Innate and Adaptive Immune Regulation During Chronic Viral Infections

Abstract

Chronic viral infections represent a unique challenge to the infected host. Persistently replicating viruses outcompete or subvert the initial antiviral response, allowing the establishment of chronic infections that result in continuous stimulation of both the innate and adaptive immune compartments. This causes a profound reprogramming of the host immune system, including attenuation and persistent low levels of type I interferons, progressive loss (or exhaustion) of CD8⁺ T cell functions, and specialization of CD4⁺ T cells to produce interleukin-21 and promote antibody-mediated immunity and immune regulation. Epigenetic, transcriptional, posttranscriptional, and metabolic changes underlie this adaptation or recalibration of immune cells to the emerging new environment in order to strike an often imperfect balance between the host and the infectious pathogen. In this review we discuss the common immunological hallmarks observed across a range of different persistently replicating viruses and host species, the underlying molecular mechanisms, and the biological and clinical implications.

INTRODUCTION

Chronic viral infections are a significant global health burden. For example, human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) are the causative agents of acquired immune deficiency syndrome (AIDS), which results in an estimated 1.5 million deaths worldwide annually. Hepatitis C virus (HCV) and hepatitis B virus (HBV) are major causes of viral hepatitis. Moreover, HCV is the major risk factor for the development of hepatocellular carcinoma, the fifth most common cancer in the world.

Viruses that cause chronic infections have evolved a range of distinct mechanisms that allow them to be retained within their host. Endogenous retroviruses, for instance, exist in the genome of every member of a host species, are present for the lifetime of the host, and are transmitted vertically from parent to offspring (1). Other strategies utilized by viruses during chronic infections include viral latency and persistent active replication. Herpes simplex virus 1 and 2 (HSV-1 and HSV-2, which cause oral and genital herpes, respectively), Epstein-Barr virus (EBV, which causes mononucleosis and a number of lymphomas), and human and mouse cytomegaloviruses are all examples of latent viruses. Initial infection with these viruses is associated with acute symptoms before the virus enters a period of latency with sporadic reactivation. During these quiescent periods, the viruses enter a transcriptionally silent state in which new infectious virions are not generated, allowing the virus to avoid immune surveillance (reviewed in 2). In contrast, other viruses—such as HIV-1, HCV, and HBV in humans; simian immunodeficiency virus (SIV) in primates; and certain variants of lymphocytic choriomeningitis virus (LCMV) in mice [e.g., Clone 13 (Cl13) and Docile]—display the capacity to continuously replicate and package new viral particles despite the presence of an antiviral immune response. Viral integration, latency, and persistent replication are all effective strategies to establish chronic infection (and indeed some viruses, such as HIV-1, utilize more than one strategy at a time).

Notably, the presence of persistent viral products, a common feature of infections with continuously replicating viruses, causes drastic and sustained alterations in the host immune system. This often results in altered susceptibility to secondary infections, cancers, and inflammatory disorders. Thus, at the risk of neglecting the unique effects of individual viruses on their respective hosts, in this review we discuss selected examples of the overlapping immune hallmarks found in infections with persistently replicating viruses, including LCMV infection in mice; SIV infection in primates; and HIV, HBV, and HCV infections in humans.

