The Immunologic and Virologic Monitoring Laboratory Core (IVMLC, Core B) is designed to provide support for assays and analyses integral to PPG projects 1, 3 and 4. The IVMLC is equipped with a highspeed 9 color FACSAria cytometer capable of simultaneous four-way cell sorting, and an additional 7- color FACSCanto flow cytometer is available for all analyses that do not involve cell sorting. The IVMLC is fully outfitted to perform validated assays to measure TREC by real-time PCR, antigen-specific T-cell proliferation, T-cell cytotoxicity and T-cell cytokine synthesis (intracellular and ELISA) that are applicable to both murine and human cells. The IVMLC also provides support for virologic assays to measure CMV viral load. IVMLC Director Dr. Edmund Waller has previously developed, validated and applied the core technologies as part of an on-going multi-center clinical trial sponsored by the National Marrow Donor Program (NMDP). Co-Director Dr. Cindy Giver and Hilary Rosenthal, MT, both have extensive experience in the application of these techniques to over 500 clinical samples analyzed for the NMDP project, and have written the standard operating procedures (SOP) for laboratory assays and QA/QC protocols. Working closely with Principal Investigators for PPG projects 1, 3 and 4, the IVMLC personnel will use existing procedures to design protocols specifically for these studies. The IVMLC will support project 1 by performing in vitro experiments to quantify alloreactive cytotoxic T-lymphocytes (CTLs), as well as murine in vivo CTL assays. The effectiveness of vaccine-induced cellular immunity will be derived from reductions in MCMV load (real-time MCMV PCR assay, and quantitative viral culture assays). For project 3, FACS analysis and T-cell sorting will be performed for preclinical experiments, and FACS analysis will also be used to monitor post-transplant immune reconstitution in patients enrolled in the phase 1/2 clinical trial. Frequencies of allo-reactive T-cells will be determined by limiting dilution MLR (LDA) and proliferation assays using CFSE labeling. Cytokine synthesis in response to specific antigens will be measured using flow-based and ELISA methods. TREC analyses of sorted CD4+ and CD8+ T-cell subsets will monitor de novo T-cell generation as a sensitive indicator of thymopoiesis, and flow-based tetramer analyses will determine the frequencies of HLA A2 and B7 restricted CMV pp65-specific T-cells. For project 4, flow cytometry assays will identify antigen-presenting cells (APC) and characterize the expression of various costimulatory molecules on the relevant APC, identify HEL specific T-cell subsets, and analyze these Tcells for activation status and effector function. Flow-based assays will also be used to determine T-cell cytokine expression status and proliferation rates. The IVMLC Director and Co-Director will continue to work with PPG Principal Investigators while experiments are ongoing to ensure that all aspects of the procedures and analyses meet their particular specifications, and to assist in developing new assays as the projects progress.
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