SELECTIVE IN-VITRO EXPANSION OF HLA CLASS I-RESTRICTED HIV-1 GAG-SPECIFIC CD8-CELLS - CYTOTOXIC T-LYMPHOCYTE EPITOPES AND PRECURSOR FREQUENCIES( T)

Objective: To identify HIV-1 Gag cytotoxic T-lymphocyte (CTL) epitopes and HLA restriction of their recognition, and to define precursor fre quencies of HIV-1 Gag-specific CTL in the blood of seropositive indivi duals. Methods: B-lymphoblastoid cell lines (B-LCL) infected with reco mbinant vaccinia viruses (rVV) containing a gene coding for HIV-1 Gag (rVV-Gag) were fixed with paraformaldehyde (PFA) and used as antigen-p resenting cells (APC) to stimulate peripheral blood mononuclear cells (PBMC) from asymptomatic HIV-seropositive individuals. Specific CTL ac tivity was determined in Cr-51-release assays using B-LCL as targets a fter infection with rVV-Gag or after pulsing with partially overlappin g peptides spanning the Gag sequence. Results: In vitro stimulation re sulted in an increased number of CD8+ T cells and CD45R0+ and HLA-DRcells. Gag-specific cytotoxicity, mediated predominantly by HLA class I-restricted CD8+ CTL, was observed in all seven individuals studied. Multiple HLA-restricted CTL epitopes were identified with a single cul ture from one of the individuals. Gag-expressing APC were successfully used as stimulator cells in limiting dilution analysis to determine C TL precursor (CTLp) frequencies. Conclusion: PFA-fixed rVV-Gag-infecte d autologous B-LCL can be used as stimulator cells in bulk PBMC cultur es to identify CTL epitopes and to determine CTLp frequencies. This me thod will facilitate the analysis of HIV-1-specific CTL responses in H IV-infected and vaccinated individuals.