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. 2001 May;8(3):628-31.
doi: 10.1128/CDLI.8.3.628-631.2001.

Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65

S A Ghanekar  1 , L E Nomura, M A Suni, L J Picker, H T Maecker, V C Maino
Affiliations

Gamma interferon expression in CD8(+) T cells is a marker for circulating cytotoxic T lymphocytes that recognize an HLA A2-restricted epitope of human cytomegalovirus phosphoprotein pp65

S A Ghanekar et al. Clin Diagn Lab Immunol. 2001 May.

Abstract

Antigen-specific CD8(+) T cells with cytotoxic activity are often critical in immune responses to infectious pathogens. To determine whether gamma interferon (IFN-gamma) expression is a surrogate marker for cytotoxic T lymphocytes (CTL), human cytomegalovirus-specific CTL responses were correlated with CD8(+) T-cell IFN-gamma expression determined by cytokine flow cytometry. A strong positive correlation was observed between specific lysis of peptide-pulsed targets in a (51)Cr release assay and frequencies of peptide-activated CD8(+) T cells expressing IFN-gamma at 6 h (r(2) = 0.72) or 7 days (r(2) = 0.91). Enumeration of responding cells expressing perforin, another marker associated with CTL, did not improve this correlation. These results demonstrate that IFN-gamma expression can be a functional surrogate for identification of CTL precursor cells.

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Figures

FIG. 1
FIG. 1
Representative cytokine and cytotoxicity responses to CMV peptides on day 0 and day 7 in one HLA A2+ donor. (A) CFC of CD3+ CD8+ lymphocytes from unstimulated PBMC (left panel), or PBMC stimulated with a pp65 HLA A2-restricted-peptide (amino acids 495 to 503, NLVPMVATV) for 6 h on day 0 (middle panel) or 7 days (right panel). (B) Four-hour 51Cr release assay of purified CD8+ lymphocytes after 7 days of peptide restimulation in the same donor as in panel A. Target cells were B-LCL or JY cells loaded with HLA A2-restricted peptide, control (ctrl.) peptide (MVATVGGGA), or no peptide. (C) Example of CFC responses to HLA-A2-restricted peptide (left panels) versus HLA B7-restricted peptide (pp65 amino acids 418 to 426, PRVTGGGAM) (right panels) in a second donor who was HLA A2+ B7+, stimulated for 6 h (top panels) or 7 days (lower panels). This donor, although HLA A2+, responded predominantly to the HLA B7-restricted epitope. (D) Four-hour 51Cr release assay of purified CD8+ lymphocytes after 7 days of HLA A2-restricted peptide stimulation in the same donor as in panel C. Target cells were B-LCL loaded with HLA A2-restricted or HLA B7-restricted peptide. Results correlated with those obtained by CFC in panel C.
FIG. 2
FIG. 2
Correlation between 6-h cytokine assay and 7-day 51Cr release assay in 12 donors. The statistical analysis was performed using GraphPad (San Diego, Calif.) software, assuming that the data are sampled from Gaussian populations with a two-tailed distribution. (A) Frequencies of CD8+ IFN-γ+ T lymphocytes following 6 h of activation with HLA A2 peptide on day 0 correlated with percent specific lysis of target cells in a 51Cr release assay performed after 7 days of in vitro stimulation of PBMC with HLA A2-restricted peptide. (B) Frequencies of CD8+ IFN-γ+ T cells obtained in the day 7 CFC assay (6-h restimulation) correlated with percent specific lysis of target cells in a 51Cr-release assay also done on day 7.
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