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Histone Deacetylase Inhibitors Impair the Elimination of HIV-Infected Cells by Cytotoxic T-Lymphocytes......HDACis exert negative effects - new study reports
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we observed that each of the HDACis tested exhibited significant impairment of the abilities of CTL clones to eliminate HIV-infected target cells. These results indicate that HDACis can exert negative effects on the CTL that may be needed for flush-and-kill approaches to eradication......HDACis suppress the ability of CTL to kill HIV-infected cells. This interaction has the potential to limit the effectiveness of combining CTL with HDACis in flush and kill approaches to HIV eradication, and should be considered in the prioritization and optimization of potential curative strategies.......We observed that romidepsin, panobinostat, and SAHA all rapidly suppressed IFN-γ production from virus-specific CD8+ T-cells
http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004287
Richard Brad Jones1,2, Rachel O'Connor1, Stefanie Mueller1,2, Maria Foley2,3, Gregory L. Szeto2,3, Dan Karel1, Mathias Lichterfeld4, Colin Kovacs5,6, Mario A. Ostrowski5,6,7, Alicja Trocha1, Darrell J. Irvine1,2,3,8, Bruce D. Walker1,4,8*
1 The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Boston, Massachusetts, United States of America,
2 Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 3 Department of Biological
Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America, 4 Massachusetts General Hospital, Boston, Massachusetts,
United States of America, 5 The Maple Leaf Medical Clinic, Toronto, Ontario, Canada, 6 Department of Medicine, University of Toronto, Toronto, Ontario, Canada, 7 Li Ka
Shing Medical Institute, St. Michael's Hospital, Toronto, Ontario, Canada, 8 Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America
"These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia."
"Here, we uncover an unexpected negative interaction between these two agents, whereby HDACis suppress the ability of CTL to kill HIV-infected cells. This interaction has the potential to limit the effectiveness of combining CTL with HDACis in flush and kill approaches to HIV eradication, and should be considered in the prioritization and optimization of potential curative strategies."
"It was initially thought that the reactivation of latent HIV by HDACis would be sufficient to eliminate infected cells through viral cytopathic effects. Recent data, showing that in vitro treatment of patient PBMCs with 500 nM SAHA failed to lead to a reduction in inducible viral reservoirs, suggests that this may not be the case, and that immune effectors, such as HIV-specific cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, or immunotoxins will likely be needed to recognize and eliminate these exposed target cells in so called 'flush-and-kill' strategies [13]. Notably, in this same study, CD8+ T-cells freshly isolated from ART-treated HIV-infected patients could eliminate infected cells in a primary cell model of latency only if pre-stimulated with peptides and IL-2 ex vivo [13], highlighting that, in the case of CTL-based flush-and-kill strategies, the functional state of virus-specific CD8+ T-cells will be important for reservoir elimination."
"Of principal importance, we observed that each of the HDACis tested exhibited significant impairment of the abilities of CTL clones to eliminate HIV-infected target cells. These results indicate that HDACis can exert negative effects on the CTL that may be needed for flush-and-kill approaches to eradication."
"losses in viability of activated CTL, as we observed in vitro with romidepsin and, to a lesser extent, panobinostat, could result in an irreversible impairment of virus-specific cellular immune responses, particularly if HIV-specific T-cell responses are primed first, for example by therapeutic vaccination. Thus, our data suggest that the potential for a given HDACi to impair CTL function both in the first few hours of treatment when viability is intact, and over longer time-lines by the gradual induction of cell death should be considered in designing flush-and-kill approaches."
"The HIV-specific cellular immune response, in addition to playing a vital role in the ongoing health of HIV-infected individuals, stands to make a powerful contribution to HIV eradication strategies. Thus we would make a case that a critical stage in evaluating potential latency reversing drugs to be used in CTL-based flush-and-kill strategies should be to test the effects of these drugs on the functions of HIV-specific T-cells. This information can both be used to help prioritize classes of candidates, and to select optimal drug candidates from within a class. We would also suggest that this should be extended to flush-and-kill strategies focused on other immune effectors. Notably, romidepsin has also been reported to both suppress the cytotoxic activity and to drive apoptosis of NK cells, which are another potential key immune effector in flush and kill strategies [24]. Information regarding how a drug influences CTL and other immune effectors should be generated along with other parameters such as EC50 and toxicity in order to prioritize candidates. Through screening efforts, or rational design based on an improved understanding of the mechanisms by which HDACis exert their immunosuppressive effects, it may also be possible to identify HDACis that retain the ability to induce the expression of latent HIV without impairing the ability of CTL or other immune effectors to eradicate these unmasked targets."
