Coinhibitory receptors and CD8 T cell exhaustion in chronic ... : Current Opinion in HIV and AIDS (original) (raw)

INTRODUCTION

Although the key role of CD8 T cells in controlling HIV/simian immunodeficiency virus (SIV) infection is well established [1–5], ultimately HIV/SIV-specific CD8 T responses are not capable of clearing the infection. Several mechanisms have been proposed to explain this failure related either to infected cells (i.e., manipulation of the T cell receptor (TCR) machinery by Nef protein) [6,7] or to intrinsic defects of HIV/SIV-specific CD8 T cells [8]. Furthermore, it is well established that ‘exhaustion’ of HIV/SIV-specific CD8 T cell responses is an important mechanism used by the virus to evade the adaptive immune response in chronic HIV/SIV [9,10]. Emerging data suggest that exhaustion of virus-specific adaptive responses could play a crucial factor for viral persistence in several chronic infections or for the failure of antigen-specific cytotoxic T lymphocytes (CTLs) to abolish tumor cells [11]. Therefore, understanding the molecular mechanisms governing this biological process will shed light on the pathogenesis of chronic infections and will provide valuable information for the development of novel immunotherapies aiming to elicit or boost effective CTL responses against chronic viral infections and cancers.

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WHAT DO WE MEAN BY ‘EXHAUSTION’ OF CD8 T CELLS? LESSONS FROM MOUSE MODELS

Virus-specific CD8 T cell exhaustion in the context of chronic viral infections was first described in the mouse Lymphocytic choriomeningitis (LCMV) model by Moskophidis _et al._[12]. Early studies revealed that exhaustion was characterized by a spectrum of functional impairments, such as loss of cytokine production [13,14] and ultimately by the physical disappearance of virus-specific CD8 T cells [15–17]. Furthermore, exhausted virus-specific CD8 T cells exert a nonflexible differentiation status compared to nonexhausted cells [18]. Therefore, exhaustion represents a dynamic process, driven by high levels of persisting virus [19,20], in which virus-specific CD8 T cells become gradually incompetent to function, proliferate, and survive. Exhausted virus-specific CD8 T cells express a unique molecular signature, characterized by major differences in signaling, transcriptional factors, and metabolism, compared to nonexhausted cells [21]. Transcriptional analysis revealed that exhaustion results from an orchestrated function of centrally connected pathways [22] although functional exhaustion appeared to be a distinct process from anergy [21].

Several reports have established the critical role of a complex network of costimulatory/coinhibitory surface receptors as regulators of CD8 T cell biology in viral infections [23] and autoimmunity [24]. The coinhibitory receptor PD-1 was found to be a key receptor involved in the exhaustion of CD8 T cells in chronic viral [25] and parasitic [26] infections as well within the tumor environment [27]. Chronic antigen-specific stimulation of TCR induces a high, sustained expression of PD-1 on virus-specific CD8 T cells through mechanisms involving factors such as T-bet [28] and Blimp-1 [29,30▪▪], whereas soluble factors such as IFN-a [31] and RANTES [32] have also been shown to play a role in the regulation of PD-1 on antigen-specific CD8 T cells. PD-1-induced signaling, in turn, affects the function and proliferation of virus-specific CD8 T cells as well as their motility within the secondary lymphoid organs [33▪▪].

The majority of chronic virus-specific CD8 T cells in the LCMV model was found to coexpress PD-1 and Tim-3 [34], a phenotype marking more severe exhausted CD8 T cells. As a consequence, in-vivo manipulation of both PD-1 and Tim-3 was more effective in restoring exhausted virus-specific CD8 T cell responses [35]. These data indicate a nonredundant role of these two coinhibitory receptors as regulators of CD8 T cell exhaustion. In line with this, a recent landmark study by Blackburn _et al._[36] described the synergistic effect of several coexpressed coinhibitory receptors in the exhaustion of LCMV-specific CD8 T cells. Apart from PD-1, other receptors such as CD160, 2B4, and LAG-3 were also found to contribute to exhaustion of chronic virus-specific CD8 T cells [36]. Therefore, coexpression of several coinhibitory receptors marks virus-specific CD8 T cells which are highly exposed to negative signals for T cell activation leading to a ‘deep-exhaustion’ profile. Although the reason for the simultaneous expression of several coinhibitory receptors is not well understood, we could hypothesize that the existence of multiple coinhibitory receptors could be because of coregulation of many of these inhibitory molecules resulting in the termination of uncontrolled CD8 T cell responses [37].