INNATE IMMUNITY: DYNAMICS AND OPPOSING EFFECTS OF TYPE I INTERFERONS DURING CHRONIC VIRAL INFECTION

The host innate immune response is a highly potent barrier of defense comprising many innate immune cells, such as dendritic cells (DCs), macrophages, and natural killer (NK) cells. These cells are capable of recognizing a wide range of viral products through a complex and overlapping molecular network of pattern-recognition receptors (PRRs), including Toll-like receptors (TLRs) as well as cytosolic sensors such as the helicases retinoic acid–inducible gene 1 (RIG-I) and MDA5 (3). Activation of innate cells through these PRRs promotes rapid viral sensing and the release of key antiviral cytokines with the potential to rapidly restrain viral replication and drive further innate and adaptive immune responses (3). In particular, the type I interferons (IFN-I) are a pleiotropic cytokine family consisting of thirteen IFNα isoforms and one IFNβ isoform that signal through a shared ubiquitously expressed receptor (IFNαβR) (4). Signaling through IFNαβR leads to both autocrine and paracrine activation, resulting in phosphorylation of signal transducer and activator of transcription 1 and 2 (STAT1 and STAT2). This induces hundreds of IFN-I-stimulated genes (ISGs) that promote an antiviral state and activate multiple immune cells (4). Despite this sophisticated innate response, many viruses, including those that achieve chronic infection, are able to suppress and/or evade these early innate host responses to favor their own replication and transmission. In this section, we focus on IFN-I to illustrate the dynamic changes that the innate immune system undergoes and the significant and broad consequences that such changes can have during chronic viral infections.

Type I Interferon Induction by Persisting Viruses

Within days after HIV detection in plasma from infected patients, and within hours after infection with LCMV Cl13 in mice, elevated levels of systemic IFN-I are detected (5, 6). Furthermore, enhanced ISG expression is evident in tissues and/or T cells after LCMV Cl13, HCV, and SIV infections (Figure 1) (7–10). In contrast, genes with innate immune function are undetectable early after in vivo HBV infection in chimpanzees (11).

Figure 1.

Type I interferon (IFN-I) dynamics and opposing effects during chronic viral infections. Upon infection with most persistent viruses, IFN-I are powerfully and systemically induced; this initial response is rapidly attenuated, although low IFN-I and IFN-I-stimulated gene (ISG) levels still persist in multiple cells and tissues. Several innate cells, including macrophages, conventional dendritic cells (cDCs), and plasmacytoid dendritic cells (pDCs) (and possibly nonhematopoietic cells, which are not depicted), sense viral products via pattern-recognition receptors (PRRs) to contribute to IFN-I levels. Although they are the most powerful IFN-I producers early after challenge with persistent viruses, pDCs show an impaired capacity to produce IFN-I during chronic stages of lymphocytic choriomeningitis virus (LCMV), human immunodeficiency virus (HIV), hepatitis C virus (HCV), and hepatitis B virus (HBV) infections. IFN-I exert both beneficial and detrimental effects on immune responses and viral control, which appear conserved among different persistent infections. The relative degree and impact on viral replication of such opposing IFN-I effects may vary for individual cases.

Several nonhematopoietic and hematopoietic cells, including macrophages, conventional DCs (cDCs), and plasmacytoid DCs (pDCs), can contribute to the initially robust IFN-I response by recognizing and responding to viral infection through PRR-mediated signaling pathways. For example, in LCMV Cl13 infection, cDCs and macrophages produce IFN-I through MDA5/MAVS-dependent pathways (8, 12–14). Although the cellular source and the specific type of IFN (e.g., IFN-I and/or IFN-III) that induces elevated ISGs in the liver upon HCV infection are still unclear, studies indicate that hepatocytes are capable of producing IFN-I via RIG-I signaling in response to HCV infection (15). Finally, monocyte-derived DCs can sense HIV-2 (and HIV-1 when its replication is not restricted by the host enzyme SAMHD1) via cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS) pathways, inducing proinflammatory cytokines and IFN-I production (reviewed in 16).

Despite the fact that almost all cell types can produce IFN-I upon viral encounter, pDCs are highly specialized for secreting these cytokines, producing up to 1,000 times more IFN-I than any other blood cell type and synthesizing a broader range of IFN-I isoforms upon TLR stimulation (17). pDCs are able to directly recognize several persistent viruses, including HIV and HCV in humans and LCMV in mice, through TLR7 (18–20). pDCs have been shown to be essential for T cell responses and viral control during chronic LCMV infection (21), although another study using a different LCMV variant and an inducible pDC deletion system observed only minimal effects (14). Notably, in contrast to IFN-I production in most cell types, pDC IFN-I production does not require viral replication, and the majority of DC IFN-I comes from uninfected pDCs early after LCMV Cl13 infection (19). Similarly, studies with HCV found that uninfected pDCs are able to sense neighboring infected hepatocytes by recognizing HCV RNA–containing exosomes (20, 22), and neither viral fusion nor replication is required for pDC recognition of HIV (16).

