Check point inhibitors as therapies for infectious diseases

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ScienceDirect Check point inhibitors as therapies for infectious diseases Maureen A Cox1, Robert Nechanitzky1 and Tak W Mak1,2 The recent successes of immune check point targeting therapies in treating cancer patients has driven a resurgence of interest in targeting these pathways in chronically infected patients. While still in early stages, basic and clinical data suggest that blockade of CTLA-4 and PD-1 can be beneficial in the treatment of chronic HIV, HBV, and HCV infection, as well as other chronic maladies. Furthermore, novel inhibitory receptors such as Tim-3, LAG-3, and TIGIT are the potential next wave of check points that can be manipulated for the treatment of chronic infection. Blockade of these pathways influences more than simply T cell responses, and may provide new therapeutic options for chronically infected patients. Addresses 1 The Campbell Family Institute for Breast Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, ON M5G 2M9, Canada 2 Department of Immunology, University of Toronto, Toronto, ON M5G 2C1, Canada Corresponding author: Mak, Tak W ([email protected])

Current Opinion in Immunology 2017, 48:61–67 This review comes from a themed issue on Host pathogens Edited by Marc Pellegrini and Elizabeth Hartland

massive; an estimated 700 000 people die each year due to both HCV and HBV related complications respectively, including cirrhosis and liver cancer. Due to the overwhelming number of patients affected by chronic viral infections as well as the inadequate tools available to treat infected patients, new therapeutic options need to be explored to deal with this global health crisis. When the immune system fails to eradicate an infection rapidly, numerous inhibitory pathways are initiated to curb the response and prevent immune-mediated tissue damage, a process termed exhaustion [3]. The best described of these inhibitory pathways are CTLA-4 and PD-1 mediated inhibition (Figure 1). Expression of these inhibitory receptors has been found on virus-specific cells from patients harboring chronic HIV, HBV, and HCV infections [3] and is associated with dysfunction of the T cell response and increased viral burden. Furthermore, blockade of PD-1 and CTLA-4 pathways in vitro restores the anti-viral capacity of exhausted T cells derived from chronically infected patients [3], and has been used successfully to rejuvenate immune responses to tumors in cancer patients [4]. Although these treatments are associated with inflammatory tissue injury, particularly in the liver and gastrointestinal tract [5], these immunotherapeutic approaches could be the next frontier in treating chronic viral infections.

http://dx.doi.org/10.1016/j.coi.2017.07.016 0952-7915/ã 2017 Published by Elsevier Ltd.

Introduction A substantial portion of the world population harbors at least one chronic infection which confers severe long term health consequences, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) [1]. Unfortunately few interventions are available to combat chronic infections. Nucleoside analogs in combination with other therapies reduce viral load in HIV and HBV infected patients, however they do not eradicate the infection, and must be continued for life. While the advent of direct-acting antiviral agents (DAA) has greatly enhanced the response rate in chronic HCVinfected patients, there are still numerous barriers to treatment, including the high cost of these drugs, as well as the risk of emergence of drug-resistant strains [2]. The global health burden of these chronic infections is www.sciencedirect.com

CTLA-4 Activation of T cells requires both T cell receptor (TCR) ligation and co-stimulation via CD28. The receptor CTLA-4 on T cells directly competes with CD28 and delivers an inhibitory signal, which is vital to prevent lethal autoimmunity; CTLA-4 deficient mice succumb to widespread autoimmunity with potent CD4 and CD8 T cell activation within 3–4 weeks of life [6]. Intriguingly, the activation of CD8 T cells that occurs following CTLA-4 loss appears to be CD4 T cell mediated, as these cells do not demonstrate any enhanced activation in the absence of CTLA-4 deficient CD4 T cells [7]. CTLA-4 is highly expressed on regulatory T cells (Treg) and is important for their survival and function, therefore this autoimmunity could be partially driven by defects in Tregs (Figure 1). In humans specific alleles of CTLA-4 are associated with enhanced activation of the immune system; one single nucleotide polymorphism (SNP) prevalent in humans is an adenosine (A) to guanine (G) substitution at residue +49 in exon 1 (+49G). This substitution is associated with a reduction in CTLA-4 mRNA [8], increased T cell activation [8–10], and an increased Current Opinion in Immunology 2017, 48:61–67

62 Host pathogens

Figure 1

Secondary Lymphoid Organs

Infected Tissues

Expanded effector T cells migrate to infected tissues CTLA-4 CTLA-4+ Tregs

T cell priming

B cell activation/survival

Expanded T cells express numerous inhibitory receptors, including PD-1 Tim3 CTLA-4+ LAG-3 Tregs CTLA-4 TIGIT

