Fig 1: Recognition of HIV+ Non-activated CD4+ T Cells by Autologous HIV-Specific CD8+ T CellsTwo hours after a Vpr-ßlam infection assay, the primary non-activated HIV+ CD4+ T cells (blue) from an elite controller (EC) were co-cultured for 30 min with an autologous Gag-specific CD8+ T cell line (green), fixed, and stained for actin with phalloidin (orange) and perforin (red) or CD107a (red), and cell contacts analyzed by confocal microscopy. A compilation of z stacks was acquired for each cell contact and analyzed with IMARIS software. NI, non-infected; HIV, HIV infected.(A) 3D reconstructions of two representative experiments with non-infected cells (left) or infected cells (right) are shown with perforin staining (top) or CD107a staining (bottom).(B and C) The percentage of points of contact displaying fluorescent signal for perforin (B) or CD107a (C) is quantitated and shown. Means ± SDs for three independent experiments using cells coming from three different HIV controllers are shown; statistics were calculated by unpaired t test. *p < 0.05.(D) For each cell contact, the actin signal was analyzed and quantified with IMARIS software in 3D. Two representative experiments are shown, demonstrating actin rings only in the HIV-infected condition.(E) The percentage of points of contact displaying actin rings is calculated for both conditions and shown as means ± SDs for three independent experiments with cells coming from three different HIV controllers. Statistics were calculated by unpaired t test.(F) Actin polarization on CD8+ T cells toward the point of contact was quantified by measuring the ratio of actin fluorescent signal at the point of contact versus the rest of the cell. The means ± SDs for three independent experiments with 10 cell contacts analyzed per experiment with cells coming from three different HIV controllers; statistics were calculated by unpaired t test.(G) CD107a staining gated on live CD3+CD8+ cells revealed the degranulation of CD8+ T cells in response to autologous HIV X4 infected cells (blue) or HIV R5 infected cells (yellow), compared to Gag peptide-sensitized target cells (red). A representative experiment is shown.(H) The means ± SDs for three independent experiments with cells coming from one HIV controller are represented. Statistics were calculated by an ANOVA multiple comparison test relative to the non-infected condition. *p < 0.05, **p < 0.01, and ***p < 0.001.(I) Antibodies to LFA-I, ICAM-2, and LFA-3 were used in an experiment similar to (G) with HIV X4. The means ± SDs for four independent experiments with cells coming from four different HIV controllers are shown, and statistics were calculated with a one-way ANOVA multiple comparison test relative to the isotype control. **p < 0.01; ns, not statistically significant.See also Video S1 and Figures S3 and S4.