QUANTITATION OF HUMAN INFLUENZA VIRUS-SPECIFIC CYTOTOXIC T-LYMPHOCYTES - CORRELATION OF CYTOTOXICITY AND INCREASED NUMBERS OF IFN-GAMMA PRODUCING CD8(+) T-CELLS

To study virus-specific cytotoxic T lymphocyte (CTL) activity at the single cell level, an IFN-gamma specific ELISPOT assay was adapted to elucidate the frequency of influenza-specific CTLs together with a standard cytotoxic Cr-51-release assay. Peripheral blood mononuclear cells (PBMCs) from human volunteers were cultured with influenza virus-infected autologous cells; following 3 or 7 days of culture, T cell subsets were assessed for IFN-gamma production by IFN-gamma-specific ELISPOT and ELISA, while IFN-gamma mRNA expression was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Influenza virus-specific CTL activity was measured in a 4 h Cr-51-release assay. Culture of PBMC with autologous A/Taiwan influenza (H1N1)-infected stimulator cells resulted in IFN-gamma spot forming cells (SFCs) at 3 days that increased after 7 days of incubation. Numbers of IFN-gamma SFCs directly correlated with levels of secreted IFN-gamma and higher levels were seen in supernatants from 7 day cultures. RT-PCR analysis (35 cycles of amplification) showed greater IFN-gamma mRNA in T cells isolated from 7 day cultures. Separate aliquots of T cells from these cultures were also assessed for virus-specific cytotoxicity and T cells from 7 day (but not from 3 day) cultures induced high Cr-51 release. Analysis indicated a significant direct correlation between level of cytotoxicity, number of IFN-gamma SFCs, and amount of IFN-gamma in culture supernatants. Studies with purified T cell subsets showed that elevated IFN-gamma SFCs, IFN-gamma synthesis, and cytotoxic activity were associated with CD4-CD8(+) T cells but not with the CD4(+)CD8(-) T cell subset. These results show that increased IFN-gamma production, including increased IFN-gamma mRNA and IFN-gamma SFCs directly correlate with increased antigen-specific, CD8(+) T cell mediated cytotoxicity. Thus, assessment of IFN-gamma SFCs may provide an alternative and quantitative means for assessment of influenza virus-specific CTL in humans.