In conclusion, these studies indicate that the majority of persistently replicating viruses can be sensed by innate cells via PRRs, resulting in a robust IFN-I response in the early stages of in vivo chronic infection.

Rapid Attenuation and Persistence of Type I Interferon Responses

Following the initial IFN-I response after infection, systemic IFN-I becomes undetectable at later stages, despite maintained viral loads (Figure 1) (5, 6). This is accompanied by a profound reduction in pDC IFN-I production capacity during LCMV, HIV, and HCV infections that often coincides with altered innate responses to secondary, unrelated pathogens (6, 18, 23, 24). However, it is important to note that in LCMV, HCV, HIV, and SIV infections, IFN-I or ISG transcripts are persistently detected in total DCs, CD4⁺ and CD8⁺ T cells, and/or tissues, albeit at lower amounts than those detected during the peak IFN-I response (7, 10, 12, 25, 26). Several mechanisms may contribute to the attenuation of IFN-I in the postacute stages of chronic viral infections, including (a) direct suppression of innate pathways by viral products and (b) host immunomodulatory mechanisms. Examples of direct mechanisms of IFN-I inhibition include HCV NS3–4A protein inhibition of TRIF (the TLR3 adaptor), RIG-I, and MAVS viral sensing pathways upstream of IFN production (27–29) as well as LCMV nucleoprotein (NP) blockade of IRF3 activation (30). Similarly, the HIV proteins Vpr and Vif induce IRF3 degradation (31), and direct HIV gp120 interaction with pDCs inhibits TLR9 (but not TLR7) responses, including pDC activation and IFNα secretion (32). The HIV capsid also interacts with selected host cofactors to evade PRR sensing in macrophages (33). The aforementioned undetectable levels of innate genes early after in vivo HBV infection (11) may be partly explained by HBV polymerase interference with IRF3 phosphorylation and/or by inhibition of MAVS signaling by the viral HBx protein (34–37). Moreover, in line with the reduced TLR2 expression in hepatocytes, monocytes, and Kupffer cells (liver-resident macrophages) from chronic HBV patients, HBV soluble antigen can reduce expression of TLR2 in hepatic cell lines (38). Similarly, HBV soluble antigen also suppresses TLR9 (but not TLR7) signaling in pDCs, and TLR9 expression is reduced in blood pDCs from chronically infected patients (39, 40). In addition to the direct IFN-I inhibitory activities of viral proteins, viruses that establish chronic infections also promote host immunomodulatory responses that may cause aberrant functioning of innate cells. For example, during chronic HCV infection, pDC IFN-I production capacity is impaired, at least in part, by monocyte-derived tumor necrosis factor α (TNFα) and interleukin (IL)-10 (24).

It should be noted that not all aspects of innate responses are attenuated during chronic viral infections; indeed, select pathways are enhanced. For instance, whereas LCMV Cl13–infected mice respond poorly to TLR9 ligand (CpG) challenge, they produce abnormally high IFN-I levels upon TLR4 ligand (LPS) stimulation, leading to rapid death (6). In line with these observations, chronic HCV infection also promotes hyperresponsive macrophages, including Kupffer cells, contributing to the chronic liver inflammation that is characteristic of chronic HCV infection (41, 42). Finally, macrophages treated with HIV and antigen-presenting cells isolated from HIV-infected patients show hyperresponsiveness to unrelated TLR ligands and commensal bacteria, respectively (43, 44).

Together, these studies highlight that although innate immune cells are capable of a robust IFN-I response in the early stages of most chronic viral infections, blockade of innate pathways by viral proteins together with host immunomodulatory molecules leads to a profound (albeit incomplete) attenuation of IFN-I at later stages of infection.