CTLA-4, PD-1, Tim3, LAG-3, TIGIT

Effector T cell function

NK cell anti-viral activity PD-1 PD-1,Tim3

Current Opinion in Immunology

Inhibitory receptors curb immune responses both during priming and in tissues. Priming of T cells in secondary lymphoid organs is inhibited directly by CTLA-4 expression on responding cells as well as indirectly by CTLA-4+ Treg cells. In secondary lymphoid tissue, PD-1 signaling can inhibit B cell survival during chronic infection. Upon activation, T cells upregulate numerous inhibitory receptors and migrate into infected tissues. In these tissues, engagement of these inhibitory receptors inhibits effector T cell and NK cell function, resulting in persistence of the pathogen. CTLA-4+ Treg cells can also inhibit effector T cell function in tissues.

incidence of multiple autoimmune disorders [10]. Interestingly, the +49G allele appears protective for development of chronic HBV, HCV [11,12], or cervical cancer development following human papilloma virus (HPV) infection [13], suggesting inhibition of CTLA-4 could facilitate control of these pathogens.

CTLA-4 in this limited number of patients was well tolerated with no patients requiring steroid therapy to deal with immune mediated adverse events, however further investigation will be required to determine the safety profile of CTLA-4 blockade in HBV and HCV infected patients.

Drugs which block CTLA-4 are already in use in human cancer patients [4], however use of these drugs in HCV or HBV infected patients has been limited due to concerns over further hepatic injury associated with CTLA-4 inhibition. To date, 8 case reports have been published evaluating treatment of either HBV or HCV infected melanoma patients with Ipilimumab (human a-CTLA-4) where viral titers were evaluated before and after treatment. Of these 8 patients, viral titers increased in 2 following treatment, while titers dropped in the other 6 patients [14]. Interestingly, the only 2 patients who did not develop progressive melanoma while on ipilimumab treatment, and therefore presumably responded to immunotherapy, experienced dramatic reductions in viral load [14]. A separate clinical trial of the human a-CTLA-4 antibody tremelimumab in hepatocellular carcinoma (HCC) patients infected with HCV demonstrated a drop in viral of greater than 10 fold in 10 out of 17 patients examined [15]. This drop in viral titers also correlated with emergence of new mutated variants of HCV, suggesting immune pressure on the virus [15]. Inhibition of

In HIV or simian immunodeficiency virus (SIV) infection CTLA-4 is expressed both by responding and infected T cells. High expression of CTLA-4 on CD4+ T cells correlates with worse disease outcome and elevated T cell exhaustion in infected individuals [16], therefore CTLA-4 blockade should theoretically improve anti-viral immunity in these patients. Conversely, CTLA-4 is highly expressed on infected T cells, along with multiple other inhibitory receptors [17], therefore blocking CTLA-4 in HIV infected patients could also increase viral replication by activating infected T cells and inducing their proliferation. This appears to be true in early infection, as blockade of CTLA-4 during acute SIV infection augmented viral replication in mucosal sites and drove an increase in expression of immunosuppressive factors, resulting in compromised anti-SIV immunity and poor response to therapeutic vaccination [18]. In contrast, treatment later during infection (90 weeks post-infection) was beneficial, reducing both viral load in lymph nodes and expression of immunomodulatory factors [19]. It is possible that inhibition of CTLA-4 at this time point,

Current Opinion in Immunology 2017, 48:61–67

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Check point blockade and chronic infection Cox, Nechanitzky and Mak 63

Table 1 Inhibitory receptor expression on numerous immune cell types. Expression and function of the inhibitory receptors PD-1, CTLA-4, Tim-3, LAG-3, and TIGIT on CD4+ and CD8+ T cells as well as B cells and NK cells PD-1

CTLA-4

Tim3

LAG-3

TIGIT

CD4+ T cells Activated and exhausted cells [21] Tregs T follicular helper cells [44–46]

Expressed upon activation [6,7] Competes with CD28 signaling Critical for T-reg function [7]

Mediates deletion of activated cells [33] Enhances control of tuberculosis [34]

Expressed on a subset of Expressed on Treg and CD25-Treg cells [37] memory cells, expression increased with activation

CD8+ T cells Activated and exhausted cells Inhibits function [21]

Expressed on activation, but not as highly as in CD4+ [6,7]

Co-expressed with PD-1 in HIV, HCV, and HBV Mediates deletion of activated cells [33] Enhances control of tuberculosis [34]