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Histone Deacetylase Inhibitors Impair the Elimination of HIV-Infected Cells by Cytotoxic T-Lymphocytes
Abstract
Resting memory CD4+ T-cells harboring latent HIV proviruses represent a critical barrier to viral eradication. Histone deacetylase inhibitors (HDACis), such as suberanilohydroxamic acid (SAHA), romidepsin, and panobinostat have been shown to induce HIV expression in these resting cells. Recently, it has been demonstrated that the low levels of viral gene expression induced by a candidate HDACi may be insufficient to cause the death of infected cells by viral cytopathic effects, necessitating their elimination by immune effectors, such as cytotoxic T-lymphocytes (CTL). Here, we study the impact of three HDACis in clinical development on T-cell effector functions. We report two modes of HDACi-induced functional impairment: i) the rapid suppression of cytokine production from viable T-cells induced by all three HDACis ii) the selective death of activated T-cells occurring at later time-points following transient exposures to romidepsin or, to a lesser extent, panobinostat. As a net result of these factors, HDACis impaired CTL-mediated IFN-γ production, as well as the elimination of HIV-infected or peptide-pulsed target cells, both in liquid culture and in collagen matrices. Romidepsin exerted greater inhibition of antiviral function than SAHA or panobinostat over the dose ranges tested. These data suggest that treatment with HDACis to mobilize the latent reservoir could have unintended negative impacts on the effector functions of CTL. This could influence the effectiveness of HDACi-based eradication strategies, by impairing elimination of infected cells, and is a critical consideration for trials where therapeutic interruptions are being contemplated, given the importance of CTL in containing rebound viremia.
Author Summary
The advent of antiretroviral therapy has greatly improved the prognosis for HIV-infected individuals with access to care. However, current therapies are unable to cure infection, committing treated individuals to a lifetime of medication with significant economic burden. Furthermore, it has become clear that antiretroviral therapy does not completely restore health, leaving treated HIV-infected individuals at increased risk of cardiovascular disease, neurological disorders, and other health issues. Thus, there is a need to develop therapies capable of curing HIV infection. It is thought that, to be successful, curative strategies will need to combine a means to flush the virus out of the latently-infected cells in which it hides, with a means to kill these unmasked targets. A front-running approach proposes to use a class of drugs called histone deacetylase inhibitors (HDACis) as flushing agents, with cytotoxic T-lymphocytes (CTL, or killer T-cells) to purge viral reservoirs. Here, we uncover an unexpected negative interaction between these two agents, whereby HDACis suppress the ability of CTL to kill HIV-infected cells. This interaction has the potential to limit the effectiveness of combining CTL with HDACis in flush and kill approaches to HIV eradication, and should be considered in the prioritization and optimization of potential curative strategies.
Introduction
Antiretroviral therapy (ART) is capable of durably suppressing viremia in HIV-infected subjects, but is unable to cure infection. The financial and psychological burden of lifelong therapy, as well as a growing appreciation for co-morbidities that occur in HIV-infected individuals on long-term therapy, such as cardiovascular disease and neurocognitive disorders, have led to the prioritization of HIV cure research [1], [2]. The best understood, and perhaps most obstinate, barrier to eradicating infection is the existence of a pool of infected resting memory CD4+ T-cells [3]-[5]. By virtue of their quiescent state, these cells are not thought to express HIV antigens, rendering them invisible to the immune system. These cells are very long-lived, with an estimated half-life of 44 months, suggesting that 60 years of uninterrupted ART would be required for full decay of the reservoir [6]. As the reservoir almost certainly replenishes itself through ongoing rounds of re-infection and homeostatic proliferation, it is unlikely that current ART regimens could cure an individual within a lifetime [7], [8].
Such theoretical and experimental analyses have led to the consensus that the eradication of HIV from an infected individual will require a means for actively depleting the resting CD4+ T-cell reservoir, most likely to be achieved by inducing viral expression that could trigger immune-mediated clearance of infected cells. While a variety of compounds have been shown to reactivate virus from CD4+ T-cells, a class of drugs known as histone deacetylase inhibitors (HDACis) has emerged as the front-runner and a number of these, including vorinostat (suberoylanilide hydroxamic acid or SAHA), romidepsin, and panobinostat, have entered into HIV clinical trials aimed at testing their abilities to reduce or eradicate viral reservoirs in the context of ART [9]-[11] (reviewed in [12]).
