cell Sorting Facility
By Paul Hallberg
The BSL3 Cell Sorting Facility currently provides core services to the University of
Paul Hallberg Pennsylvania CFAR community of investigators, outside academia and commercial clients
for the sorting and analysis of infectious and biohazardous materials. A state-of-the art
high-speed cell sorter has been installed in the existing BSL3 Suite in the Abramson
Research Center (ARC) of The Children’s Hospital of Philadelphia and represents a joint
hallberg@email. initiative with the Abramson Cancer Center of the University of Pennsylvania.
The facility is equipped with a BD Biosciences FACSVantage SE with DiVa Option® flow
cytometer from BD Biosciences (San Jose, CA) with an array of useful options. The
TurboSort Plus option allows for sorting at higher sheath pressures (up to 60psi) and
generating higher drop drive frequencies (up to 99K), permitting high-speed cell sorting.
The FACSDiVa Option takes advantage of the latest digital electronics, permitting faster
signal processing, full interbeam compensation, four-way sorting (QuadraSort), software
compensation (via a matrix of spillover coefficients) and new sort masks for improved
conflict resolution (resulting in better purity and yield sorts). The sorter is equipped with
the BD Aerosol Management option to rapidly evacuate aerosolized particles within the
enclosed sort chamber.
The cell sorter is equipped with three lasers and eight fluorescence detectors. Along with
two detectors for light scatter and time, 11 parameters are available allowing performance
of a wide variety of services including: eight color detection, auto-cloning (single
cell/well), immunophenotyping, fluorescent protein (CFP, GFP, YFP, DsRed), 4-way sorting.
New clients continue to discover the BSL3 Cell Sorting Facility as a valued resource for a
variety of applications and services to facilitate their research with live biohazardous and
infectious materials. CFAR member, Dr. Una O’Doherty, Department of Pathology and
Laboratory Medicine, relies on our expertise to isolate both naïve and memory T-cells in
HIV+ PBMC’s from human donors, to facilitate her research on HIV-1 latency. Dr. Carl
June’s lab and associates continue to utilize the BSL3 Sorting Facility to ultra-purify CD4+
T-cells from HIV+ donors, for use in bioassays to characterize cytokines and chemokines.
Dr. David Roos, Penn Genomics Institute, has found our services an asset to cloning
toxoplasmodium parasites transfected with the reporter gene YFP, for studies on drug
resistance. Dr. Jun Zhu, Laboratory of Microbiology, uses the BSL3 Sorting Facility to study
differential gene expression by sorting Vibrio cholerae transfected with the reporter gene
GFP. Dr. Kyong-Mi Chang, Department of Medicine, Division of Gastroenterology, continues
to utilize our services for isolation of CD4+CD25+ regulatory T cells in HCV donors,
facilitating her studies of immune mechanisms of viral persistence.
Conveniently located on campus at the Leonard and Madlyn Abramson Pediatric Research
Institute, our core facility is designed to address the needs of investigators working with
pathogenic agents. We provide high quality support, technical expertise and through our
association with the Abramson Cancer Center facility, expert consultation on experimental
design that will allow you to use the full range of cytomic applications for your study of
Let us help you find new and efficient ways of utilizing high content technology in your
CFAR Immunology Core Page 7
Primary Human Cells
By Tatiana Golovina
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
Tatiana entire Penn community our prices are below what you could do yourself and you
Golovina don’t have to waste your time preparing the cells. Below is a list of Frequently
215-573-6219 Asked Questions from this service. If you have a question that we should include
on the list, please contact me.
upenn.edu Do I need IRB approval to use your facility? Yes, but you will only need to fill
out an expedited form that we have partially filled out for you. For Penn
investigators go to the forms page
download the partially filled out IRB expedited review forms and fill in the
missing information and send these forms to
Joseph Sherwin, Ph.D.
133 S. 36th Street
Philadelphia, PA 19104-3246
Mail Code 3246
These forms should only take 10-15 minutes to fill out and most investigators get
approved within a month. Note for each funding source you will need a separate
IRB approval that uses that Grant’s Title.
For Wistar and CHOP investigators, please consult your local IRBs. We are happy
to provide whatever paperwork they require.
Who are the donors? Can I request a specific donor?
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
most ethnicities. We generally schedule donors 2 months ahead, and we will do
our best to schedule a requested donor. HIV infected individuals can be
scheduled but will require special pricing. 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. Other patient groups can also be
scheduled but will need prior IRB approval. When recruiting donors, please let
them know that they need to be at Ravdin 3 at 7:30 in the morning. Please send
the proposed donor’s email address to us and we will make arrangements. The
identity of the donor will remain confidential.
Page 8 CFAR Immunology Core
Primary Cells from page 7
How much do cells costs? Price is per million cells. We had to modestly raise our prices for the
T Cells** $2.00
CD4+ T** $2.25
CD8+ T** $2.75
B Cells** $12.00
Unpurified Apheresis Product $0.75
Mononuclear Cell-Enriched Apheresis Product $775.00
What days of the week are primary cells available?
PBMCs, PBLs, and elutriated monocytes are generally ready by 2pm on Tuesdays and Thursdays.
Cell types that require negative selection are usually ready by 6 pm. We have found cells stored
overnight work well for most applications. We have recently started getting apheresis products on
the first and third Wednesday of every month. We usually have extra cells of these days so large
orders are more likely to get filled on these days.
How do I place an order? Investigators must have IRB approval prior to receiving cells from this
facility. Registered investigators should email requests to email@example.com. You will get
an email confirmation that your order was received.
When is the last call for cells?
After 9 am on the day the cells are being prepared, no more orders will be accepted.
I placed an order how come I didn’t get cells? This is question we wish there was no reason to
ask. But unfortunately, there are a few complications that preclude us from giving you cells when
1. Our donor does not show up. While we certainly do everything to prevent this from happening
(email reminders, phone calls etc…) it does happen. Occasionally, we can find a replacement
on short notice but sometimes we can’t. We are always looking for emergency donors i.e.
those that can get to the blood bank and bleed for 2 hours on short notice. If you think that
you can do this, we would love to have your contact info.
2. Apheresis unit gets flooded with patients. As a general rule the apheresis unit has been very
accommodating to our needs. However, HUP is a hospital and the patient needs supersede
research so every once and a while we are unable to get a blood product.
3. Too much demand; too few cells. There is certain randomness in how many cells particular
labs want each day. Thus, there is a potential that people will request more cells than we
have. If this happens, we try to do the most good for the most number of people. If for some
reason you get bump, we will give you first priority the next time we purify cells.
CFAR Immunology Core Page 9
Primary Cells from page 8
Exactly how are the cells purified?
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. Please inquire if need to know this information prior to placing an order
Can I request that my cells be placed in specific media?
We have RPMI w 10% FCS. Any other specific media will have to be provided by the requestor.
What concentration are the cells stored at?
Monocytes are ~10e6/ml. CD8 T cells and B cells are generally 3-5e6/ml, CD4 T cells are at
5e6/ml, PBMCs are at 20e6/ml. As enumeration techniques tend to vary among labs, we
recommend that you count the cells prior to use.