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. 2019 Nov 26;29(9):2783-2795.e5.
doi: 10.1016/j.celrep.2019.10.101.

Latency-Reversing Agents Induce Differential Responses in Distinct Memory CD4 T Cell Subsets in Individuals on Antiretroviral Therapy

Marion Pardons  1 , Rémi Fromentin  2 , Amélie Pagliuzza  2 , Jean-Pierre Routy  3 , Nicolas Chomont  4
Affiliations

Latency-Reversing Agents Induce Differential Responses in Distinct Memory CD4 T Cell Subsets in Individuals on Antiretroviral Therapy

Marion Pardons et al. Cell Rep. .

Abstract

Latent proviruses persist in central (TCM), transitional (TTM), and effector (TEM) memory cells. We measured the levels of cellular factors involved in HIV gene expression in these subsets. The highest levels of acetylated H4, active nuclear factor κB (NF-κB), and active positive transcription elongation factor b (P-TEFb) were measured in TEM, TCM, and TTM cells, respectively. Vorinostat and romidepsin display opposite abilities to induce H4 acetylation across subsets. Protein kinase C (PKC) agonists are more efficient at inducing NF-κB phosphorylation in TCM cells but more potent at activating PTEF-b in the TEM subset. We selected the most efficient latency-reversing agents (LRAs) and measured their ability to reverse latency in each subset. While ingenol alone has modest activities in the three subsets, its combination with a histone deacetylase inhibitor (HDACi) dramatically increases latency reversal in TCM cells. Altogether, these results indicate that cellular HIV reservoirs are differentially responsive to common LRAs and suggest that combination of compounds will be required to achieve latency reversal in all subsets.

Keywords: CD4 T cells; HIV reservoir; HIV-Flow; LRA; NF-κB; P-TEFb; histone acetylation; latency; latency-reversing agent; memory subsets.

