Fig. 1. Phenotypic characterization and permissiveness to HIV-1 infection of CD4⁺CTLA-4⁺ T cells. Total CD4⁺ T cells were isolated by negative selection using magnetic beads, and stimulated with immobilized CD3 (1 µg ml⁻¹) and soluble CD28 antibodies (0.5 µg ml⁻¹) for 72 h. (a) Representative surface staining for surrogate activation markers co-expressed with CTLA-4 (intracellular) on activated CD4⁺ T cells. Analysis was performed on gated CD3⁺CD4⁺ T cells that excluded the viability dye LIVE/DEAD. (b) Enrichment of CTLA-4^high T cells by sorting CD25⁺ T cells using flow cytometry. Left panel: CTLA-4 and CD25 levels on activated T cells prior to sorting. Right panels: purity of the two populations CTLA-4^hi and CTLA-4^lo after cytometry sorting. (c) Representative HIV p24 intracellular staining in CD25⁺CTLA-4^hi (upper panels) and CD25^–CTLA-4^lo (lower panels) T cells infected with HIV-1^Nef+ or HIV-1^ΔNef at 48 h post-infection. SSC, side scatter. (d) Mean±sd frequency of HIV-infected cells in CD25⁺CTLA-4^hi and CD25^–CTLA-4^lo T cells when cells were infected with HIV-1^Nef+ or HIV-1^ΔNef (n = 3). Paired t-test P values are indicated.