OBJECTIVE: To identify HIV-1 Gag cytotoxic T-lymphocyte (CTL) epitopes and HLA restriction of their recognition, and to define precursor frequencies of HIV-1 Gag-specific CTL in the blood of seropositive individuals. METHODS: B-lymphoblastoid cell lines (B-LCL) infected with recombinant vaccinia viruses (rVV) containing a gene coding for HIV-1 Gag (rVV-Gag) were fixed with paraformaldehyde (PFA) and used as antigen-presenting cells (APC) to stimulate peripheral blood mononuclear cells (PBMC) from asymptomatic HIV-seropositive individuals. Specific CTL activity was determined in 51Cr-release assays using B-LCL as targets after infection with rVV-Gag or after pulsing with partially overlapping peptides spanning the Gag sequence. RESULTS: In vitro stimulation resulted in an increased number of CD8+ T cells and CD45R0+ and HLA-DR+ cells. 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 culture from one of the individuals. Gag-expressing APC were successfully used as stimulator cells in limiting dilution analysis to determine CTL precursor (CTLp) frequencies. CONCLUSION: PFA-fixed rVV-Gag-infected autologous B-LCL can be used as stimulator cells in bulk PBMC cultures to identify CTL epitopes and to determine CTLp frequencies. This method will facilitate the analysis of HIV-1-specific CTL responses in HIV-infected and vaccinated individuals.

0 (Antigens, CD45), 0 (Epitopes), 0 (Gene Products, gag), 0 (HLA Antigens), 0 (Peptide Fragments), 0 (Recombinant Proteins), Antigen-Presenting Cells/immunology, Antigens, CD45/immunology, B-Lymphocytes/immunology, Cell Differentiation, Cell Line, Cells, Cultured, Epitopes/immunology, Gene Products, gag/*immunology, HIV-1/*immunology, HLA Antigens/*immunology, Human, Leukocytes, Mononuclear/*immunology, Lymphocyte Activation, Peptide Fragments/immunology, Recombinant Proteins/immunology, Support, Non-U.S. Gov't, T-Lymphocytes, Cytotoxic/*immunology, Vaccinia virus/genetics
Erasmus MC: University Medical Center Rotterdam

van Baalen, C.A, Klein, M.R, Geretti, A.M, Keet, R.I.P.M, Miedema, F, van Els, C.A.C.M, & Osterhaus, A.D.M.E. (1993). Selective in vitro expansion of HLA class I-restricted HIV-1 gag-specific CD8+ T cells: cytotoxic T-lymphocyte epitopes and precursor frequencies. AIDS, 7, 781–786. Retrieved from http://hdl.handle.net/1765/3469