Flow cytometric determination of intracellular or secreted IFNgamma for the quantification of antigen reactive T cells.

The detection of antigen-induced IFNgamma secretion at the single cell level can be used to identify and enumerate antigen-reactive T cells from peripheral blood. This study was performed to analyze the suitability of T cell enumeration by flow cytometry in comparison with the ELISPOT assay. Peripheral blood mononuclear cell (PBMC) samples from six HLA-A2+ healthy subjects were analysed for the frequency of influenza-reactive CD8+ T cells by flow cytometry detecting either intracellular IFNgamma (IC-FC) or secreted IFNgamma (S-FC). All samples were also analysed by IFNgamma ELISPOT assay. The frequency of influenza peptide-reactive T cells determined by IC-FC was 0.01 to 0.34% of CD8+ T cells and by ELISPOT assay 0.02 to 0.23% of CD8+ T cells (n=6 subjects) with a high inter-assay reproducibility and a close correlation between the assays (r=0.77, P<0.001). Little or no IFNgamma production was observed in unstimulated PBMC samples using either the IC-FC or the ELISPOT assay. In contrast, using S-FC large numbers of IFNgamma-secreting CD8+ T cells (0.37% to 5.55%, n=6 subjects) were detected in unstimulated PBMC. The frequency of influenza-reactive CD8+ T cells (0.57-5.19%, n=6 subjects) determined by S-FC did not correlate with the values from the IC-FC or ELISPOT assays. This comparative study shows the suitability of the determination of frequencies of antigen reactive T cells in PBMC by IC-FC. The advantage of IC-FC is the possibility to phenotype simultaneously antigen-reactive T cells.