Cytokine regulation of TRAF1 levels in T cells. (a) Purified CD8 T cells from healthy donors were stimulated with either anti-CD3 (1 µg/ml)/CD28 (10 µg/ml) as a positive control or 20 ng/ml of IL-2, IL-7, IL-15, or IL-21, or media alone for 6 d. At day 6, cells were analyzed by flow cytometry. The right graph shows summary data, gated on CD45RA⁻ CD8⁺ T cells, reported as the difference in median fluorescence intensity (dMFI) of TRAF1 compared with FMO controls, with each symbol representing a different donor. Statistical analysis was performed using one-way ANOVA. (b) PBMCs from HIV-infected donors were CFSE-labeled and incubated with cytokines as described in a. Data are shown gated on CD45RA⁻CFSE^low (divided) CD8 T cells except for unstimulated samples, which were gated on CD45RA⁻ CD8 T cells, as they did not undergo division. Data are reported as dMFI relative to FMO controls averaged for four different donors per group. Statistical analysis was performed using one-way ANOVA within each group of HIV donors (early, viral controller, and chronic). (c) TRAF1 levels in HIV-specific CD8 T cells in response to IL-7. To detect HIV-specific CD8 T cells over time, purified CD8 T cells were expanded in response to autologous monocytes pulsed with respective HIV peptides and 4-1BBL-AdV for 8 d with or without IL-7, as described in Materials and methods. The dMFI of TRAF1 against FMO in HIV-specific CD8 T cells for four donors, two early and two chronic, are reported. (d) Mice were infected with LCMV clone 13. At day 21-after infection, mice were treated with 10 µg/mouse of IL-7 or PBS. Tetramer⁺ and PD-1⁻ CD8 T cells were sorted on day 23 as described in Fig. 4 and lysed for Western blot. CD8 T cells from uninfected TRAF1^−/− and WT mice were used as controls. The left plot shows representative data and the bottom right plot shows the summary of TRAF1/actin ratio on the sorted cells which each data point representing an individual mouse. Data in d are the summary of two independent mouse experiments.