Inhibition of antigen-specific CD8+ T cell proliferation using peptide pools- possible mechanisms

Abstract

Studying T cell proliferation in response to peptide pool stimulation is an important readout in vaccine development and epitope discovery studies. We have shown that increased concentration as well as number of peptides per pool inhibits the proliferation of antigen-specific CD8 T cells (GASL, 2008). Understanding the possible mechanisms underlying this would help improving the methodological limitations of using peptide pools. The main objective of this study was to examine whether the cytotoxicity of antigen-specific CD8 T cells and the proliferation of high affinity T cells were affected by peptide pools. PBMCs from HLA-A2-positive individuals responding either to HCV-NS31073 (CINGVCWTV), Influenza-MA (GILGFVFTL), CMV-pp65 (NLVPMVATV) or EBV-BMLF1259-267 (GLCTLVAML) peptides were studied. Cytotoxicity (CD107 staining) and proliferation of high- and low-affinity CD8 T cells were studied by staining with multimeric peptide: MHC complexes and CFSE by employing two different strategies: peptide titration (1μg/ml to 0.001μg/ml) and tetramer titration (0.5μl to 0.05μl). Additionally, expression of 14 different surface markers were analyzed by flowcytometry. Cytotoxicity was not affected by adding peptide pools. In the different experimental set up we observed the low expansion of cells in the presence of unrelated peptide pools secondarly impairs cytotoxicity. Further, low- but not high-affinity CD8+ T cell proliferation was affected. Surface expression of activation markers like CD69, CD25 and CCR5 were down regulated while the expression of T cell negative regulatory marker CTLA-4 was upregulated on Ag-specific-CD8 T cells in the presence of peptide pools. Conclusion: No effect of peptide pools on the cytotoxicity. Low affinity T cells are mainly affected by the peptide pools. Expression levels of several surface markers were altered. Experiments are ongoing to investigate whether the inhibition of proliferation is a consequence of these phenotypic changes.