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 in vitro. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence in vivo, ex vivo, and in vitro; pSTAT5 strongly correlates with increased levels of integrated viral DNA in vivo, and in vitro 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 in vivo, 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 in vitro and ex vivo. 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.

HIV persists in infected hosts in a small number of CD4 + memory T cells as latently infected proviruses. Homeostatic cytokines, which play a major role in the maintenance of T cell memory, also enable the persistence of latently infected cells. The pathways downstream of these homeostatic cytokines are well known and drugs that target these pathways have been developed and have been safely used in inflammatory diseases and in myelofibrosis. We have used these drugs to inhibit the maintenance and the spread of HIV infected cells carrying latent forms of the virus. We show that ruxolitinib and tofacitinib will inhibit the expansion of these cells and their capacity to infect other cells upon reactivation. This class of drugs is currently being tested in clinical trials.

Competing interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: JHB is an employee of GlaxoSmithKline. The affiliation of SS with Unconditional Love, Inc., and JHB with GlaxoSmithKline does not alter our adherence to all the PLOS Pathogens policies on sharing data and materials.

Funding: This work was generously supported by NIH-NIMH grant 1R01MH100999 and NIH-NIAID grant 5P30AI050409 (to RFS); Center for AIDS Research, NIH-NIAID grant 5P01AI076174 and NIH-NIAID grant 5U19AI096109 (to RPS); amfAR fellowship 108261-51-RFRL (to FPD), and the amfAR Impact grant 109222-58-RGRL (to CC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We previously reported that ruxolitinib and tofacitinib blocked reactivation of HIV in a primary T cell latency model at physiologic concentrations, underscoring its potential to block HIV reservoir expansion and viral dissemination from latent stores [ 34 ]. Herein, we monitored the impact of these clinically approved and extensively evaluated Jak-STAT inhibitors on several stages of HIV persistence including seeding of bystander cells and HIV reactivation from latency. Overall, we show ex vivo and in vitro that these Jak inhibitors use several mechanisms to impede the seeding and maintenance of the HIV reservoir.

Tofacitinib and ruxolitinib are two FDA-approved Jak inhibitors for long-term use for the treatment of rheumatoid arthritis, myelofibrosis, or polycythemia vera [ 28 – 30 ]. Ruxolitinib demonstrates potent inhibition of pro-inflammatory cytokines in vivo, including IL-6, IL-1α/β, and TNF-α [ 29 – 32 ], all of which have been shown to enhance HIV replication in vitro [ 33 , 34 ]. Inhibition of Jak-STAT signaling by ruxolitinib was shown to significantly impede T cell homeostasis, reducing CD4 T cell numbers as well as decreasing numbers of T regulatory cells and activated (HLA-DR + ) CD4 T cell populations after a few weeks of treatment [ 28 ]. Tofacitinib showed only small changes in CD3 + , CD4 + and CD8 + counts and an increase in B cell counts after 24 weeks of treatment [ 29 , 30 , 35 ]. Attenuated activation and proliferation were not specific to CD4 T cells but were also reported for Natural Killer (NK) cells treated with ruxolitinib in vitro and also in ruxolitinib or tofacitinib treated patients where the number of mature NK cells was reduced [ 28 , 35 ]. Ruxolitinib treatment was further shown to block monocyte–derived DC differentiation, DC-derived IL-12 production and activation marker expression triggered by exposure to lipopolysaccharide (LPS) [ 28 ], demonstrating the impact of Jak inhibitors on innate and adaptive immune responses.

Phosphorylation of STAT-5 (pSTAT5) is triggered following the engagement of IL-2, IL-7 or IL-15 cytokines with their receptors leading to pro-survival signaling and increased proliferation [ 15 , 20 , 21 ]. Given the presence of multiple binding sites for pSTAT5 within the HIV long terminal repeat (LTR) [ 22 ], IL-2, IL-7 and IL-15 enhanced viral expression from productively infected cells [ 14 , 15 , 20 , 21 , 23 ]. Interestingly, a dominant negative STAT5 inhibited Jak-induced HIV LTR activity and decreased productive HIV infection while overexpression of STAT5 enhanced virus production in unstimulated primary T cells [ 22 ]. Together, these events underscore the relationship between activation of the STAT5 pathway and production of HIV, including events that impact the establishment of latency, its maintenance, and /or expansion of the HIV viral reservoir [ 24 – 27 ].