The expression pattern of inhibitory receptors may also indicate a ‘compartmentalization’ of the exhaustion process with respect to anatomical site. The expression level (molecules per cell), an important factor underlying the preexhausted function of a coinhibitory receptor [38], as well as the function of particular inhibitory receptors could be affected by the local cytokine milieu within secondary lymphoid organs and inflamed peripheral tissues [39]. To this end, a comprehensive analysis of coinhibitory receptors and their ligands with respect to the anatomical localization of the virus-specific CD8 T cells could be very informative.

Despite the current paradigm, recent studies have suggested an alternate view of ‘exhaustion’. These studies suggest that exhaustion represents a unique differentiation status with effector function optimized to control viral replication and limit disease, rather than an inactive status of virus-specific CD8 T cells leading to their disappearance [40,41,42▪▪]. Given the high heterogeneity of the memory CD8 T cell pool in a chronic viral infection, associated with a continuous recruitment of ‘new’ responders [43], it is important to distinguish between an ‘activation’ and ‘exhausted’ status of particular virus-specific CD8 T cells expressing coinhibitory receptors. However, it is very difficult to distinguish activated effector T cells from ‘exhausted’ T cell simply on the basis of surface expression of coinhibitory receptors because these molecules that are expressed on ‘exhausted’ T cells are also expressed on activated T cells, albeit the levels may differ. Collectively, these studies point to the importance of manipulating more than one negative receptor in the context of novel immunotherapies aiming to enforce the CTL arm of the immune system.

EXHAUSTION OF HIV-SPECIFIC CD8 T CELLS

It is well accepted that the quality rather than the breadth of the HIV-specific CD8 T cell responses is impaired in chronic HIV [44]. Among the biological features considered in early studies, as mediators of CD8 T cells exhaustion in HIV was telomere length, a surrogate of replicative senescence [45]. Other studies have described a skewed differentiation [46,47] and impaired cytokine production [48] and survival [49] of HIV-specific CD8 T cells in chronic HIV. It is important to emphasize that these defects characterize selectively HIV-specific CD8 T cells compared with other virus specificities even from the same individuals [46,48,49]. This specific profile could provide a mechanistic explanation for the failure of HIV-specific CD8 T cells to clear HIV. Furthermore, differentiation, cytokine dysfunction, and compromised survival could represent different aspects of HIV-specific CD8 T cell ‘exhaustion’. Whether these different ‘exhaustion phenotypes’ characterizing chronic HIV-specific CD8 T cell responses are centrally orchestrated is not known.

More recently, a significant upregulation of PD-1 was reported selectively in HIV-specific CD8 T cells from viremic individuals [50–52]. This phenotype was associated with impaired cytokine production [50,52], proliferation, and survival of HIV-specific CD8 T cells [51]. In vivo, this exhausted profile was associated with increased turnover rates of PD-1high SIV-specific CD8 T cells [53]. In line with the data described in chronic LCMV [36], HIV-specific CD8 T cells coexpress several coinhibitory receptors even compared with cytomegalovirus-specific CD8 T cells from the very same individuals [54,55]. Furthermore, only the simultaneous expression of PD-1, CD160, and 2B4 on HIV-specific CD8 T cells was significantly correlated with their impaired poly-functionality and the viral load [55], pointing to a ‘deeply-exhausted’ status of HIV-specific CD8 T cells in advanced disease. In-vitro manipulation of more than one receptor had an additive effect on the proliferation of HIV-specific CD8 T cells, supporting a synergistic activity between the coinhibitory receptors [54,55] and arguing against a redundant role of different coexpressed coinhibitory receptors.

