Figure 1: Effects of metformin on viral outgrowth in memory CD4⁺ T-cells from ART-treated PWH.

(A) Shown is the flow chart for the viral outgrowth assay (VOA). Briefly, memory CD4⁺ T-cells from ART-treated PWH were stimulated with CD3/CD28 Abs, in the presence (n=11) or the absence (n=6) of ARVs and in the presence/absence of metformin (1 mM) or INK128 (50 nM) for 3 days. Supernatants were collected, cells were split in two news wells, and media containing IL-2 and metformin or INK128 was refreshed every 3 days. Experiments were performed in 4–8 original replicates per condition. One original replicate at day 0 generated 8 final replicates at day 12. At the end of the experiment, T-cells derived from the same original replicate were merged for PCR and flow cytometry analysis. Shown are (B) integrated HIV-DNA levels quantified by real-time nested PCR and (C) levels of HIV-p24 in cell-culture supernatants quantified by ELISA. Shown are (D) representative flow cytometry dot plot of intracellular HIV-p24 and surface CD4 expression; (E) statistical analysis of the frequency of CD4^lowHIV-p24⁺, (F) the frequency of CD4^highHIV-p24⁺ cells; and (G) the geometric MFI (GeoMFI) of HIV-p24 expression in CD4^low HIV-p24⁺ and CD4^high HIV-p24⁺ T-cell subsets. Each symbol represents one donor; bars indicate the median ± interquartile range. Kruskal-Wallis test and uncorrected Dunn’s multiple comparison p-values are indicated on the graphs.