Impairs function in exhausted murine CD8 T cells [28] and anti-tumor T cells [37]

Co-expressed with PD-1 in HIV [35] Correlates with progression

B cells

Naı¨ve and resting Expression on murine memory B cells B cells activated in a Downregulated in T-dependent manner germinal center Inhibits activation [21]

Not expressed

Expression on murine B cells activated in a Tdependent manner [37]

Not expressed

NK cells

Expressed on a subset of mature NK cells in HCMV infected individuals, is associated with loss of function [49]

Expressed on most mature NK cells in humans Enhances IFNg [51] Can inhibit cytotoxicity [52]

Marker of NK cell maturation [37] Suppresses cytotoxicity

Expressed on mature NK cells in humans, inhibits cytotoxicity [53]

Expressed on IL-2 expanded murine NK cells

and subsequent reactivation of infected CD4+ T cells results in elimination of the latent viral reservoir by allowing these cells to cycle and then die. In one HIV infected patient treated with ipilimumab for metastatic melanoma, there was an increase in memory and effector CD4 T cells, however there was also a 19.6 fold increase in unspliced viral RNA following treatment [20], suggesting an increase in active viral replication. However, the plasma HIV RNA decreased during this time frame [20], for unknown reasons. The authors suggest that this decrease in plasma HIV RNA may be due to elimination of latently infected cells re-activated by CTLA-4 inhibition, however they could detect no change in cell-associated HIV DNA. Further investigation is necessary to determine whether a-CTLA-4 treatment boosts viral replication and infection in HIV-positive patients, or enhances anti-HIV immunity to control the infection and eliminate latently infected cells (Table 1).

PD-1 PD-1 expression is induced on multiple immune cell types following infection, including T cells, B cells, natural killer (NK) cells, and NK T cells. Upon engaging either of its ligands PD-L1 or PD-L2, PD-1 dampens TCR or B cell receptor (BCR) signaling, inhibits proliferation, and suppresses cytokine production [21]. Human studies evaluating the capacity of checkpoint inhibitors to resuscitate exhausted T cells have been conducted primarily on peripheral blood mononuclear cells (PBMC), however this does not accurately reflect the populations www.sciencedirect.com

present in the infected tissues. In contrast to PBMCs, liver-derived CD8 T cells isolated from chronically infected HCV patients co-express CTLA-4 and PD-1, and inhibition of either of these pathways alone is insufficient to induce anti-viral activity in vitro [22]. Blockade of both pathways, however, restored the functionality of liver-derived exhausted cells [22]. This may in part explain the underwhelming response of HCV infected patients to Nivolumab, a human a-PD-1 antibody. In the 20 patients treated with the highest dose (10 mg/kg), 3 had a substantial reduction in viral load of greater than 4 logs [23]. It is possible that co-blockade of PD-1 and CTLA-4 would increase the fraction of patients that responded to checkpoint blockade. Blockade of CTLA-4, and the subsequent expansion of the anti-viral response, could also synergize with PD-1 blockade independently of co-inhibition in effector cells. In a chronic HCV model in chimpanzees, only 1 of 3 study subjects responded to a-PD-1 treatment with a reduction in viral load, and this individual had the largest and most diverse anti-HCV response prior to therapy [24], suggesting a-PD-1 efficacy in HCV treatment may be limited by the robustness of the pre-existing response. Currently patients are being recruited to the clinical trial NTC01658878, which will evaluate the response of HBV and HCV infected hepatocellular carcinoma patients to combination a-PD-1 and a-CTLA-4 therapy. This study should reveal whether co-blockade of these pathways can bolster anti-viral immunity in addition to anti-tumor responses. Current Opinion in Immunology 2017, 48:61–67

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Inhibition of PD-1 signaling largely appears protective in animal models of HIV infection. In a humanized mouse model, PD-1 blockade reduced viral titers and bolstered CD8 T cell numbers in vivo [25]. In SIV infected macaques, blockade of PD-1 increased the number of anti-viral T cells in the blood and gut, as well as increasing the expression of effector molecules and cytokines [19,26]. In addition to bolstering T cell responses, treatment with anti-PD-1 also improved the B cell response in these infected macaques, both in the expansion of memory B cells and an increased a-HIV antibody titer. These effects of anti-PD-1 treatment were observed irrespective of whether treatment was initiated early (10 weeks postSIV infection) or late (90 weeks post-SIV infection), although the pro-immune effects were more impressive in the late treatment group [19]. Significantly, SIV titers were also reduced following a-PD-1 treatment, despite high levels of PD-1 on infected CD4+ T cells, indicating PD-1 blockade does not facilitate viral replication in infected PD-1high cells. Due to the proven ability of PD-1 blockade to improve immune responses to tumors, and its potential to incite better immunity to HIV, two clinical trials have been proposed to evaluate the impact of PD-1 blockade in patients with HIV-associated malignancies, both alone (NCT02595866) and in combination with a-CTLA-4 (NCT02408861).

