Loss of TRAF1 protein from HIV-specific T cells during chronic HIV infection. Freshly thawed PBMCs from HIV-infected individuals were stained for CD8, CD3, TRAF1, HIV-tetramer, and CD38 (a-d), and flow cytometry data were analyzed with the gating strategy described in Fig. S1. (a) Frequency of TRAF1⁺ HIV-specific CD8 T cells in recently or chronically infected donors or viral controllers (groups defined in Table S1). (top) Summary of the frequency of TRAF1⁺ HIV-tetramer⁺ cells for all donors analyze with each symbol representing a single epitope, with one to two epitopes per donor. Statistical analysis was performed by one-way ANOVA. (bottom) Representative histograms from each group, with shaded panels indicating FMO controls, open histograms indicating TRAF1 staining on CD3⁺CD8⁺tetramer⁺ T cells. Note: some donors showed bimodal staining for TRAF1 on their HIV-specific T cells. (b) Correlation between the frequency of TRAF1⁺ HIV-specific T cells and the level of activation as measured by the frequency of CD38⁺ HIV-specific T cells. Both viral controllers and chronic progressors are included in this analysis. Statistical analysis was performed using linear regression (P = 0.009). (c) TRAF1 expression in HIV-specific T cells using longitudinal samples from three donors with five HIV-specific epitopes. (top) Representative TRAF1 staining during early and later time point within the same donor (shown for two donors); (bottom) Summary plot with each donor represented by a different symbol and filled and open symbols distinguishing two epitopes within the same donor. FMO controls are indicated in the shaded histograms. (d) Frequency of TRAF1⁺ T cells of total CD8 T cells for the three groups of donors. (e) TRAF1 expression in total CD8⁺CD45RA⁻ T cells before and after activation. Each symbol represents an individual donor. PBMCs were labeled with CFSE and stimulated with 1 µg/ml of anti-CD3 and 10 µg/ml of anti-CD28 for 6 d, and then stained for CD3, CD8, and TRAF1. The frequency of TRAF1⁺ T cells on the CFSE low (divided) CD8⁺CD45RA⁻ population is reported for the stimulated cells (right axis). For the unstimulated cells, the frequency of TRAF1⁺ in the CD8⁺CD45RA⁻ T cell population is reported (left axis). VC, viral controller.