Latency reversal agents affect differently the latent reservoir present in distinct CD4+ T subpopulations
Fig 2
Detection of cells expressing HIV-1 RNA after viral reactivation with different LRAs.
Freshly isolated CD4+ T cells from 9 ART-suppressed HIV-infected individuals were reactivated with different LRAs for 22h and subjected to the RNA FISH/flow protocol for detection of HIV transcripts. Drug concentrations were as follows: 40 nM Romidepsin (RMD), 30 nM Panobinostat (PNB), 1 μM JQ1, 100 nM Ingenol (ING), 10 nM Bryostatin-1 (BRY-1), 81 nM PMA plus 1 μM Ionomycin (IONO) or media alone. A. Proportion of cells expressing HIV-1 RNA in CD4+ T cells for each condition normalized to the medium control from individual patients are shown. Medians and min and max ranks are represented and statistical comparisons with the control medium were performed using the Wilcoxon test. *p<0.05, **p<0.01. B. Percentage of patients showing synergistic, antagonistic or additive effects (Bliss independence model) on HIV reactivation are shown as individual ring graphs for each combination of different LRA families studied. C. Proportion of HIV-transcribing cells relative to the positive control PMA/Ionomycin. Pies for individual patients normalized to the positive control and median values for all patients are represented in a box and whisker plot graph. HIV-1 RNA+ and HIV-1 RNA- fractions are shown in orange and blue, respectively. D. Fraction of the HIV-reservoir susceptible to HIV reactivation after LRA treatment in CD4+ T cells from 9 ART-suppressed patients. Lower pies show the proportion of reactivated and non-reactivated cells. Upper pies show the fraction of reactivated cells after treatment with each compound depicted in the adjacent legend. We next calculated the percentage of the HIV-transcriptionally active viral reservoir after exogenous reactivation with the LRAs. We observed that between 3 and 31% (median value of 16.28%) of the total cells that encompass the viral reservoir (measured as the number of cells containing proviral DNA) were capable of transcribing HIV-1 RNA after viral reactivation (Fig 2D). The potency of each LRAs and their combinations in each individual patient is also shown in Fig 2D. Thus, while in general only a fraction of cells harboring HIV-1 DNA can be reactivated by current available LRAs, differences in terms of strength and consistency of this viral reactivation are observed in the whole population of CD4+ T cells from different ART-treated individuals.