Beyond CTLA-4 and PD-1: next generation inhibitory pathways While PD-1 and CTLA-4 blockade has made the most clinical progress to date, there are redundant inhibitory pathways induced during chronic infections, and evidence of compensation by these alternate pathways during immunotherapy [27]. In addition to PD-1 and CTLA-4, T cells responding to a persistent infection can express the inhibitory receptors Tim-3, LAG3, and TIGIT (Figure 1). Co-expression of inhibitory receptors is additive in restricting effector activity; the greater the level of co-expression, the more profound the exhaustion in mice [28] and human T cells [29–32]. Interaction of Tim-3 and its ligand Galectin-9 (Gal-9) results in deletion of effector CD4+ and CD8+ T cells in autoimmune or cancer models [33], respectively, and could mediate deletion of exhausted T cells that occurs with chronic infection. The inhibitory functions of Tim-3 are inhibited by the expression of human leukocyte antigen B associated transcript 3 (Bat3), and loss of Bat3 is also associated with enhanced exhaustion [33]. Interestingly, Tim-3 signaling to Gal-9 on macrophages is critical for their ability to control Mycobacterium tuberculosis (Mtb) infection, a clinically relevant co-morbidity for HIV patients. As expected, blockade of Tim-3 impaired the ability of T cells from healthy donors to facilitate macrophage killing of Mtb in vitro, however blockade of either Tim-3 or Gal-9 improved Mtb killing when the donor T cells were derived from HIV+ patients [34], suggesting blockade of Tim-3 Current Opinion in Immunology 2017, 48:61–67

in HIV+ individuals would actually reduce their susceptibility to Mtb infection. It will be important to determine whether this is also true for patients harboring other chronic infections, such as HBV and HCV, prior to initiation of Tim-3 targeted therapies in these patients. TIGIT expression on T cells in HIV infection correlates with progressive disease and marks profoundly exhausted cells [35]. Chew et al. found that the frequency of TIGIT+ CD4+ T cells correlated with the HIV DNA content in CD4+ cells [35], which could be due to the fact that TIGIT+PD-1+LAG3+ CD4+ T cells are enriched for HIV-infected cells [17]. This raises a conundrum, where inhibition of these checkpoints in patients could enhance viral replication in the infected CD4+ T cells. Further study will be required to determine the impact of checkpoint blockade in the context of HIV infection. The majority of the TIGIT+ CD8+ T cells in HIV patients coexpress PD-1, which further inhibits the functionality of the cells [35]. Significantly, blockade of TIGIT and PDL1 together synergized to restore T cell proliferation and IFNg production in vitro [35]. In a mouse model of chronic infection, co-blockade of PD-1 and TIGIT in vivo restored T cell responses to much greater degree that either treatment alone [36]. The mechanism by which TIGIT inhibits T cell function is still poorly described; however, there appear to be both T cell-intrinsic and extrinsic roles for TIGIT in modulating immunity. TIGIT can engage with CD155 on dendritic cells to shift their cytokine production away from the inflammatory IL-12 towards the immunomodulatory IL-10. Further, TIGIT+ Treg cells appear uniquely capable of suppressing antiviral Th1 type responses, as opposed to Th2 responses. In tumors and animal models of chronic infection, LAG3 contributes to CD8 T cell dysfunction [28,37], however the findings in chronically infected human patients are mixed. In HCV infected patients LAG3 is induced on liver-derived T cells as compared to healthy controls, and knock down of LAG3 in these exhausted cells restored some function [38]. Similarly, LAG3 expression correlates with CD8 T cell dysfunction in HBV-infected patients [39], however LAG3 does not appear to be a dominant mediator of T cell exhaustion in HIV infection [40].

