Kinetics of Antiviral Activity by Human Immunodeficiency Virus Type 1-Specific Cytotoxic T Lymphocytes (CTL) and Rapid Selection of CTL Escape Virus In Vitro

Titration of peptides recognized by the Rev-specific clone TCC108. Chromium-labelled autologous B-LCL cells were incubated with the peptide Rev(65-75) GRSAEPVPLQL, Rev(66-75) RSAEPVPLQL, or Rev(67-75) SAEPVPLQL for 1 h at the concentrations indicated. Subsequently, the target cells were washed and cocultivated with TCC108 cells for 4 h. The effector-to-target cell ratios were 10:1. The average percent specific lysis (with standard error) for triplicates is shown. No lysis of B-LCL cells without peptide was observed (data not shown).

Kinetics of HIV-1 production and lysis of infected cells by Rev- and RT-specific CTL. (A) CD4⁺ TCL2H7 cells were infected as described in Materials and Methods, and culture supernatants were harvested at the indicated times for analysis of virus production. The TCL2H7 cells were analyzed for p55 expression and for susceptibility to CTL-mediated lysis by Rev-specific clone TCC108, RT-specific clone TCL1C11, and non-HIV-specific clone TCC112. The effector-to-target ratios were 10:1. Lysis of uninfected CD4⁺ TCL2H7 cells was below 5% in all assays (data not shown). The chromium release data are plotted as the average (with standard error) for triplicates at the time point at which the chromium release assay was terminated, i.e., 6 hours after the addition of chromium. This time was required for the chromium labelling (1 h), washing of the target cells and preparing the cocultures of the effector and target cells (1 h), and incubation (4 h). (B) p55 expression (closed symbols) and virus production (open symbols) by TCL2H7 cells in the presence of TCC108 cells, TCL1C11 cells, or TCC112 cells. Effector and target cells were discriminated by flow cytometric analyses of CD8 and CD4 expression, respectively. The population of p55-expressing TCL2H7 cells is expressed as a percentage of the CD8⁻ cells and not of the CD4⁺ cells, since CD4 was down-regulated in a major fraction of the infected cells. (C) Infected TCL2H7 cells were analyzed in chromium release assays after incubation without peptide or with the relevant peptides at 10 μM: SAEPVPLQL for TCC108 cells and IVLPEKDSW for TCL1C11 cells. The average specific lysis (with standard error) for triplicates is shown. The dashed line shows the percentage of p55-expressing cells at 48 h after infection. Lysis of uninfected TCL2H7 cells without peptides was always below 5% (data not shown), and peptide-pulsed uninfected TCL2H7 cells were lysed as efficiently as peptide-pulsed infected cells (data not shown).

Inhibition of HIV-1 replication by Rev-specific CTL. HLA-B14-expressing PBMC from an HIV-seronegative individual were infected with HIV-1 IIIB as indicated in Materials and Methods. PBMC (5 × 10⁶) were cocultivated without CTL, with 5 × 10⁵ Rev-specific TCC108 cells, or with 5 × 10⁵ non-HIV-specific TCC112 cells. Both types of TCC cells had been stimulated 7 days before addition to the PBMC. (A) Virus production was analyzed by quantification of the RT activity in culture supernatants. The average RT activity (with standard error) for triplicates is shown. (B) The fates of the CD4⁺ PBMC and the CD8⁺ TCC108 and TCC112 cells were determined by counting of the cells and flow cytometric analysis of membrane-expressed CD4, CD8, and HLA-A2. HLA-A2 was included to discriminate between CD8⁺ PBMC (expressing HLA-A2) and the added TCC108 and TCC112 cells (both expressing HLA-A1 and -A28). (C) CD8⁺ cells were recovered from the cultures on days 6 and 11 by magnetic bead selection and analyzed for Rev-specific CTL activity on autologous B-LCL cells infected with rVV-rev or rVV-control. The average percent specific lysis (with standard error) for triplicates is shown.