Beneficial and Deleterious Type I Interferon Effects on Antiviral Immunity

IFN-I antiviral effects are mediated by induction of several ISGs that function to impair viral replication through multiple mechanisms [e.g., protein translation inhibition, viral RNA degradation (reviewed in 4)]. IFN-I also orchestrate both innate and adaptive immune cells such as DCs, macrophages, NK cells, and T cells following viral infections, providing activation and survival signals (4, 45). Consistently, studies using persistent LCMV infection, in which the virus is cleared from the blood and most tissues by 2–3 months postinfection, demonstrate that mice lacking IFNαβR establish lifelong viremia (46). Furthermore, Sandler et al. (47) demonstrated that treatment of rhesus macaques with IFNαβR neutralizing antibody during acute SIV infection resulted in enhanced CD4⁺ T cell depletion, decreased antiviral gene expression, reduced proportions of cytotoxic NK cells, and increased SIV reservoirs, all correlating with faster progression to simian AIDS. In addition, treatment with recombinant IFN-I can often boost the host defense and enhance viral control. Wang et al. (14) showed that treatment with recombinant IFNα5 and IFNβ between days 2 and 5 after LCMV Cl13 infection (perhaps equivalent to delaying the IFN-I attenuation described above) resulted in early viral containment accompanied by enhanced virus-specific CD8⁺ T cell responses, although the same treatment in the chronic phase of infection had no effect. Similarly, prophylactic treatment with pegylated IFNα2a (pIFNα) prior to SIV mucosal challenge led to increased resistance to infection mediated in part by an increase in CD56⁺ NK cells; however, continuous pIFNα treatment resulted in an IFN-I desensitization state with reduced ISG levels and increased cell-associated viral loads (47). Notably, pIFNα also enhances expression of several ISGs known to have direct anti-HIV activity (reviewed in 48). However, treatment with pIFNα in HIV-infected patients has yielded mixed results; some studies have shown improved viral control as a result of increased ISG levels, whereas other studies have reported only modest or null effects (49–52). Finally, current therapeutic strategies for HCV infection include pIFNα administration, resulting in a success rate that varies from 40% to 80% depending on time of treatment, HCV genotype, basal IFN-I signature, and polymorphisms associated with the IFN-III cytokines (reviewed in 53).

In addition to the beneficial roles of IFN-I, several studies in HCV and HIV/SIV demonstrate that persisting IFN-I and ISGs correlate with poor disease prognosis (25, 54, 55). Moreover, comparative studies of SIV-infected sooty mangabeys and African green monkeys (which remain healthy despite high viral replication) versus rhesus macaques (which progress to simian AIDS) have shown that a reduced IFN-I signature in both tissues and T cells correlates with better clinical outcome (7, 9, 56). Further studies have also demonstrated that sustained IFN-I signatures correlate with reduced viral control in chronic stages of HIV infection (57, 58). Recently, two reports using LCMV Cl13–infected mice demonstrated that treatment with neutralizing antibody to IFNαβR prior to or during infection led to accelerated viral control despite inducing early enhancement in viremia (likely due to the attenuation of the IFN-I-mediated antiviral effects described above). This transient blockade of IFNαβR resulted in reduced expression of IL-10 and PDL1 (both of which contribute to CD8⁺ T cell exhaustion, as described below) as well as improved splenic architecture and virus-specific CD4⁺ T cell responses (10, 59). Previous studies have shown that IFN-I can also impair the development of cDCs in both LCMV and HIV (60, 61) and can mediate HIV-induced apoptosis of CD4⁺ T cells (62). These deleterious IFN-I effects may contribute to the improvement in immune responses upon IFNαβR blockade.

In conclusion, whereas IFN-I have easily demonstrable effects in controlling replication of persistent viruses in many different chronic infections, they can also limit antiviral immune responses and viral control. Although the relative contributions of positive and negative IFN-I effects may vary depending on the nature of the viral infection, gaining a better understanding of how IFN-I production and silencing are regulated in the context of persistent viral replication may allow manipulation of endogenous IFN-I levels and selected cellular sources to improve viral containment.

