Fig. 4. CTLA-4 downregulation is associated with decreased T-cell activation threshold and enhanced HIV-1 replication upon CTLA-4 triggering. CD4⁺ T cells were activated for 48 h as in Fig. 1, exposed to HIV for an additional 24 h, and restimulated via beads coated with CD3/CD28 and CTLA-4 antibodies or matched isotype control for an additional 24 h (for IL-2 quantification), 4 days (for HIV DNA quantification) or 9 days (for HIV p24 quantification). (a) Levels of IL-2 production measured by ELISA in a representative subject. Simultaneous TCR and CTLA-4 cross-linking was carried out using decreasing concentrations of CD3 and CD28 antibodies (CD3: 280–35 ng/20×10⁶ beads; CD28: 56–7 ng/20×10⁶ beads; four beads per cell) and a fixed concentration (1 µg/20×10⁶ beads) of CTLA-4 antibodies on cells infected with HIV-1^Nef+ or HIV-1^ΔNef. (b) Fold increase in IL-2 production from HIV-1^Nef+ over HIV-1^ΔNef under simultaneous TCR and CTLA-4 cross-linking (mean±sd from four subjects). (c) CTLA-4-dependent inhibition of viral replication as monitored by quantification of total HIV DNA at day 4 following TCR (CD3 and CD28 antibodies 35/7 ng/20×10⁶ beads, respectively; four beads per cell) and CTLA-4 (1 µg/20×10⁶ beads) cross-linking (or isotype control: Iso-IgG) on HIV-1^Nef+- and HIV-1^ΔNef-infected cells (n = 4). (d) HIV p24 production measured by ELISA at day 6 following simultaneous TCR and CTLA-4 cross-linking on cells infected with eGFP-HIV-1 virus expressing Nef compared with the ΔNef virus. Shown are the p24 production levels from both viral infections under simultaneous TCR and CTLA-4 cross-linking relative to TCR⁺ Isotype activation (n = 3). Prior to TCR/CTLA-4 triggering, CD4⁺ T cells infected for 24 h with eGFP viruses were treated with and maintained in 5 µM azidothymidine-containing medium to prevent reinfection. Paired t-test P values are indicated.