Fig. 2. HIV-1 Nef downregulates CTLA-4 expression in HIV-infected primary CD4⁺ T cells. CD4⁺ T cells were activated for 48 h as in Fig. 1 and exposed to HIV for an additional 48 h. (a) Representative dot-plots showing the percentage of HIV p24⁺ cells (left panels) expressing CTLA-4 (intracellular and extracellular), CD4 and CD3 after infection with HIV-1^ΔNef (upper panels) or HIV-1^Nef+ (lower panels). Numbers in each quadrant refer to the percentage of HIV p24⁺ T cells expressing CTLA-4, CD4 and CD3. (b) Percentages (upper panels) and MFI (lower panels) of CTLA-4, CD4 and CD3 following infection with HIV-1^Nef+ or HIV-1^ΔNef (n = 22). Paired t-test P values are indicated. (c) Left panels: representative FACS data for CTLA-4 (upper panels) and CD3 expression (lower panels) on total GFP⁺ and GFP^– cells after infection with GFP-HIV-1^ΔNef, GFP-HIV-1^Nef+, GFP-SIVcpz^Nef+ and GFP-SIVmac239^Nef+ (representative of n = 3). Right panels: histograms showing the frequency of CTLA-4⁺ and CD3⁺ cells from the GFP⁺ subpopulations (infected cells) for the four different viruses at 48 h post-infection (n = 3). Paired t-test P values are indicated; ns, not significant.