CD8⁺ T CELLS: KEY FEATURES AND DRIVERS OF FUNCTIONAL EXHAUSTION DURING CHRONIC VIRAL INFECTION

CD8⁺ T cells are specialized in the elimination of intracellular pathogens. Their cytotoxic function includes the secretion of cytolytic proteins such as granzyme B and perforin and of cytokines such as IFNγ and TNFα. Consistently, CD8⁺ T cell cytolytic responses are required for direct control of persistently replicating viruses such as LCMV in mice and SIV or HBV in macaques (63–67). Furthermore, robust CD8⁺ T cell responses correlate with disease protection in HIV-, HCV-, and HBV-infected patients (68–71), and human leukocyte antigen class I haplotypes correlate with disease protection in HIV (72). In addition, vigorous CD8⁺ T cell responses can rapidly drive viral escape mutations in both persistent LCMV and HIV infections (73, 74). However, CD8⁺ T cell functions are typically inferior during chronic viral infections in comparison with the potent effector and memory T cells generated during acute infections. This state of T cell hyporesponsiveness has been termed exhaustion and is common to numerous chronic viral infections in mice and humans as well as cancer (75). In this section we give an overview of the general features of CD8⁺ T cell exhaustion during chronic viral infection and highlight the most recent advances in understanding the underlying transcriptional, epigenetic, and metabolic mechanisms as well as potential therapies designed to restore CD8⁺ T cell functions (Figure 2).

Figure 2.

CD4⁺ and CD8⁺ T cell adaptation during chronic viral infections. Persistent antigen drives recalibration of T cell responsiveness by inducing transcriptional (e.g., reduced TBET and increased EOMES), epigenetic (e.g., DNA methylation of the PD1 gene locus), and metabolic (e.g., reduced glycolysis and spare respiratory capacity) changes that result in limited functional properties [e.g., CD8⁺ T cell cytotoxicity, T helper 1 (Th1) cytokine production] and/or functional specialization [e.g., IL-21 elevation and T follicular helper (T_FH) cell differentiation]. Recent studies have begun uncovering the heterogeneity in the CD4⁺ and CD8⁺ T cell compartment during chronic viral infection. Importantly, although they are partially hyporesponsive, CD4⁺ and CD8⁺ T cells are essential to control persistently replicating viruses in mice and humans.

Cardinal Features of Exhausted CD8⁺ T Cells

Exhaustion in CD8⁺ T cells from LCMV Cl13–infected mice was first characterized as a progressive loss of antiviral functions, including the antigen-specific secretion of IL-2, followed by secretion of TNFα, and finally by IFNγ secretion upon cognate peptide recognition (75–77). Although exhausted T cells retain expression of the cytolytic molecules granzyme A and B, their cytotoxic capacity is progressively reduced (77–79). Exhausted CD8⁺ T cells also fail to differentiate into potent memory cells (80, 81), although a memory-like CD8⁺ T cell precursor during chronic LCMV infection has recently been proposed (82).

The cellular phenotype of exhaustion is characterized by the surface expression of inhibitory receptors—most prominently PD1, but also TIM-3, LAG3, 2B4, and CD160—which directly contribute to CD8⁺ T cell dysfunction in several chronic viral infections (83–89). Exhaustion also encompasses a reduced response to cytokines including IL-7 (81, 90), IL-2 (91), and IL-15 (92) that normally promote proliferation and survival in effector and/or memory CD8⁺ T cells. In addition, CD8⁺ T cell responsiveness to IL-12, IL-18, and IL-21 has recently been shown to be reduced during chronic LCMV infection (93). In contrast, virus-specific CD8⁺ T cells show enhanced production of the suppressive cytokines IL-10 and/or TGFβ in LCMV-, HCV-, and HIV-infected hosts (94–98).

Importantly, chronic antigen stimulation maintains the exhausted state of CD8⁺ T cells in LCMV Cl13 infection (80, 92, 99); the amount of antigen correlates with the degree of exhaustion (100, 101). Similarly, levels of exhaustion mirror viremia in HIV- and HCV-infected patients (102–104). Consistently, CD8⁺ T cells specific for LCMV nucleoprotein (the most abundant viral protein) are more susceptible to deletion, an extreme form of exhaustion (77). This is similar to the loss of high-avidity T cells that occurs early after HIV infection (105).