Current antiretroviral therapy (ART) has yielded significant success in achieving long-term suppression of viral load and in improving survival of HIV infected subjects [ 1 – 4 ]. Even so, ART fails to eliminate a small number of cells harboring integrated, replication competent viral DNA. This HIV reservoir has represented a major limitation in eradicating HIV. The HIV reservoir has been shown to persist in central memory T CM , transitional memory T TM and effector memory T EM CD4 T cells which require exposure to γ-C receptor cytokines for their long-term persistence [ 5 , 6 ]. IL-2, IL-7 and IL-15 are γ-C receptor, homeostatic cytokines involved in the maintenance of T cell memory and which activate STAT5 mediated signaling [ 7 ]. In addition, the Jak-STAT pathway is also triggered by type I and type II Interferons, two important mediators of inflammation in viral infections including HIV [ 8 – 11 ]. Initial attempts to purge HIV involved the use of IL-2; results of these studies while promising, since virus was undetectable, did not reach their objective as viral load rebounded upon cessation of therapy [ 12 ]. We have previously shown that IL-7 driven homeostatic proliferation contributes to HIV persistence by promoting the survival and proliferation of latently infected cells [ 13 , 14 ]. Further highlighting the role of IL-7 in the expansion and maintenance of the viral reservoir, an ACTG sponsored trial (ACTG protocol number 5214; www.clinicaltrials.gov as # NCT00099671 ) demonstrated that IL-7, also leads to a 70% increase in the absolute numbers of CD4 T cells harboring integrated viral DNA [ 14 ], suggesting that this intervention would not be compatible with an HIV eradication strategy. IL-15, which also signals through STAT5, has also been demonstrated to induce homeostatic proliferation of CD4 T cell subsets [ 15 – 18 ]. Furthermore, recent in vitro and ex vivo studies with IL-15, the IL-15 superagonist (ALT-803) and IL-2 illustrated that not only did these γ-C cytokines increase viral reactivation, but they also primed the reservoir within CD4 T cells for recognition by autologous HIV-specific CD8 T cells [ 19 ].

