Background: Degranulation activity, as measured by CD107a mobilization, is commonly used to define cytotoxic CD8 T-cells. However, the relationship between granule's content and cytotoxic capacity of virus-specific CD8 T-cells was never investigated. Methods: A variety of virus-specific CD8 T-cell responses including CMV (n = 16), EBV (n = 21) and Flu (n = 17) were identified using tetramer complexes or peptide stimulation and analyzed for CD107a mobilization, as well as for the simultaneous expression of perforin (Perf), granzyme (Grz) A, GrzB and GrzK by polychromatic flow cytometry combined with CD127 (i.e. IL-7Ra), CCR7 and CD45RA, that were used to assess T-cell differentiation.
Results: Polychromatic flow cytometric analyses combining GrzA, GrzB, GrzK and Perf on CD8 T-cells identified 16 distinct populations. Highly differentiated cells (CCR7-CD127-CD45RA±) mostly contained Perf and GrzB, but not GrzA or GrzK, which in contrast were mostly found in poorly differentiated CD8 T-cells (CCR7+CD127+CD45RA-). Interestingly, analysis of the different viruses showed typical virusspecific patterns. CMV-specific T-cells were mostly composed of four distinct subsets and >35% of cell were Perf+GrzB+GrzA+GrzK±. Three distinct subsets were observed in EBV-specific T-cells and >40% of cell were Perf-GrzK+GrzB±GrzA±. Whereas above 60% of Flu-specific T-cells were Perf-GrzK+GrzB-GrzA-. Differences between the viruses were highly significant (All P <0.01). Of note, Flu-specific CD8 T cells, i.e. lacking perf and GrzB, did not show direct ex vivo cytotoxicity despite the fact that a similar level of degranulation activity was observed in Flu-, EBV- and CMV-specific CD8 T cells (ranging from 40-60%, P >0.05) following stimulation with the same peptides.
Conclusions: The composition of granules is highly heterogeneous in CD8 T-cells and associated with differentiation. However, typical patterns of granule's content are observed for the different viruses and lack of perforin is associated with the absence of cytotoxic activity. In contrast, degranulation activity is consistently observed in all models of virus infection (including non cytotoxic responses) and therefore represents an irrelevant marker of CD8 T-cells with cytotoxic capacity.