A systematic comparison of methods to measure HIV-1 specific CD8 T cells.

Several methods are now available to evaluate the frequencies of virus-specific CD8 T cells but require a systematic comparison to help at choosing the best strategy for evaluation. First, we compared the ELISpot-IFNgamma assay, intracellular IFNgamma staining and HLA class I tetramer-binding assay to quantify the HIV-specific CD8 T cells. Second, we determined the frequency of recognition of HIV antigens and evaluated whether the mode of antigen presentation might influence the results: We compared HIV antigen presentation in the same ELISpot-IFNgamma assays by using recombinant vaccinia viruses (rVVs) encoding for HIV-LAI Gag, Pol, Env, Nef, Tat and Vif proteins, or a panel of 49 synthetic 8-11 amino acid length peptides tested either individually or pooled. Third, we compared the antigens recognized by memory CTL analysis using chromium release assay (CRA) on CTL lines and by effector CD8 cell analysis using ELISpot assay. Our results show that: (1) Flow cytometry and ELISpot assay measuring IFNgamma production give the same frequency of HIV-specific CD8 T cells; (2) tetramer-binding assay detects more HIV-specific CD8 T cells than other methods; (3) pools of optimal peptides and sum frequencies of individual optimal peptides give similar results in ELISpot assay; (4) ELISpot assays using peptides are more sensitive than those using rVV; and (5) CRA and ELISpot assay when using rVV provide a comparable profile of HIV antigen recognition by memory CTLs (CRA) and effector CTLs (ELISpot) in two thirds cases. These results have important implications for the choice of immunological methods to evaluate CD8 T cells responses to vaccines.