Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors

<div><p>Despite advances in the treatment of HIV infection with ART, elucidating strategies to overcome HIV persistence, including blockade of viral reservoir establishment, maintenance, and expansion, remains a challenge. T cell homeostasis is a major driver of HIV persistence. Cytokines involved in regulating homeostasis of memory T cells, the major hub of the HIV reservoir, trigger the Jak-STAT pathway. We evaluated the ability of tofacitinib and ruxolitinib, two FDA-approved Jak inhibitors, to block seeding and maintenance of the HIV reservoir <i>in vitro</i>. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence <i>in vivo</i>, <i>ex vivo</i>, and <i>in vitro</i>; pSTAT5 strongly correlates with increased levels of integrated viral DNA <i>in vivo</i>, and <i>in vitro</i> Jak inhibitors reduce the frequency of CD4⁺ T cells harboring integrated HIV DNA. We show that Jak inhibitors block viral production from infected cells, inhibit γ-C receptor cytokine (IL-15)-induced viral reactivation from latent stores thereby preventing transmission of infectious particles to bystander activated T cells. These results show that dysregulation of the Jak-STAT pathway is associated with viral persistence <i>in vivo</i>, and that Jak inhibitors target key events downstream of γ-C cytokine (IL-2, IL-7 and IL-15) ligation to their receptors, impacting the magnitude of the HIV reservoir in all memory CD4 T cell subsets <i>in vitro</i> and <i>ex vivo</i>. Jak inhibitors represent a therapeutic modality to prevent key events of T cell activation that regulate HIV persistence and together, specific, potent blockade of these events may be integrated to future curative strategies.</p></div

Multivariate model of the <i>ex vivo</i> Jak-STAT pathway markers that best predict the size of the HIV reservoir.

<p>Multivariate model of the <i>ex vivo</i> Jak-STAT pathway markers that best predict the size of the HIV reservoir.</p

Jak inhibitors reduce frequency of cells harboring integrated viral DNA and IL-15-induced reactivation of latent HIV-1 in CD4 T cells.

<p>CD4 T cells were isolated from viremic donors and incubated with CD3/CD28 plus 0.01, 0.1, 1.0 or 10 μM of Jak inhibitors with or without EC₉₉ of ART (180 nM zidovudine, 100 nM efavirenz, 200 nM Raltegravir) (A and B). After six days, integrated viral DNA was quantified using ultra sensitive Alu PCR <i>versus</i> DMSO controls (n = 5). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Error bars represent S.E.M. and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 for A and B. In panel C-E, memory CD4⁺ T cells were isolated from ART treated aviremic donors (n = 3), activated with 10 ng/ml IL-15 (panel D) or CD3/CD28 (panel E) and maintained with or without 1 μM ruxolitinib in the presence of ART. Six days post reactivation, extracellular viral RNA copies were quantified by qRT-PCR (*p < 0.01, one-way ANOVA).</p

Jak inhibitors block cytokine-induced STAT5 phosphorylation and Bcl-2 expression.

<p>STAT5 phosphorylation (% in CD4+ T cells) or Bcl-2 expression (MFI in CD4+ T cells) was measured by flow cytometry in PBMC isolated from HIV negative donors and stimulated for 15 min (pSTAT studies; A, C) or 6 days (Bcl-2 studies; B, D) with IL-2 (left panels), IL-7 (middle panels) and IL-15 (right panels) (n = 3) and increasing concentrations (0.01, 0.1, and 1.0 μM) of ruxolitinib or tofacitinib (A-D). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. (-) indicates no cytokine was added. Error bars represent S.E.M. and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: **p < 0.01 and ****p < 0.0001.</p

Ruxolitinib does not inhibit normal TCR function and signaling that is independent of HIV-1 infection.

<p><b>(A)</b> Mean CD3 zeta and SLP76 phosphorylation (MFI in CD4 cells) as quantified by flow cytometry in CD4⁺ cells isolated from HIV negative donors and stimulated with anti-CD3/CD28 in the presence of increasing concentrations of Ruxoltinib <i>versus</i> DMSO treated control cells (n = 3). Statistical significance was determined by an upaired t-test corrected for multiple comparisons using the Holm-Sidak method. <b>(B)</b> Mean cytokine production (% of IL-2⁺, TNF-α⁺ and IFN-γ⁺ triple positive cells) in CD3+CD8- cells or CD3+CD8+ cells as measured by flow cytometry in PBMC isolated from HIV negative donors and stimulated for 6 hr with aCD3/CD28, Brefeldin A (5 μg/ml) and increasing concentrations of Ruxoltinib <i>versus</i> DMSO treated cells (n = 3). Statistical significance for <b>(B)</b> determined by two-way ANOVA followed by Sidak’s multiple comparison post-test: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. <b>(C)</b> Mean cytokine production (% of TNF-α⁺IFN-γ⁺ double positive cells) in CD3+CD4+ cells (n = 6) or CD3+CD8+ cells (n = 3) as measured by flow cytometry in PBMC isolated from stably treated, HIV positive donors and stimulated for 6 hr with 1 μg/ml gag-peptide, Brefeldin A (5 μg/ml) and increasing concentrations of Ruxoltinib <i>versus</i> DMSO treated cells. 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Statistical significance for <b>(C)</b> determined by paired Wilcoxon rank sum test.</p

Immunologic mechanisms of viral persistence and impact of Jak inhibitors on the viral reservoir.

<p>Positive correlation between pSTAT5 and negative correlation with IL-7R (A) is associated with increased levels of integrated viral DNA (B). PD-1 is associated with levels of integrated viral DNA and homeostatic proliferation (B). T cell activation promotes productive viral replication and increases viral co-receptor, HLA-DR, and CD38, as well as increase in proliferation (C). Pro survival signal Bcl-2 promotes survival of the viral reservoir (A), and IL-15, IL-7, and TNF-α induce reactivation of latent HIV-1, thereby reseeding the viral reservoir (D). HIV LTR shows multiple binding sites for pSTAT5 (E), demonstrating that binding of this transcription factor to the LTR could promote pro-HIV transcripts. Bystander infection in activated cells promotes priming uninfected cells for infection, recruitment of uninfected cells to the site of infection, and reseeding of reservoirs with (D, E, F). STAT5-driven homeostatic proliferation could increase absolute numbers of T_regs and lead to further immune dysregulation (G). Red bars indicate pro-HIV events that are blocked by Jak inhibitors.</p

Ruxolitinib inhibits bystander infection.

<p>Uninfected CD4⁺ T cells were incubated with or without cell trace violet (CTV) dye. Cells with CTV dye were stimulated with CD3/CD28 and various concentrations of ruxolitinib or DMSO for 3 days (A, top). Cells without CTV dye were incubated with CD3/CD28 for 3 days followed by a 2 hours spinoculation with a replication competent eGFP Nl4-3 X4 HIV-1 (A, bottom). After spinocualtion on Day 3, both cultures (traced and untraced) were co-incubated for two days in the absence of ruxolitinib. Representative dot plots for bystander infection quantification are demonstrated in panel B. Ruxolitinib inhibits bystander infection (GFP and CTV double positive) of uninfected bystander cells (CTV⁺) in a dose dependent manner (B and C, n = 3). Ruxolitinib blocks proliferation (CTV-lo) of bystander cells in a dose dependent manner with all concentrations tested (D). 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Error bars represent mean with S.E.M (C) or mean with standard deviation (D) and statistical significance determined by two-way ANOVA followed by Sidak’s multiple comparison post-test (C; ****p < 0.0001) or a two-tailed paired T test (D; *p < 0.005).</p