It was initially thought that the reactivation of latent HIV by HDACis would be sufficient to eliminate infected cells through viral cytopathic effects. Recent data, showing that in vitro treatment of patient PBMCs with 500 nM SAHA failed to lead to a reduction in inducible viral reservoirs, suggests that this may not be the case, and that immune effectors, such as HIV-specific cytotoxic T-lymphocytes (CTL), natural killer (NK) cells, or immunotoxins will likely be needed to recognize and eliminate these exposed target cells in so called 'flush-and-kill' strategies [13]. Notably, in this same study, CD8+ T-cells freshly isolated from ART-treated HIV-infected patients could eliminate infected cells in a primary cell model of latency only if pre-stimulated with peptides and IL-2 ex vivo [13], highlighting that, in the case of CTL-based flush-and-kill strategies, the functional state of virus-specific CD8+ T-cells will be important for reservoir elimination.
In evaluating strategies predicated upon coordinating CTL with latency-reversing drugs it is critical to consider potential side effects of these drugs on CTL function. This is particularly true in the case of HDACis, which are known to exert potent and diverse effects on both the innate and adaptive immune system (reviewed in [14] and [15]). A number of HDACis, including SAHA, have been shown to suppress the production of inflammatory cytokines by both T-cells and innate immune cells, in vitro and in vivo [16]-[21]. SAHA, romidepsin, and other HDACis have also been shown to interfere with the differentiation of monocytes into dendritic cells (DCs), as well as to block the ability of DCs to upregulate CD1a, CD80, CD83 and other co-stimulatory molecules, resulting in impaired priming of T-cells [22]-[26]. These immunosuppressive activities of HDACis have been associated with therapeutic benefits in murine models of graft-versus-host disease (GVHD) and autoimmune/inflammatory disorders such as autoimmune lymphoproliferative syndrome, experimental autoimmune encephalomyelitis (EAE, a model of multiple sclerosis), and diabetes mellitus [27]-[32].
While it is tempting, based on this body of evidence, to generally characterize HDACis as immunosuppressive agents, this would not be an accurate assessment. The HDACi panobinostat (LBH589), which is also in clinical trials for HIV eradication (CLEAR trial, ClinicalTrials.gov Identifier NCT00256139), has been reported to enhance T-cell activation in vivo, resulting in elevated serum Th1 cytokines and accelerated progression in a murine model of graft-versus-host disease [33]. Even within an individual, the effects of a given HDACi have been reported to be divergent depending upon the particular facet of the immune response being studied. For example, in a murine model of allogeneic bone marrow transplantation it has been shown that SAHA decreased levels of serum cytokines and GVHD while having no effect on donor T-cell proliferation or killing of host cells [20]. In a second example, co-administration of the HDACi MS-275 with a viral-vectored vaccine served to suppress the immune response to the vector, while enhancing the response to the viral vector insert and suppressing autoimmune pathology [34].
This diversity of effects of HDACis on immune cells is likely rooted in two sources. First, there are 18 different HDAC enzymes in humans, divided into four different families. Different HDACi drugs interact with different subsets of these enzymes, depending upon the dose being used [35]. Second, in addition to altering histone acetylation status, and thus chromatin structure, HDACis can interact with multiple transcription factors, including NF-κB, AP-1, and others either by interfering with co-repressor HDAC enzymes that are recruited to transcription factor binding sites resulting in enhanced transcription, or by directly blocking deacetylation of the transcription factor itself [36]-[40]. Foxp3, for example, requires acetylation of lysine residues for maximal activation. HDACis, by preventing deacetylation of Foxp3, enhance its activity and thus boost the numbers and function of Tregs in vivo [41]. Other nonhistone proteins that serve as direct HDAC substrates include p53, GATA-1, STAT3, and STAT5 [42], [43]. This high degree of complexity, both in terms of immunological outcomes and underlying mechanisms, necessitates that HDACis be studied in a context that is matched to their intended utility. Thus, there is a need to understand the impact of HDACis being taken forwards in flush-and-kill eradication strategies on the abilities of HIV-specific CTL to eliminate infected target cells.
