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Related Collections Immunobiology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 March 2002, Vol. 99, No. 5, pp. 1730-1740 IMMUNOBIOLOGY Quantitation, selection, and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon- production and growth of cytotoxic precursors Guenther Koehne, Katherine M. Smith, Teresa L. Ferguson, Roxanne Y. Williams, Glenn Heller, Eric G. Pamer, Bo Dupont, and Richard J. O'Reilly From the Department of Pediatrics, Bone Marrow Transplantation Service; the Department of Clinical Laboratories, Cellular Immunology Laboratory; the Department of Epidemiology and Biostatistics; the Department of Medicine, Infectious Diseases Service, Memorial Hospital; and the Immunology Program, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY. Techniques for the quantitation of virus-specific and alloantigen-reactive T cells vary in their measurement of clinically relevant T-cell effector populations, their sensitivity and quantitative accuracy, and the time required to obtain measurable results. We compared frequencies of Epstein-Barr virus (EBV)-specific and major alloantigen-reactive T cells as measured by flow cytometric analysis of responding T cells producing intracellular interferon- (IFN-) and by limiting-dilution analysis (LDA) of cytotoxic T-cell precursors (CTLp) at sequential time points during the generation of EBV-specific T-cell lines. The expansion of EBV-specific T lymphocytes and the depletion of alloreactive T cells in cultures of T cells sensitized with autologous EBV-transformed targets followed similar kinetics when measured by either method. Frequencies of EBV- specific T cells generating intracellular IFN- exceeded by 25- to 90-fold the frequencies of responding CTLp at each stage of expansion, whereas the frequencies of alloreactive T cells generating intracellular IFN- exceeded by 30- to 220-fold those detected by LDA. The assay that quantitated T cells producing IFN- yielded more reproducible and precise results than LDA. Furthermore, frequencies detected by the enumeration of T cells responding to immunodominant EBNA 3a and EBNA 3c peptides by IFN- production or their capacity to bind peptide-HLA tetramers were strikingly similar and represented significant fractions of T cells generating IFN- in response to autologous EBV B lymphoblastoid cell line. Functional analysis of responding viable T cells, fractionated on the basis of their secretion of IFN-, demonstrated that EBV-specific and alloantigen cytotoxic T cells were predominately or exclusively detected in the CD8+IFN-+ fraction of T cells. Strikingly, the CD4+IFN-+ cell fractions were not cytotoxic against EBV-transformed or allogeneic targets. © 2002 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: O. Beck, M. S. Topp, U. Koehl, E. Roilides, M. Simitsopoulou, M. Hanisch, J. Sarfati, J. P. Latge, T. Klingebiel, H. Einsele, et al. Generation of highly purified and functionally active human TH1 cells against Aspergillus fumigatus Blood, March 15, 2006; 107(6): 2562 - 2569. [Abstract] [Full Text] [PDF] D. Trivedi, R. Y. Williams, R. J. O'Reilly, and G. Koehne Generation of CMV-specific T lymphocytes using protein-spanning pools of pp65-derived overlapping pentadecapeptides for adoptive immunotherapy Blood, April 1, 2005; 105(7): 2793 - 2801. [Abstract] [Full Text] [PDF] E. S. Doubrovina, M. M. Doubrovin, S. Lee, J.-H. Shieh, G. Heller, E. Pamer, and R. J. 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