Fig. 3. CTLA-4 downregulation by Nef coincides with enhanced IL-2 production in primary CD4⁺ T cells. CD4⁺ T cells were activated for 48 h as in Fig. 1, exposed to HIV-1 for an additional 48 h and restimulated via CD3/CD28 antibodies (plate-coated CD3 antibodies, 0.1 µg ml⁻¹; soluble CD28 antibodies, 0.1 µg ml⁻¹). (a) Left panel: frequency of CTLA-4⁺ cells on the HIV p24⁺ subpopulations from HIV-1^ΔNef- compared with HIV-1^Nef+-infected cells at 48 h post-infection (n = 5). Percentages (middle panel) and MFI (right panel) of IL-2-expressing T cells measured by intracellular staining in the total CD4⁺ T cells (including both HIV p24⁺ and p24^–) infected with HIV-1^Nef+ or HIV-1^ΔNef upon 15 h of TCR restimulation (the same donors as in the left panel). (b) Representative dot-plots showing the frequency of IL-2-expressing cells in the HIV p24⁺ and p24^– populations from cells infected with HIV-1^Nef+ and HIV-1^ΔNef compared with non-infected control cells. (c) Percentages (left panel) and MFI (right panel) of IL-2⁺ cells within the p24⁺ or p24^– populations in cells infected with HIV-1^Nef+ or HIV-1^ΔNef. (d) IL-2 production was measured by ELISA at 24 h following TCR restimulation (plate-coated CD3 antibodies, 0.1 µg ml⁻¹; soluble CD28 antibodies, 0.1 µg ml⁻¹) of cells infected with HIV-1^Nef+ or HIV-1^ΔNef and uninfected cells (Mock) from three different donors. (e) Levels of IL-2 production measured by ELISA after TCR and CTLA-4 simultaneous cross-linking on CD4⁺ T cells infected with either HIV-1^Nef+ or HIV-1^ΔNef in eight donors. Simultaneous TCR and CTLA-4 cross-linking was carried out using beads coated with CD3 and CD28 antibodies (35 and 7 ng/20×10⁶ beads, respectively; four beads per cell) and a fixed concentration (1 µg/20×10⁶ beads) of CTLA-4 antibodies. Relative IL-2 values were calculated by dividing the concentration of IL-2 produced upon CTLA-4 and TCR simultaneous cross-linking by the concentration of IL-2 produced after TCR stimulation alone. Nef-mediated CTLA-4 downregulation in cells infected with HIV-1^Nef+ (as shown in Fig. 2) was confirmed in each experiment prior to CTLA-4 triggering. Paired t-test P values are indicated; ns, not significant.