Here, we report a series of experiments designed to assess the effects of the HDACis currently being tested in HIV eradication clinical trials on the in vitro function of virus-specific T-cells. We show that, while the HDACis tested did not exhibit detectable toxicity to ex vivo bulk CD8+ T-cells over the doses and time-courses tested, romidepsin and, to a lesser extent, panobinostat exhibited delay toxicity to activated CD8+ T-cells and to CTL clones. Each of the HDACis tested rapidly suppressed the production of IFN-γ from PMA/ionomycin by ex vivo stimulated CD8+ and CD4+ T-cells at an early time-point not associated with losses in viability. The production of IFN-γ in response to peptides representing viral epitopes was rapidly and durably suppressed by treatment of both CTL clones and ex vivo CD8+ T-cells with HDACis. Treatment with romidepsin abrogated the proliferation of HIV-Gag- and CMV-pp65-specific CD8+ and CD4+ T-cells, while panobinostat and SAHA only significantly impaired proliferation of CMV-pp65-specific CD8+T-cell responses. Of principal importance, we observed that each of the HDACis tested exhibited significant impairment of the abilities of CTL clones to eliminate HIV-infected target cells. These results indicate that HDACis can exert negative effects on the CTL that may be needed for flush-and-kill approaches to eradication.
Discussion
In this study we explored the potential for HDACi latency-reversing drugs to impact upon multiple functions of virus-specific CTL. We observed that romidepsin, panobinostat, and SAHA all rapidly suppressed IFN-γ production from virus-specific CD8+ T-cells. The proliferation of both CD8+ and CD4+ T-cells in response to either CMV-pp65 or HIV-Gag peptide pools was abrogated by treatment with romidepsin, while the effects of SAHA and panobinostat on proliferation were only significant for the CD8+ T-cell response to CMV-pp65. Critically, each of these HDACis also impaired the ability of HIV-specific CTL to eliminate infected CD4+ and peptide pulsed BLCL as measured in liquid culture and collagen matrices respectively.
The overall data support the idea that there are mechanisms both involving and independent of losses in T-cell viability at play, and that these make differential contributions depending on the functional assay being tested, drug concentrations, exposure/rest times, and the type of effector cell (ex vivo T-cells, or in vitro expanded CTL clone). The data presented in Figs. 1 and 2 clearly demonstrate that romidepsin and, to a lesser extent, panobinostat are disproportionately toxic to activated T-cells as compared to their resting counterparts. However, data presented in Fig. 3 demonstrate that all 3 HDACis suppress IFN-γ production from viable T-cells. In the case of romidepsin, our data support the idea that these two modes of suppression are linked, with early suppression of functionality followed by later onset of apoptosis. However, in the case of SAHA, functional suppression appears to occur in the absence of any impact on cell viability. Treatment with panobinostat appears to be intermediate between these two situations, depending also upon the exposure concentration.
We propose that the net effects of HDACi treatment on HIV-specific T-cells in the majority of our functional assays involved contributions from both of these modes of suppression, particularly in the case of romidepsin. For example, the impaired abilities of romidepsin-treated CTL to eliminate HIV-infected target cells over 16 hour co-cultures likely resulted both from rapid suppression of CTL function (CTL are capable of killing target cells within 2-10 minutes [46]-[48]) as well as apoptosis at later time-points post-exposure. This is supported by the observed exacerbation of this impairment following a 14 hour romidepsin wash-out period (Fig. 7). This is also directly visualized in Fig. 8A, and Supporting Movie S3. Here, following romidepsin treatment, apoptotic CTL are visible, but a CTL also remains viable (excludes sytox dye) throughout the 5:34 (h:mm) imaging, while failing to kill a target cell that it engages from 1:03 to 2:26. These two modes of HDACi-induced CTL impairment - inhibition of function in viable T-cells and reduction in T-cell viability (with the former leading to the latter in some cases) - have the potential to have different impacts in a therapeutic setting. It may be possible to mitigate the impact of transient CTL impairment on flush-and-kill eradication strategies by designing dosing schedules that target a temporal therapeutic window, whereby either HIV antigen expression occurs before CTL are substantially impaired, or persists while CTL are given time to functionally recover. A better understanding of the kinetics and durability of HIV antigen presentation from reactivated latently-infected cells is required to evaluate the plausibility of this approach. On the other hand, losses in viability of activated CTL, as we observed in vitro with romidepsin and, to a lesser extent, panobinostat, could result in an irreversible impairment of virus-specific cellular immune responses, particularly if HIV-specific T-cell responses are primed first, for example by therapeutic vaccination. Thus, our data suggest that the potential for a given HDACi to impair CTL function both in the first few hours of treatment when viability is intact, and over longer time-lines by the gradual induction of cell death should be considered in designing flush-and-kill approaches.