Given the critical role of PD-1 in regulating virus-specific CD8 T cell responses, it is of special interest to understand its own regulation during T cell activation. An association between the expression level of PD-1 and the differential regulation of T cell functions was recently reported [38]. Chronic antigen-specific stimulation of TCR is a requirement for the sustained, high expression of PD-1 on HIV-specific [56] and SIV-specific [57] CD8 T cells. In line with this observation, successful antiretroviral treatment reduced the expression of PD-1 on HIV-specific CD8 T cells [55,58]. Although the general immune activation and the function of certain cytokines could affect the expression of PD-1 on bulk CD8 T cells [10,59], this generalized stimuli is not sufficient to explain the selective upregulation of PD-1 in HIV-specific CD8 T cells compared with other virus-specific CD8 T cells in the same individuals [51,55]. At a molecular level, stimulation of T cells induces the recruitment of transcription factors such as Notch [60] and NFATc-1 [61] at the PD-1 promoter leading to upregulation of its transcription. Similar to chronic LCMV-specific CD8 T cells [62], the epigenetic regulation of the PD-1 promoter shapes the expression profile of PD-1 on HIV-specific CD8 T cells [63]. At the signaling level, cross-linking of PD-1 reduced the expression of Bcl-xl [64], an important antiapoptotic factor for activated T cells [65], and inhibited the cytokine production and proliferation of HIV-specific CD8 T cells through the induction of the basic leucine ATF-like transcription factor [66].

Tim-3, another member of the coinhibitory receptor family, was first described by Monney et al. in 2002 [67] in murine IFN-g-producing T cells. In-vivo studies in autoimmunity tolerance mouse models revealed a negative role for T cell activation [68,69]. Its suppressor activity can be mediated either directly by promoting cell death and exhaustion of CD8 T cells [68] or indirectly by inducing myeloid-derived suppressor cells [70]. Tim-3 and PD-1 cooperate for the induction of CD8 T cell exhaustion in chronic viral infections [34] and cancer [27,71,72]. Coexpression of Tim-3 and PD-1 marks virus-specific CD8 T cells capable of producing IL-10 [34]. HIV-specific CD8 T cells are characterized by increased expression of Tim-3 [73,74], a profile associated with impaired cytokine production and in-vitro proliferation [73]. Furthermore, in-vitro blocking of Tim-3 boosted the degranulation and cytotoxic capacity of HIV-specific CD8 T cells [73,75]. More recently, a direct role of Tim-3 in the stable synapse formation as well as the proximal TCR signaling [76▪▪] was shown, providing a mechanistic basis for the negative regulation of CD8 T cell function by Tim-3. Highly Active Anti-Retroviral Therapy (HAART) treatment significantly suppressed Tim-3 on HIV-specific CD8 T cells [74] indicating that, like PD-1, Tim-3 expression is dependent on the chronic antigen-specific TCR stimulation. Further phenotypic analysis of circulating CD8 T cells revealed that the combined expression of CD56 and Tim-3 can discriminate between individuals that naturally control HIV and individuals under HAART [77]. Although a synergistic activity between PD-1 and Tim-3 has been shown [34], analysis of Tim-3 and PD-1 in vertically infected young individuals revealed a selective dependence of Tim-3 expression on both HIV replication and aging [78]. These findings indicate that Tim-3 could be a novel target for immunotherapies in HIV and with respect to aging.

Chronic HIV-specific CD8 T cells are characterized by the expression of multiple coinhibitory receptors, including PD-1, Tim-3 as well as CD160 and 2B4 [55]. Use of CD160 can discriminate between ‘exhausted’ (PD-1highCD160high) and ‘activated’ (PD-1high) HIV-specific CD8 T cells [54]. It was found that only the coexpression of CD160 and PD-1 marks functionally impaired HIV-specific CD8 T cells compared with cells expressing only PD-1 [54]. A negative role of 2B4 as regulator of virus-specific CD8 T cell responses selectively in HIV-infected individuals was recently shown [79].