Checkpoint inhibitors and non-T cells Checkpoint inhibitors target pathways known to repress T cells, however T cells act in concert with numerous immune cell populations, including B cells and NK cells, to combat infection and control chronic pathogens. B cells are heavily involved in repressing viral titers in chronic infections, and are vital in development of sterilizing immunity to vaccination or acute infection. In human patients harboring chronic viral infections, temporary depletion of B cells using the CD20 targeting chemotherapeutic Rituximab results in resurgence of virus, including previously controlled infections, such HCV, HBV, www.sciencedirect.com

Check point blockade and chronic infection Cox, Nechanitzky and Mak 65

varicella zoster virus (VZV), cytomegalovirus (CMV), and parvovirus. In HIV, depletion of B cells using Rituximab in one patient resulted in an increase in viral titer that directly correlated with the decrease in B cell numbers and serum antibody levels [41]. Further, infection with HIV depletes CD27+ memory B cell populations, while elite controllers maintain a greater number of memory B cells [42]. In SIV infected macaques, the depletion of memory B cells is strongly associated with rapid progression to AIDS [43], and this loss appears related to PD-1 expression; blockade of PD-1 enhanced survival of these memory B cells and increased the time to progression in treated animals, and the activated memory B cells that were selectively deleted during early SIV infection expressed PD-1 [43]. Therefore, it is possible that PD-1 blockade itself is protecting these memory B cells from deletion during SIV, and potentially HIV infection (Figure 1). Alternatively, PD-1 is also highly expressed on T follicular helper cells (Tfh), a specialized subset of CD4+ T cells that promote B cell germinal responses [44]. During persistent infection, CD4 T cells predominantly adopt a PD-1hi CXCR5+ ‘Tfh-like’ phenotype in both mouse models [45] and in HIV infected patients [44]. These accumulated Tfh cells in HIV are less functional, and this is thought to be mediated by PD-1/PD-L1 interactions in the germinal center [44,46]. In vitro blockade of PD-L1 in HIV+ patient-derived PBMCs enhanced the proliferation and ability of exhausted CD4 T cells to produce numerous cytokines, including the hallmark Tfh cytokine IL-21 [47]. Furthermore, in vitro blockade of PD-1 enhanced anti-HIV antibody production in patient derived PBMC [46,48], suggesting the blockade of PD-1 enhances the humoral response to HIV, potentially by allowing Tfh populations to better engage antiviral B cells. The effects of checkpoint inhibitors has been largely attributed to restoration of exhausted T cell responses, however innate immune cells, including NK and NKT cells, are underappreciated actors in combating viral infection, and are also functionally impaired by expression of inhibitory receptors (Figure 1). In NK cells, PD-1 expression is exclusively found on mature CD56dim CD57+ cells, and these cells exhibit poor cytokine production and cytolytic activity compared to PD-1 mature NK cells derived from the same donor [49]. These defects are particularly striking when target cells expressed PD-L1 or PD-L2, and can be partially reversed by antibody blockade of PD-1 [49]. Tim-3 regulation of NK cell function is more complicated; Tim-3 is expressed on the preponderance of circulating NK cells in humans [50], and interaction with its ligand Gal9 can facilitate activation and IFNg production from these cells. Downregulation of Tim-3 on NK cells is thought to contribute to their dysfunction in HIV-1 infection [51], however Tim-3 is upregulated in chronic HBV patients, and blockade of Tim-3 in vitro improved the cytolytic response of NK www.sciencedirect.com

cells derived from HBV infected patients [52]. TIGIT is also expressed on the majority of mature NK cells in humans, and interferes with immune activation in several ways which have been reviewed recently [53]. Briefly, TIGIT expression on NK cells inhibits cytotoxicity, potentially by disrupting polarization of cytotoxic granules, and TIGIT may inhibit IFNg production by NK cells; overexpression in mouse NK cells inhibited IFNg production, while TIGIT-deficient NK cells demonstrated enhanced IFNg production when stimulated with target cells. Finally, TIGIT signaling to dendritic cells polarized these populations to produce more immunomodulatory IL-10, and less inflammatory IL-12p40. Furthermore, invariant NKT cell populations express LAG3 in HIV-infected individuals, which restricts their capacity to produce IFNg [40]. While checkpoint blockade clearly has an impact on exhausted T cell populations, it will be interesting to determine how much of the response to immunotherapy is driven by restoration of NK cell responses versus the T cells.

Discussion Development of new therapeutics to treat chronic infections is vital due to the overwhelming numbers of patients affected and the severe long-term health impact of these persistent infections. Immunotherapy to revitalize exhausted responses has the potential to drastically change how we treat chronic infections and dramatically improve patient outcomes. Care will need to be taken to evaluate unexpected side effects, however, such as resurgence of virus in HIV infected patients due to proliferation of infected T cells.

Conflict of interest The authors have no conflict of interest to declare.

Acknowledgements This work was supported by Canadian Institutes of Health Research funding to T.W.M.

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