Our findings are limited to ex vivo and in vitro experiments, and the extent to which HDACis impact CTL function in HIV-infected patients is presently unknown. On one hand, the clearance of drugs in vivo may serve to mitigate some of the effects of HDACis on HIV-specific CTL, although we attempted to account for this by washing out drug in most assays. On the other hand, our in vitro experiments incorporated only single doses of HDACis whereas the SAHA and panobinostat clinical trials have incorporated repeated dosing. This could potentially exacerbate any effects, particularly if these drugs cause lasting changes to the T-cell functional profile, or compromise the survival of T-cells. In the CLEAR trial, for example, patients received panobinostat on days 1, 3, and 5 every other week for 8 weeks. An additional challenge to predicting whether a therapeutic window between latency reversal and CTL inhibition exists is that infected cell elimination assays, which utilized highly functional CTL clones, at relatively high effector:target ratios in most experiments, and activated target cells expressing high levels of HIV-Gag, can be interpreted as an idealized environment for CTL killing of target cells. Thus, it is reasonable to speculate that even a subtle difference in killing efficiency observed in our in vitro assays may manifest as a critical difference in vivo where CTL are likely to be exhausted or otherwise functionally impaired, and where CTL encounters with reactivated target cells are rare. Following from this, while we did not observe significant impairment of infected cell killing at 1 nM or 5 nM of panobinostat, we cannot rule out that more subtle impairments in function may lead to reductions in abilities of CTL to eliminate exposed natural reservoir cells in vivo. Based on the magnitudes of the effects observed in vitro, along with consideration of the pharmacokinetic and pharmacodynamic properties of these drugs, at the dosing regimens being taken forwards into HIV clinical trials we propose that HDACis are differentially likely to impact upon relevant T-cell functions in vivo, with the following hierarchy: romidepsin>panobinostat>SAHA. Ultimately, however, we hope that the primary outcome of our study will be to motivate the incorporation of assays measuring ex vivo T-cell function into ongoing and planned HDACi clinical trials, and that immunosuppression will be considered as a potential factor limiting the effectiveness of any observed outcomes.
We must also highlight, in a more general sense, the potential risk of treating HIV-infected subjects, whose immune systems do not fully recover even with ART, with HDACis. In addition to the ex vivo and in vitro data presented in the current study, a number of studies have observed in vivo immunosuppressive activities of HDACis, including romidepsin and SAHA [16]-[20]. While opportunistic infections have not been reported in HIV-infected subjects receiving SAHA and panobinostat, this is a greater concern for romidepsin. We observed greater impairment of CTL function, and toxicity with romidepsin treatment as compared to panobinostat and SAHA at the doses tested. Lymphopenia is also known to be a common side-effect of romidepsin treatment, and serious and sometimes fatal infections, including pneumonia and sepsis, have been reported in clinical trials in oncology settings [49]. Notably though, these side-effects were observed at higher doses than those planned for HIV eradication trials. The incorporation of immunological end-points as important safety parameters in the early stages of testing romidepsin in HIV-infected subjects would help to address concerns regarding a potential impact on the immune system in general. The possibility that romidepsin or other HDACis could impair HIV-specific T-cell responses in vivo should also be ruled out before any therapy interruptions are contemplated as part of eradication trials, given the importance of CTL in containing any rebound viremia.
The HIV-specific cellular immune response, in addition to playing a vital role in the ongoing health of HIV-infected individuals, stands to make a powerful contribution to HIV eradication strategies. Thus we would make a case that a critical stage in evaluating potential latency reversing drugs to be used in CTL-based flush-and-kill strategies should be to test the effects of these drugs on the functions of HIV-specific T-cells. This information can both be used to help prioritize classes of candidates, and to select optimal drug candidates from within a class. We would also suggest that this should be extended to flush-and-kill strategies focused on other immune effectors. Notably, romidepsin has also been reported to both suppress the cytotoxic activity and to drive apoptosis of NK cells, which are another potential key immune effector in flush and kill strategies [24]. Information regarding how a drug influences CTL and other immune effectors should be generated along with other parameters such as EC50 and toxicity in order to prioritize candidates. Through screening efforts, or rational design based on an improved understanding of the mechanisms by which HDACis exert their immunosuppressive effects, it may also be possible to identify HDACis that retain the ability to induce the expression of latent HIV without impairing the ability of CTL or other immune effectors to eradicate these unmasked targets.
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