Apart from the complex network of costimulatory/coinhibitory receptors and its role in HIV-specific CD8 T cell exhaustion, a ‘cross-talk’ between this network and other immune-suppressor mechanisms, such as IL-10 and TGF-β, is highly possible. Recent data have shed light on this cooperation of negative signals with respect to CD4 T cell responses in HIV [80]. Do similar interactions apply to CD8 T cells too? To further add to the complexity of the ‘exhaustion’ process of HIV-specific CD8 T cells, a recent study revealed that this process could be mediated by an altered function of the mitogen activated protein kinase/extracellular signal regulated kinase pathway independently of the expression of PD-1 [81▪▪].

EXHAUSTION OF NON-HIV VIRUS-SPECIFIC CD8 T CELLS

Apart from HIV, exhaustion is a process characterizing virus-specific CD8 T cells in other chronic infections too. Although liver is a tolerogenic environment [82], strong hepatitis B virus (HBV)-specific CD8 T cell responses can be detected during chronic hepatitis B infection [83]. Hepatic virus-specific CD8 T cells express higher levels of PD-1 compared to their counterparts in circulation, a profile associated with a stronger effect of the in-vitro anti-PD-L1 treatment on the hepatic cells [84,85]. Interestingly, the exhaustion of circulating HBV-specific CD8 T cells was less apparent in young compared to adult individuals with chronic hepatitis [86], indicating a connection between aging and exhaustion of virus-specific CD8 T cells. In-vivo blocking of the PD-1/PD-L1 interaction restored the function of HBV-specific CD8 T cells [87,88▪], a treatment that could be complementary to other antiviral therapies [88▪]. A selective upregulation of Tim-3 on hepatic HBV compared to cytomegalovirus-specific CD8 T cells was associated with increased levels of its ligand galectin-9 in the liver [89]. In-vitro blocking experiments revealed a nonredundant role for PD-1 and Tim-3 in HBV-specific CD8 T cell exhaustion [89]. Similarly to chronic HBV, exhausted hepatic hepatitis C virus (HCV)-specific CD8 T cells were characterized by increased expression of PD-1 [90–92], Tim-3 [91], 2B4, and CD160 [90]. Like in HIV/SIV [56,57], the simultaneous expression of coinhibitory receptors on HCV-specific CD8 T cells was because of chronic antigen-specific stimulation [90] and was associated with skewed differentiation of these cells [90]. Surprisingly, hepatic HCV-specific CD8 T cells were found to express CTLA-4 in addition to PD-1 [92], and their in-vitro restoration required the inhibition of both CTLA-4 and PD-1 [92]. The breadth of the preexisting hepatic HCV-specific CD8 T cells determined their response to an in-vivo manipulation of the PD-1/PD-L1 interaction [93]. A comparison between hepatic and circulating HCV-specific CD8 T cells revealed that the liver environment plays a critical role for the exhaustion of these cells in chronic HCV [94▪]. Furthermore, HCV/HIV coinfection was characterized by increased frequency of exhausted CD8 T cells [95,96] and accelerated loss of ‘effector’ CD8 T cells expressing PD-1 and Tim-3 [96]. Overall, the chronic hepatitis studies have provided valuable information regarding the role of coinhibitory molecules in exhaustion of virus-specific CD8 T cells with respect to their tissue distribution. The expression of their ligands as well as the presence of other innate immunity cells [87] could play an important role in this process.

IMMUNOTHERAPIES TARGETING THE COINHIBITORY RECEPTORS

Accumulating data suggest that in-vivo manipulation of coinhibitory receptors could be a promising way to rejuvenate the exhausted antigen-specific CD8 T cell responses against viruses [97] as well as tumors [98]. Studies using nonhuman primate models of SIV infection have shown that the in-vivo manipulation of PD-1/PD-L1 interaction can restore the exhausted SIV-specific CD8 T cell responses [99,100] and extend the survival of infected animals [99]. Interestingly, the blocking of PD-1/PD-L1 interactions in the context of antiretroviral therapy interruption significantly suspended the viral load rebound [100,101]. In line with the SIV data, in-vivo treatment with an anti-PD-1 antibody significantly reduced the HIV viral load in a humanized mouse model [102]. However, the manipulation of coinhibitory receptors is expected to affect not only the antigen-specific CD8 T cells but also the CD4 T cell and B cell responses [100,101]. This is of particular interest for the design of novel strategies in which such interventions could be a promising tool for HIV eradication.

