Quantitation, selection, and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon-gamma production and growth of cytotoxic precursors.
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-gamma (IFN-gamma) 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-gamma 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-gamma exceeded by 30- to 220-fold those detected by LDA. The assay that quantitated T cells producing IFN-gamma 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-gamma production or their capacity to bind peptide-HLA tetramers were strikingly similar and represented significant fractions of T cells generating IFN-gamma 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-gamma, demonstrated that EBV-specific and alloantigen cytotoxic T cells were predominantly or exclusively detected in the CD8(+)IFN-gamma(+) fraction of T cells. Strikingly, the CD4(+)IFN-gamma(+) cell fractions were not cytotoxic against EBV-transformed or allogeneic targets.
Koehne
Guenther
G
Department of Pediatrics, Bone Marrow Transplantation Service, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA. koehneg@mskcc.org
Smith
Katherine M
KM
Ferguson
Teresa L
TL
Williams
Roxanne Y
RY
Heller
Glenn
G
Pamer
Eric G
EG
Dupont
Bo
B
O'Reilly
Richard J
RJ
D015415:Biomarkers / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D002469:Cell Separation / Q000379:methods; D003602:Cytotoxicity, Immunologic / Q000276:immunology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods; D004854:Herpesvirus 4, Human / Q000276:immunology*; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods; D007371:Interferon-gamma / Q000032:analysis* / Q000096:biosynthesis; D018655:Lymphocyte Count; D016131:Lymphocyte Subsets / Q000166:cytology / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology* / Q000378:metabolism
D015415:Biomarkers; D019309:Epstein-Barr Virus Nuclear Antigens; D007371:Interferon-gamma