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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.
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