Generation and characterization of HLA-A0201 restricted HIV-1 peptide-specific T cell lines from uninfected individuals. Binding of HIV-1 peptides to A0201 was tested using the T2 cell line (panel A). These HIV-1 peptide-stimulated T cells generated antigen-specific proliferative responses (panel B). ELISPOT responses to HIV-1 peptide-loaded donor PBMCs or PHA stimulation (positive control) as well as to non-peptide loaded donor PBMCs (negative control) (Panel C). Flow cytometric analysis of CD4 and CD8 T cells in HIV-1 peptide-specific T cell lines and indicated control T cell lines (Panel D). CD8+ T cells were sorted from the HIV-1 specific T cell line after multiple stimulations in vitro and their peptide specificities determined by individual peptide-loaded DimerX staining (Panel E). In Panel E, the negative control straining was obtained using the HIV-1 specific T cell line and a non-relevant peptide loaded DimerX. The EBV positive control was obtained using donor PBMC's stimulated with 1uM BMLF-1 EBV peptide for one week and then stained with EBV peptide loaded Dimer X. One to nine percent of CD8 T cells were specific for each one of the selected HIV-1B peptides (Panel F).