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    CFAR Immunology Core CFAR Immunology Core Document Transcript

    • CFAR Immunology Core Aug, 2008 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. Immunological Assays 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 (FAX) 215-573- the full service option, you simply hand off the sample to our highly trained 8590 staff, and they perform the assay for you. rileyj@mail.med 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 investigators. 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 (stewartg@mail.med.upenn.edu) 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 (betts@mail.med.upenn.edu). 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 figure. 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. 215-662-2382 The induction of a cellular immune response is an intricate web of boyerj@mail.med. 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. Epitope Mapping 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. Tetramers 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. 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.
    • 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 • ELISA Don Campbell • Flow based Assays and Service (215) 590-2353 • Flow cytometric, single platform CD4 Counts, Multi- campbelld@email.chop.edu 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 V staining
    • 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 267-426-7177 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. chop.edu 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 infectious diseases. Let us help you find new and efficient ways of utilizing high content technology in your research.
    • 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. cells@mail.med. 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 (http://www.med.upenn.edu/bmcrc/immune/forms.shtml?immune) and 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 Mezzanine Level 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 upcoming year. PBMC $0.80 Monocytes $1.75 PBL $0.50 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 cells@mail.med.upenn.edu. 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 you wish. 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 for cells. 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.