Screening for HCV-specific T cell responses: the use of peptide pools impair the proliferation of antigen specific CD8 T- cells

Abstract

Proliferation of antigen-specific CD4/CD8 T-cells, in response to stimulation using peptide pools, has been routinely used in epitope discovery and vaccine development research. However, neither the optimal concentration of peptides nor the number of peptides per pool has been defined. Thus, the main objective of this study was to examine whether the number of peptides per pool or different peptide concentrations influence different effector functions of T-cell responses against defined CD8 T-cell epitopes. For this purpose, PBMCs isolated from HLA-A2-positive individuals responding to either to the influenza matrix peptide (GILGFVFTL), the CMV peptide pp65 (NLVPMVATV) or the HCV peptide NS3-1073 (CINGVCWTV) were studied. PBMC were cultured with these peptides and varying concentrations of different unrelated overlapping peptide pools specific for the hepatitis D and E viruses. Proliferation of peptide-specific CD8 T-cells was studied by staining with multimeric peptide-MHC complexes as well as by CFSE. Furthermore, cytokine secretion (IFN-gamma and TNF-alpha) was investigated by ELIspot and intra-cellular cytokine staining (ICS). Importantly, both HEV and HDV peptide pools inhibited the proliferation of Flu, CMV and HCV-specific CD8 T-cells in a dose dependent manner. In addition, the number of peptides per pool also influenced T cell responses as the number of proliferating Flu-, CMV and HCV-specific CD8 T cells was significantly lower when PBMCs were stimulated with pools containing 25 peptides when compared with 10 peptides. Cell death was excluded as an explanation for this phenomenon. The inhibition of proliferation was independent from the addition of IL-2. In contrast, neither IFN-gamma production nor TNF-alpha production of peptide-specific CD8 T-cells was affected by unrelated peptide pools. These results show that the total number of peptides in a pool and that the peptide concentration may largely influence CD8 T cell proliferation and warrants caution while using T cell proliferation in epitope discovery studies.