Figure 2. Expression of HIV in T_CD32^dim reservoir cells after latency disruption and susceptibility to natural killer (NK) immune responses.

Data from the direct ex vivo reactivation of the natural HIV reservoir in ART-suppressed PLWH. (A) Percentage of HIV-RNA expressing cells, measured by the RNA FISH-flow assay, within the T_CD32^dim and T_CD32⁻ subsets after viral reactivation with Ingenol, PMA/ionomycin, or romidepsin (n=9). Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test (comparison between CD32^dim and CD32⁻ within each drug condition), and the ANOVA Friedman with Dunn’s multiple comparison test (comparison between different drug conditions of the cell subset). Median with interquartile range is represented. (B) Percentage of p24⁺ cells after 18 hr viral reactivation with PMA/ionomycin (n=17). Each participant is represented by a different color. Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test. (C) Frequency of viral reactivation within the total pool of T_CD32^dim and T_CD32⁻ cells (n=17). Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test. (D) NK killing assays against viral reactivated cells. Number of p24⁺ cells per million CD4⁺ T cells after the addition of NK cells only or together with the autologous plasma is shown (n=17). Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test. (E) Percentage of ADCC in p24⁺ cells normalized to the NC control (ART + LRA + NK). Statistical comparisons were performed using one-sample t-test. (F) Percentage of CD32 expression within the total p24⁺ pool before and after HIV reactivation (n=17). Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test. (G) Percentage of T_CD32^dim within p24⁺ cells after HIV reactivation and functional NK-mediated assays (n=17). Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test. (H) Percentage of NK-mediated killing by ADCC of the reactivated T_CD32^dim or total p24⁺ cells (n=17). ADCC was calculated as the reduction of p24⁺ cells after the co-culture with NK and plasma and normalized to the condition with NK cells alone. Statistical comparisons were performed using the Wilcoxon matched-pairs signed-rank test. *p<0.05; **p<0.01; ***p<0.001.

Figure 2—figure supplement 1. Expression of HIV-RNA and viral protein p24 in T_CD32^dim reservoir cells after viral reactivation.

(A) Flow cytometry gating strategy used for the identification of T_CD32^dim cells expressing HIV-RNA after the RNA FISH-flow protocol. (B) Percentage of CD32 expression within the total CD4⁺ T cells before and after LRA treatment, using samples from ART-suppressed PLWH subjected to RNA FISH-flow assay. Statistical comparisons consisted of the Wilcoxon matched-pairs signed-rank test. **p<0.01. (C) p24⁺ cells in samples from ART-suppressed individuals at baseline and after viral reactivation with PMA/ionomycin (LRA). The limit of detection is set up at 53 copies/million cells calculated by the formula 3*SD of the mean percentage of p24⁺ cells detected in healthy donor (HD) samples. (D) Representative flow cytometry plots of viral reactivation levels (measured as p24⁺ cells) after LRA treatment and natural killer (NK) functional assays.