FIG 4.

Primary CD4⁺ T cells infected with dCA-resistant viruses express more HIV antigens and are more susceptible to CTL-mediated killing. (A) Experimental design of the CTL killing assay in which target cells are isolated from CD4⁺ T cells purified out of PBMCs from HIV-negative donors and expanded in culture for 2 weeks, followed by activation with anti-CD3 and anti-CD28 antibodies and in vitro infection by spinoculation with wild type NL4-3 virus or dCA-resistant viruses. The viruses are allowed to grow in the CD4⁺ T cell culture for 4 days, and the killing assay is performed by coculturing these cells with HIV-specific CD8⁺ T cell clone in the presence of ARVs. The killing efficiency is calculated by the percent decrease in p24-expressing cells (assessed by flow cytometry) or HIV DNA (assessed by RT-qPCR) after the coculture compared to a control incubated without CTL clone. PHA, phytohemagglutinin; MOI, multiplicity of infection. (B) Representative plots of p24-expressing cells after 3 days of in vitro infection with wild-type NL4-3 virus and dCA-resistant viruses in primary human CD4⁺ T cells from three different donors. (C) Ratio between frequency of p24-expressing cells and total HIV DNA is significantly higher in cells infected with dCA-resistant viruses than in cells infected with wild-type NL4-3 virus. (D) CTL killing results showing percent decrease in frequency of CD4⁺ T cells expressing p24 after coculture with HIV-specific CTL clone. (E) CTL killing results showing percent decrease in total HIV DNA from CD4⁺ T cells after coculture with HIV-specific CTL clone. Data in panels C to E are presented as means ± SEM (n = 13). Paired ANOVA (Friedman’s test) was used for statistical comparison.