Fig. 1.
Influenza virus stimulation increased the expression of GrB mRNA and the activity of GrB in human PBMC. A, RT-PCR electrophoresis of GrB, Perf and the housekeeping gene GAPDH in human PBMC is shown. Human PBMC were fresh (0h) or stimulated with A/Panama/2007/99 [A/H3N2] for 6 hours (6h), 20 hours (20h), 4 days (4d) or 5 days (5d). The expression of GrB mRNA increased with influenza virus stimulation while the level of Perf mRNA expression remained high at all time points under the same conditions. B, Real-Time PCR confirmed the increase of GrB mRNA expression with virus stimulation. Mean ratio for the GrB/GAPDH copy number in PBMC stimulated under the same conditions, showed a significant increase over time (N=3, ANOVA, p<0.001). Error bars represent standard error. C, Mean GrB activity showed a significant 3-folder increase from 20 hours to 5 days in PBMC stimulated with influenza virus strains, A/Panama/2007/99 [A/H3N2] and A/New Caledonia/20/99 [A/H1N1](N=5, ANOVA, p<0.001), with no significant difference between the two virus strains. Error bars represent standard error. D, The proportion of Perf+ and Perf+GrB+ CD8 T cells increased with virus stimulation. Compared to fresh PBMC (0h), CD8 T cells in PBMC stimulated with A/Panama/2007/99 [A/H3N2] for 5 days (5d) contained a higher proportion of both Perf+GrB− (arrow 1) and Perf+GrB+ (arrow 3) CD8 T cells and a reciprocal lower proportion of PerflowGrB− (arrow 2).