Results

Ex vivo and in vitro inhibition of HIV replication by ruxolitinib and tofacitinib Our group previously demonstrated that ruxolitinib and tofacitinib confer submicromolar anti-HIV-1/2 activity in human PBMCs and in macrophages without demonstrable toxicity [34], however further mechanistic information about how these drugs modulate key events involved in the expansion and maintenance of the HIV viral reservoir have not been explored. Herein we show that both inhibitors significantly (p < 0.0001) reduced p24 production by CD4+ T cells isolated from viremic donors and stimulated with CD3/CD28 when compared to DMSO controls (Fig 2A and 2B and S3 and S4 Figs for data on individual donors). This inhibition was observed when tested in the absence of ART (to observe the effect of the Jak inhibitors on viral replication and de novo infection of cells) and in the presence of ART (to observe the effect of the Jak inhibitors when spreading of HIV infection is inhibited, hence on viral production). These results showed that Jak inhibitors prevented uninfected primary CD4 T cells from HIV infection (described in detail under bystander infection assay) and inhibited the production of HIV from infected cells (ruxolitinib antiviral potency with ART; EC 50 0.17 μM; EC 90 6.2 μM; ruxolitinib antiviral potency without ART; 0.007 μM; EC 90 0.26 μM). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 2. Jak inhibitors block HIV-1 replication and production ex vivo and in vitro. Viral production was measured by ELISA p24 in cell-free supernatants of enriched CD4+ T cells isolated from 5 viremic donors and stimulated for 6 days with anti-CD3/28 in the presence of increasing concentrations of Jak inhibitors without (A) or with (B) ART. Viral production was measured by ELISA p24 in cell-free supernatants of in vitro infected CD4+ cells after 6 days culture in the presence of increasing concentrations of Jak inhibitors (C) and by the frequency of HIV-GFP+ expressing CD4+ cells after 3 day culture (D). To account for inter-patient variability in baseline values, results are reported as the fold change versus DMSO controls. 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. https://doi.org/10.1371/journal.ppat.1006740.g002 Since ruxolitinib and tofacitinib decreased γ-C-cytokine induced Bcl-2 expression (Fig 1), we monitored cell viability of CD4+ T cells isolated from viremic donors and cultured for 6 days with CD3/CD28 and increasing concentrations of Jak inhibitors. Decreased viability was observed in 3 of the 5 donors at 0.1 μM or greater ruxolitinib or tofacitinib (S15 Fig, Panel A); using a linear regression model, addition of either inhibitor to anti-CD3/CD28 stimulated cell cultures led to significantly lower frequency of viable cells (%AnnexinV-LIVE/DEAD-) compared to DMSO taking into account inhibitor concentration and adjusting for donor. In contrast, we did not observe a loss in viability of in vitro infected cells after 3 day culture with Jak inhibitors plus ART (S15 Fig, Panel B); and no loss in viability was observed after 6 day culture of uninfected cells treated with IL-2 or IL-7 (n = 3) and in 2 of 3 uninfected donors treated with IL-15 in the presence of Jak inhibitors (S15 Fig, Panel C). As HIV-1 infected subjects are known to express lower levels of Bcl-2 [41], decreased viability as a result of inhibition of Bcl-2 expression may be a potential mechanism of Jak inhibitors on blocking viral persistence along with HIV infection in CD4 cells from viremic donors. The inhibition of viral replication and production mediated by ruxolitinib and tofacitinib ex vivo was confirmed in an in vitro HIV infection model using a CXCR4-tropic, GFP tagged virus (eGFP NL4-3 replication competent HIV-1 reporter virus) that was used to identify productively infected cells by flow cytometry. At day 6 post infection, ruxolitinib and tofacitinib (0.1, 1.0, and 10 μM) significantly (p < 0.05) reduced extracellular p24 production (Fig 2C) as well as the frequency of HIV-GFP+ CD4+ cells (Fig 2D). Both inhibitors significantly (p < 0.01) decreased CCR5 surface expression on CD4 T cells that was upregulated as a result of anti-CD3/CD28 stimulation in viremic subjects (n = 5) whereas the levels of CXCR4 were not impacted by the addition of these inhibitors (S5 and S6 Figs). Since CXCR4 expression remained unchanged, decreased viral production and viral spread may be mediated through a mechanism beyond entry such as reduced T-cell activation (S16 Fig). As CCR5 dependent viral strains are mostly prevalent in vivo [42–44], these results indicate that inhibition of anti-CD3/28 induced CCR5 expression by ruxolitinib could have an impact on viral spread and dissemination in an infected untreated host [42, 43] and specifically in tissues from ART treated subjects where HIV replication still prevails in spite of the presence of ART [33, 45]. Since γ-C cytokines promote the activation of the Jak-STAT pathway (Fig 1) [8, 9, 11], paracrine or autocrine inhibition of IL-2, IL-7, and IL-15 induced signaling by ruxolitinib could be in part responsible for the inhibitory effects exerted by this compound on p24 production. Addition of an exogenous source of IL-7 (30 ng/ml) in ex vivo CD4 T cell cultures from viremic donors reversed the block by ruxolitinib at an antiviral EC50 concentration of 33 nM conferred on extracellular HIV production (p < 0.01), measured by p24 after 6 days in culture (S7 Fig; n = 4). More specifically, exogenous IL-7, which signals largely through STAT5, reverses latency, underscoring the link between IL-7, the ability to control latency, and STAT5-mediated signaling.

