Fig 1.
HIV reactivation kinetics in the latently infected cell line J-Lat.
J-Lat cells were incubated for 50h with medium (R10), single LRAs or the combination of different families of these compounds; Panobinostat (PNB, 30 nM), Romidepsin (RMD, 40 nM), Ingenol (ING, 100 nM), Bryostatin-1 (BRY-1, 10 nM) and JQ1 (1 uM). Percentage of GFP+ (in green) and Annexin V+ (in red) cells was monitored each hour using the IncuCyte ZOOM live cell imaging system (Essen Bioscience). Dashed lines show the effect at 22h for the single drugs Panobinostat, Romidepsin and JQ1 (in yellow) and for Ingenol and Bryostatin-1 (in blue). Dotted lines represent 22h.
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.
Fig 3.
Proportion of CD4+ T cells expressing the viral protein p24 after viral reactivation with different LRAs.
A. The proportion of HIV-transcribing CD4+ T cells that simultaneously produce the viral protein p24 is shown. Comparisons with the control medium were performed using the Wilcoxon test. *p<0.05, **p<0.01. B. Calculation of synergistic, antagonistic or additive effect after the combination of LRAs using the Bliss independence model. White symbols correspond to patients in which production of p24 was not detected. Medians and min to max ranks are represented in panels A and B. C. Normalization of the percentage of HIV-transcribing cells expressing p24 relative to PMA/Ionomycin. Pies for individual patients and median values for all patients are represented in a box and whisker plot graph. Fractions of HIV-1 RNA+ cells expressing p24 and lacking the expression of p24 are shown in orange and blue, respectively.
Fig 4.
Proportion of cells expressing HIV-1 RNA and p24 after viral reactivation in different CD4+ T cell subpopulations.
A. Proportion of cells transcribing HIV-1 RNA after viral reactivation with single LRAs in the following populations: TNA, TSCM, TCM, TTM, TEM and TTD. Medians are shown. B. Proportion of HIV-1+ cells per million cells in each CD4+ T cell subpopulation with all tested drugs and their combinations. C. Proportion of patients showing synergistic, antagonistic or additive effects (Bliss independence model) after HIV reactivation with combinations of LRAs are shown for each CD4+ T cell subset. Percentage of patients responding to LRAs interactions are shown for each cell subset. D. Proportion of HIV-1 RNA+ TCM cells expressing the viral protein p24. Black asterisks denote statistical significance compared with the negative control (media) using a Wilcoxon test. Red asterisks denote statistical significance compared with the combination of Romidepsin plus Ingenol using a Friedman test followed by Dunn’s post hoc tests. Medians and min to max ranks are represented in panels B and D. *p<0.05, **p<0.01.