Using reagents targeting coinhibitory receptors carries a possibility for the development of uncontrolled autoimmune reactions. Given that the coinhibitory receptors, PD-1 and Tim-3, have a role in regulating autoimmunity [103], the possibility of releasing undesirable responses in individuals treated with antibodies against these molecules is a great concern. However, recent data from clinical trials testing anti-PD-1/anti-PD-L1 treatments in cancer patients [104,105▪▪,106] or individuals with chronic hepatitis C infection [107] have shown mild-to-modest adverse effects. Moreover, therapies that target Tim-3 would be predicted to elicit less autoimmune side-effects given that its expression on T cells is more restricted than that of PD-1.

Previous studies have indicated that more than one coinhibitory receptor should be targeted in the context of such therapies. This could be an important issue particularly for chronic infections in which the role of the ‘infected’ tissue could be a critical factor for the exhaustion of virus-specific responses [94▪] or aging could affect the outcome of these treatments [78,86]. Alternative approaches such as the combined manipulation of coinhibitory molecules with depletion of regulatory CD4 T cells (Tregs) [108] or cytokine coadministration [109] should be further explored.

CONCLUSION

Investigation of the diversity of the ‘exhausted’ pool of CD8 T cells and the intracellular signals underlying the development or progression to ‘exhausted’ phenotype is critical for our understanding of the pathogenesis of chronic infections like HIV and the development of successful therapeutic interventions for cancer.

Acknowledgements

Work in the author's laboratories is supported by the Intramural Research Program of the Vaccine Research Center, NIAID, National Institutes of Health, and CAVD grant #OPP1032325 from the Bill and Melinda Gates Foundation.

Conflicts of interest

There are no conflicts of interest.

Papers of particular interest, published within the annual period of review, have been highlighted as:

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This study provides a novel mechanistic basis for the negative regulation of TCR signaling by Tim-3.

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81▪▪. Chiu YL, Shan L, Huang H, et al. Sprouty-2 regulates HIV-specific T cell polyfunctionality. J Clin Invest 2014; 124:198–208.

This study describes an alternative, PD-1 independent, mechanism of HIV-specific CD8 T cell exhaustion.

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This study shows that the manipulation of ‘exhaustion’ receptors in the presence of other antiviral treatments could further increase the efficacy of the immune system to control virus.

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94▪. Kroy DC, Ciuffreda D, Cooperrider JH, et al. Liver environment and HCV replication affect human T-cell phenotype and expression of inhibitory receptors. Gastroenterology 2014; 146:550–561.

This study reveals the role of the local tissue environment in the expression of coinhibitory receptors on virus-specific CD8 T cells.

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103. Joller N, Peters A, Anderson AC, et al. Immune checkpoints in central nervous system autoimmunity. Immunol Rev 2012; 248:122–139.

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105▪▪. Hamid O, Robert C, Daud A, et al. Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 2013; 369:134–144.

This study describes the efficacy and the low-grade adverse effects of an anti-PD-1 antibody administration in patients with advanced melanoma.

106. Topalian SL, Hodi FS, Brahmer JR, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012; 366:2443–2454.

107. Gardiner D, Lalezari J, Lawitz E, et al. A randomized, double-blind, placebo-controlled assessment of BMS-936558, a fully human monoclonal antibody to programmed death-1 (PD-1), in patients with chronic hepatitis C virus infection. PLoS One 2013; 8:e63818.

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109. West EE, Jin HT, Rasheed AU, et al. PD-L1 blockade synergizes with IL-2 therapy in reinvigorating exhausted T cells. J Clin Invest 2013; 123:2604–2615.

Keywords:

CD160; CD8; exhaustion; HIV; PD-1; Tim-3

© 2014 Lippincott Williams & Wilkins, Inc.