Jak inhibitors reduce the frequency of cells harboring integrated HIV viral DNA ex vivo and block IL-15 induced reactivation of latent HIV ex vivo We monitored the frequency of cells harboring integrated viral DNA in cultures of T cells obtained from viremic donors that were activated by T cell receptor (TCR) engagement. These experiments aimed at measuring the impact of ruxolitinib on the maintenance of the existing reservoir as cultures were generated in the presence of ART. Cultures were also performed in the absence of ART to measure the seeding of the viral reservoir, as under these conditions infected cells that can produce virions will infect new T cells. Ruxolitinib and tofacitinib significantly (p < 0.05) decreased the frequency of cells with integrated DNA in cultures of T cells activated by TCR in the presence or absence of ART (Fig 3A and 3B) with doses of ruxolitinib as low as 0.01 μM. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 3. Jak inhibitors reduce frequency of cells harboring integrated viral DNA and IL-15-induced reactivation of latent HIV-1 in CD4 T cells. 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 EC 99 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 versus 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). https://doi.org/10.1371/journal.ppat.1006740.g003 IL-15, which signals through STAT5, also regulates memory T cell homeostasis [17, 18, 21], and could be involved in modulating the HIV reservoir size by promoting the persistence of cells with integrated DNA or by enhancing HIV reactivation and dissemination. Since ruxolitinib and tofacitinib inhibited STAT5-mediated signaling triggered by IL-7 and IL-15 (Fig 1) we sought to define the impact of ruxolitinib on IL-15 induced reactivation of latent HIV in CD4 T cells from aviremic subjects (Fig 3C–3E). Memory CD4+ T cells were isolated from ART treated aviremic donors (n = 3), activated with a concentration of IL-15 that is known to activate Jak-STAT signaling (10 ng/ml) (Fig 3D) or CD3/CD28 (Fig 3E) and maintained with or without 1 μM ruxolitinib in the presence of ART. We showed in preliminary experiments that 10 ng/ml of IL-15 triggered optimal STAT5 phosphorylation and viral reactivation. Extracellular viral RNA copies were quantified by qRT-PCR six days post IL-15 activation of purified memory CD4 T cells. Indeed, IL-15 increased reactivation of latent HIV (200 to > 1,000-fold versus non stimulated controls), although to a lesser extent when compared to the CD3/28 control (> 30,000 fold versus non stimulated controls; Fig 3C–3E). However, ruxolitinib was found to significantly reduce (p < 0.01) IL-15 and CD3/28 induced reactivation of HIV from latently infected cells, resulting in values similar to unstimulated controls. Additionally, our results demonstrated that ruxolitinib and tofacitinib decreased anti-CD3/28 induced T cell proliferation (dilution of Cell Trace Violet; S8 and S9 Figs) and activation (CD25, CD38/HLA-DR and PD-1 expression; S8 Fig and S10–S12 Figs) and more importantly, led to a decrease in the frequency of p24+ cells (Fig 2) as well as a reduction in cells harboring integrated HIV provirus (Fig 3) and decreased IL-15 induced HIV reactivation (Fig 3). Each of these events mechanistically signals through STAT5, further highlighting the role of STAT5, and subsequent block by Jak inhibitors, in controlling these key events that drive viral persistence. Altogether these results indicate that Jak-STAT inhibitors can negatively impact de novo seeding and the maintenance of the HIV reservoir.

Ruxolitinib inhibits HIV bystander infection in human PBMCs The impact of ruxolitinib on the magnitude of the HIV reservoir could result from the inhibition of TCR triggered CD4 T cell activation and proliferation (S8–S12 Figs). It could also result from the direct antiviral activity of the Jak-STAT inhibitors (Fig 2A and 2B). Finally, decreased frequencies of cells harboring HIV integrated DNA could result from the diminished infection of bystander activated T cells due to the effects of these compounds on T cell activation and viral replication. We sought to determine if the presence of ruxolitinib could prevent the transfer of infectious viral particles to uninfected bystander cells or upon the formation of a virological synapse [44]. We developed an in vitro model to assess the impact of ruxolitinib on infection of bystander cells (schematic: Fig 4A; representative dot plots: Fig 4B) where bystander cells were labeled with cell trace violet (CTV+) and stimulated with CD3/28 in the presence of increasing concentrations of ruxolitinib to inhibit their activation. Unlabeled cells, (CTV-), were stimulated with CD3/28 in the absence of ruxolitinib, prior to infection with eGFP NL4-3. Infected CTV-negative cells and bystander CTV-positive cells were then co-cultured for two days to determine the number of ruxolitinib treated bystander cells susceptible to infection by eGFP virus (CTV+GFP+). As expected Ruxolitinib blocked proliferation (CTV-lo) of bystander cells (range 23% - 94%; p < 0.01 compared to no drug controls; paired t test) at all concentrations tested (Fig 4D). Cultures that did not include ruxolitinib led to the infection of fifty percent of bystander CTV+ cells (CTV+GFP+ cells) while addition of ruxolitinib led to a 75–80 percent reduction (p < 0.001) in CTV+GFP+ infected bystander cells. Our results show that ruxolitinib inhibition of Jak-STAT signaling impacts the susceptibility of uninfected cells to infection by HIV-1 at concentrations ≥ 0.01 μM (Fig 4B: representative dot plots, and Fig 4C: graphical representation) which are comparable to the ex vivo EC 50 of 0.007 μM for ruxolitinib in CD4 T cells from viremic donors and well below the EC 90 of 0.26 μM (Fig 2A). These findings suggest that Jak-STAT inhibitors could synergize with ART and decrease seeding of the HIV reservoir [34] in uninfected bystander cells. PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 4. Ruxolitinib inhibits bystander infection. 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). https://doi.org/10.1371/journal.ppat.1006740.g004

