Ex vivo differentiation of resting enriched CD4+ T cells prior to QVOA increases detectable reactivation of latent HIV-1.

a, Schematic of assay outline for QVOA and differentiation QVOA (dQVOA). The relative distribution of central memory (T_CM), effector memory (T_EM), and transitional memory (T_TM) cells during each phase of the assays are represented as blue, orange, and green characters. b, Flow cytometric analysis showing the memory T cell subset gating strategy on samples from RAVEN participant 2274-R either after resting enrichment (top panel, cells that enter QVOA) or after 7 days of differentiation (bottom panel, cells that enter dQVOA). The numbers in each gate represent the percentage of events arising from its parental population. c, Column plot showing the proportions of naïve, T_CM, T_TM, T_EM and T_EMRA cells as a percentage of live CD3+Lineage-CD8- lymphocytes before and after 7 days of differentiation. NIH 2–20 and NIH 2–37 were assessed for memory subset distribution using a different flow cytometry antibody panel compared to the RAVEN participant samples and therefore were not included in this analysis. d, XY plots showing HIV-1-Gag expression level in each well from QVOA (top panel) and dQVOA (bottom panels) over time. The rCD4+ T cell source was RAVEN participant 2147-R. Stimulation was performed on day 0 for QVOA and dQVOA and when noted ART (EFV/RAL) was included in all media in the assay. Limiting dilutions performed for each assay type are shown. e, Bar graph showing the proportions of QVOA wells (black) found to be HIV-1-Gag+ compared to dQVOA wells (grey). f, Column graph showing the latent HIV-1 reservoir measured as infectious units per million rCD4+ T cells (IUPM) through QVOA and dQVOA. Limit of detection for each assay is represented by red bars. Wilcoxon matched-pairs signed rank tests were used and * denotes p < 0.05, ** denotes p < 0.01, and *** denotes p < 0.001.