Quantitation, selection, and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon-γ production and growth of cytotoxic precursors Journal Article
| Authors: | Koehne, G.; Smith, K. M.; Ferguson, T. L.; Williams, R. Y.; Heller, G.; Pamer, E. G.; Dupont, B.; O'Reilly, R. J. |
| Article Title: | Quantitation, selection, and functional characterization of Epstein-Barr virus-specific and alloreactive T cells detected by intracellular interferon-γ production and growth of cytotoxic precursors |
| Abstract: | 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-γ (IFN-γ) 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-γ 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-γ exceeded by 30-to 220-fold those detected by LDA. The assay that quantitated T cells producing IFN-γ yielded more reproducible and precise results than LDA. Furthermore, frequencies detected by the enumeration of T cells responding to immuno-dominant EBNA 3a and EBNA 3c peptides by IFN-γ production or their capacity to bind peptide-HLA tetramers were strikingly similar and represented significant fractions of T cells generating IFN-γ 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-γ demonstrated that EBV-specific and alloantigen cytotoxic T cells were predominately or exclusively detected in the CD8+IFN-γ+ fraction of T cells. Strikingly, the CD4+IFN-γ+ cell fractions were not cytotoxic against EBV-transformed or allogeneic targets. © 2002 by The American Society of Hematology. |
| Keywords: | controlled study; functional assessment; human cell; flow cytometry; cd8-positive t-lymphocytes; t-lymphocytes; quantitative assay; reproducibility; accuracy; biological markers; gamma interferon; cd4-positive t-lymphocytes; cytotoxic t lymphocyte; cell fractionation; cytotoxicity, immunologic; precursor; cell separation; immunotherapy, adoptive; epstein barr virus; lymphocyte count; cell selection; lymphoblastoid cell line; herpesvirus 4, human; alloimmunity; lymphocyte subsets; interferon type ii; dilution; humans; human; priority journal; article; virus cell transformation; epstein-barr virus nuclear antigens |
| Journal Title: | Blood |
| Volume: | 99 |
| Issue: | 5 |
| ISSN: | 0006-4971 |
| Publisher: | American Society of Hematology |
| Date Published: | 2002-03-01 |
| Start Page: | 1730 |
| End Page: | 1740 |
| Language: | English |
| DOI: | 10.1182/blood.V99.5.1730 |
| PUBMED: | 11861290 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 14 November 2014 -- Source: Scopus |
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