Ruxolitinib does not inhibit TCR downstream proximal signaling and function An HIV cure will involve the elimination of residual persistently infected cells by immune effector mechanisms that include HIV specific T cells. Therefore therapeutic strategies such as Jak-STAT inhibition should not have an impact on T cell effector functions. We monitored the impact of ruxolitinib on the early and late signaling events downstream of TCR activation (n = 3). Fig 5A evaluated the effect of ruxolitinib on phosphorylation of SLP76 and CD3 zeta, two early events of the T cell receptor-signaling cascade [16, 46]. Fig 5A shows that ruxolitinib did not significantly alter CD3 zeta and SLP76 phosphorylation at all physiological concentrations (steady-state plasma concentrations found in vivo for all doses of ruxolitinib) tested (0.01, 0.1, and 1.0 μM) (S13 Fig- gating strategy and Fig 5A). PPT PowerPoint slide

PowerPoint slide PNG larger image

larger image TIFF original image Download: Fig 5. Ruxolitinib does not inhibit normal TCR function and signaling that is independent of HIV-1 infection. (A) 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 versus 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) 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 versus DMSO treated cells (n = 3). Statistical significance for (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. (C) 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 versus DMSO treated cells. 0.0 μM represents the average of all assays completed using % DMSO equivalent to Jak inhibitor concentrations. Statistical significance for (C) determined by paired Wilcoxon rank sum test. https://doi.org/10.1371/journal.ppat.1006740.g005 We next monitored the impact of ruxolitinib on late TCR signaling events such as the capacity of CD4+ T cell to produce effector cytokines (TNF-α, IL-2 and IFN-γ) when stimulated with CD3/CD28 (Fig 5B and S14 Fig) Ruxolitinib concentrations of 0.01, 0.1 and 1.0 μM did not alter cytokine production of IL-2, TNF-α, or IFN-γ single positive cells or IL2+TNFα+IFNγ+ polyfunctional CD4+ and CD8+ T-cells, as quantified by intracellular flow cytometry (Fig 5B and S14 Fig). Decreased cytokine production was observed only at 10 μM, which is above the C max and steady-state concentrations of ruxolitinib found in vivo [31]. Similarly, upstream events of TCR signaling and production of TNF-α, IL-2 and IFN-γ were inhibited only upon addition of a supra-physiological concentration (10 μM) of ruxolitinib, [32]. When we monitored cytokine production after gag peptide stimulation of PBMCs from stably treated, HIV-infected subjects, statistically significant differences in gag-specific antigenic responses were observed only at 10 μM ruxolitinib for TNF-α and at 0.1 μM for TNFα+IFNγ+ in CD4+ cells (Fig 5C and S18 Fig). There were no significant differences in gag-specific responses in CD8+ T-cells at the different concentration of ruxolitinib, however only 3 of the 6 subjects tested responded to gag-peptide stimulation and two subjects demonstrated decreased cytokine production at ruxolitinib concentrations ≥ 0.1 μM (Fig 5C and S18 Fig). These results demonstrate that ruxolitinib does not inhibit initial TCR function in both CD4 and CD8 T-cells nor HIV specific responses in CD4 T-cells with some donor-specific responses in CD8 T-cells, highlighting the ability of ruxolitinib to specifically inhibit signal transduction pathways that alter T cell proliferation and expansion of the HIV reservoir without interfering with the development of effector antiviral T cell functions.