Figure 9. Drug-mediated mTOR blockade counteracts the enhancing effects of ATRA on HIV reactivation in a viral outgrowth assay.

(A) HIV reactivation was measured in a viral outgrowth assay (VOA), as summarized in the flowchart. (B–D) Briefly, total memory CD4⁺ T cells (n = 5) and (E and F) FACS-sorted memory CCR6⁺ and CCR6^– T cells (n = 3) were isolated from PBMCs of HIV+ART individuals (Supplemental Table 5; ART 1–6). Cells were activated via CD3/CD28 and cultured in the presence/absence of ATRA (10 nM) and/or INK128 (50 nM). (B and C) Intracellular HIV-p24 expression in total memory CD4⁺ T cells and (E and F) sorted memory CCR6⁺ and CCR6^– T cells quantified by FACS as well as (D) HIV-p24 levels quantified by ELISA in cell culture supernatants of total memory CD4⁺ T cells harvested at day 12 after activation. Results are depicted in individual donors. Each point represents one experimental triplicate (mean ± SD of triplicate wells). Repeated-measures 1-way ANOVA with Dunnett’s multiple comparisons test (relative to cells stimulated via the TCR the presence of ATRA) (C and D) and ordinary 1-way ANOVA with Tukey’s multiple comparisons test (F) (****P < 0.0001).