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Figures

Figure 1.
Figure 1.. HDAC inhibitors display variable activities in subsets of memory CD4 T cells.
Levels of acetylated histone H4 were determined by intracellular flow cytometry in CD4 T cells (A-D) and gated memory subsets (E-I) from n=8 ART-suppressed individuals. (A) Representative histograms showing the expression of acetylated histone H4 in response to the indicated HDACi (maximal dose, see Star Methods) compared to the non-stimulated condition (NS). (B) Dose-response curves of acetylated histone H4 in response to the 3 HDACi indicated. (C) EC50 and (D) fold induction at the top plateau in total CD4 T cells were determined from the dose-response curves. (E) Representative histograms from one participant showing the expression of acetylated histone in the absence of stimulation in gated CD4 T cell subsets and (F) expression levels in subsets from 8 participants. (G) Representative dose-response curves of acetylated histone H4 in gated CD4 subsets following exposure to the 3 HDACi indicated. (H) EC50 and (I) fold induction at the top plateau in CD4 subsets were determined from the dose-response curves. Tables on the right indicate the subsets in which each drug exerts maximal potency (low EC50, in green) and maximal efficacy (high fold induction, in green). VOR: vorinostat; PNB: panobinostat, RMD: romidepsin. Horizontal bars represent median values. For statistical analyses, Friedman tests with Dunn’s correction for multiple comparisons were used (*adjusted p value <0.05; ** <0.01; ***<0.001). See also Figures S1, S2, S7 and Table S1.
Figure 2.
Figure 2.. PKC agonists display variable activities in subsets of memory CD4 T cells.
Levels of pNF-κB were determined by intracellular flow cytometry in CD8-CD3+ T cells (A-D) and gated memory subsets (E-I) from n=8 ART-suppressed individuals. (A) Representative histograms showing the expression of pNF-κB in response to the indicated agonists of PKC (maximal dose, see Star Methods) compared to the non-stimulated condition (NS). (B) Dose-response curves of pNF-κB in response to the 4 PKC agonists indicated. (C) EC50 and (D) fold induction at the top plateau in total CD4 T cells were determined from the dose-response curves. (E) Representative histograms from one participant showing the expression of pNF-κB in the absence of stimulation in gated CD4 subsets and (F) expression levels in subsets from 8 participants. (G) Representative dose-response curves of pNF-κB in gated CD4 subsets following exposure to the 4 PKC agonists indicated. (H) EC50 and (I) fold induction at the top plateau in CD4 subsets were determined from the dose-response curves. The tables on the right indicate the subsets in which each drug exerts maximal potency (low EC50, in green) and maximal efficacy (high fold induction, in green). Bryo: bryostatin, Ing: ingenol, Pro: prostratin. Horizontal bars represent median values. For statistical analyses, Friedman tests with Dunn’s correction for multiple comparisons were used (*adjusted p value <0.05; ** <0.01; ***<0.001). See also Figure S7 and Table S1.
Figure 3.
Figure 3.. PKC agonists are more potent at activating P-TEFb in differentiated subsets.
Levels of pCDK9 (pS175) were determined by intracellular flow cytometry in CD4 T cells (A-D) and gated memory subsets (E-I) from n=8 ART-suppressed individuals. (A) Representative histograms showing the expression of pCDK9 in response to the indicated agonists of PKC (maximal dose, see Star Methods) compared to the non-stimulated condition (NS). (B) Dose-response curves of pCDK9 in response to the 2 PKC agonists indicated. (C) EC50 and (D) fold induction at the top plateau in total CD4 T cells were determined from the dose-response curves. (E) Representative histograms from one participant showing the expression of pCDK9 in the absence of stimulation in gated CD4 subsets and (F) expression levels in subsets from 8 participants. (G) Representative dose-response curves of pCDK9 in response to the 2 PKC agonists indicated in gated CD4 subsets. (H) EC50 and (I) fold induction at the top plateau in CD4 subsets were determined from the dose-response curves. The tables on the right indicate the subsets in which each drug exerts maximal potency (low EC50, in green) and maximal efficacy (high fold induction, in green). Bryo: bryostatin, Ing: ingenol. Horizontal bars represent median values. For statistical analyses, Friedman tests with Dunn’s correction for multiple comparisons were used (*adjusted p value <0.05; ** <0.01; ***<0.001). See also Figure S7 and Table S1.
Figure 4.
Figure 4.. miR125b and miR155 are expressed at different levels in distinct memory subsets.
The indicated miRNAs were quantified by RT-qPCR in subsets of CD4 T cells obtained by flow cytometry cell sorting from n=5 virally suppressed individuals. Levels of expression of each miRNA were normalized to the levels of GAPDH reference gene. Means and standard deviations are represented. For each miRNA, Friedman tests with Dunn’s correction for multiple comparisons were used (*adjusted p value <0.05; ** <0.01). See also Table S2.
Figure 5.
Figure 5.. Combinations of LRAs are more potent at reactivating HIV than LRAs alone.
HIV-Flow was used to measure the frequency of p24+ cells upon reactivation of latent proviruses. CD4 T cells isolated from n=9 ART-suppressed individuals were exposed to LRAs (PNB: 100nM panobinostat, RMD: 100nM romidepsin, ING: 500nM ingenol). Results are represented as the relative percentage of maximal reactivation obtained with PMA/ionomycin (100%). Medians and interquartile ranges are shown. For statistical analyses, non-parametric Wilcoxon tests were used; statistically significant p values are indicated on the graphs. See also Figure S3 and Table S1.
Figure 6.
Figure 6.. PKC agonists in combination with HDACi have enhanced capacity to reactivate HIV in TCM cells.
HIV-Flow was used to characterize the phenotype of p24+ cells upon reactivation of latent proviruses. CD4 T cells isolated from n=9 ART-suppressed individuals were exposed to LRAs (PNB: 100nM panobinostat, RMD: 100nM romidepsin, ING: 500nM ingenol). (A) Frequency of p24+ cells per million TCM, TTM, TEM cells. Grey columns depict median values. (B) Percentage of maximal reactivation obtained with PMA/ionomycin in memory CD4 T cell subset. The level of reactivation obtained with PMA/ionomycin (100%) is represented by a dotted horizontal line. Grey columns depict median values. For statistical analyses, non-parametric Wilcoxon tests were used; statistically significant p values are indicated on the graphs. See also Figures S4, S5, S6 and Table S1.
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