Thus, a combination of diminished effector functions, expression of inhibitory receptors, and cytokine hyporesponsiveness characterizes a unique exhausted state in CD8⁺ T cells during chronic viral infections.

Transcriptional Regulation of Exhausted CD8⁺ T Cells

CD8⁺ T cell exhaustion has also been defined at the transcriptional level (106, 107). It is now well established that CD8⁺ T cell exhaustion is transcriptionally distinct from other forms of T cell hyporesponsiveness, such as senescence and anergy as well as memory, even in the presence of continuous antigen stimulation (106, 108, 109). Importantly, these findings in mice translate to human chronic infection, as global gene patterns in CD8⁺ T cells of HIV-positive elite controllers and progressors were found to resemble CD8⁺ T cells from mice infected with acute and persistent LCMV variants, respectively (110).

Exhausted CD8⁺ T cells are characterized by many alterations in transcription factors that normally drive CD8⁺ T cell cytolytic function, including reduced T-BET expression and NFATc1 nuclear translocation and upregulation of EOMES, BLIMP1, and BATF (66, 111–115). Reduction in T-BET expression correlates with CD8⁺ T cell dysfunction during chronic HIV, HCV, HBV, and LCMV infections (114–118), and its presence is necessary to limit the expression of multiple inhibitory receptors as well as to retain partial CD8⁺ T cell functionality during chronic LCMV infection (114). In contrast, the T-BET homologous T-box transcription factor EOMES promotes CD8⁺ T cell exhaustion. However, EOMES also mediates the antigen-induced conversion (and proliferation) of T-BET^hiEOMES^loPD1^int CD8⁺ T cells into T-BET^loEOMES^hiPD1^hi terminally differentiated cells and is critical to maintaining a pool of partially effective CD8⁺ T cells during chronic LCMV infection (66). Notably, the aforementioned low T-BET and high EOMES expression is predominant in CD8⁺ T cells from chronic (versus resolved) HCV infection (66), and HIV-specific CD8⁺ T cells maintain this phenotype for years after initiation of antiretroviral therapy (115).

BLIMP1, a transcriptional repressor, is known to promote CD8⁺ T cell terminal differentiation and effector functions (112). However, high levels of BLIMP1 promote expression of inhibitory molecules, including PD1 and likely IL-10 in CD8⁺ T cells from LCMV Cl13–infected mice (112, 119). Interestingly, the reduced amounts of BLIMP1 found in hemizygous mice appear sufficient to promote partial effector functions but insufficient to maximize inhibitory receptor expression in exhausted CD8⁺ T cells. Such mice exhibit enhanced control of LCMV Cl13 (112). Furthermore, monomeric NFAT transcription factors (devoid of AP-1 binding) directly induce BLIMP1 and inhibitory receptors in exhausted CD8⁺ T cells (120). In addition, while suppressing CD8⁺ T cell responses by attenuating T cell receptor (TCR) signaling (121) and decreasing cell motility (122), PD1 also induces BATF transcriptional activity, which decreases proliferation as well as IFNγ and IL-2 production in CD8⁺ T cells from HIV-infected patients (111).

In conclusion, the state of CD8⁺ T cell exhaustion does not depend on a master transcriptional regulator but appears to be sustained by a unique cohort of transcription factors that regulate different genes during acute versus chronic viral infection (66, 106, 123).

Metabolic Changes in Exhausted CD8⁺ T Cells

There has been increasing interest in the metabolic state of exhausted CD8⁺ T cells, and recent microarray analysis suggested a metabolic adaptation conserved among different persistent viral infections, including HIV and LCMV (124, 125). LCMV-specific CD8⁺ T cells exhibit decreased PI3K-AKT-mTOR signaling upon antigen stimulation, likely due to desensitization of TCR signaling by PD1 (121), which underlies defects in both glycolysis and spare respiratory capacity (123). Reduced glycolysis may increase the availability of glycolytic enzymes, such as GAPDH. When not engaged in glycolysis, this dual-functional enzyme diminishes translation of IFNγ by binding to its mRNA 3′ untranslated region, which may explain the posttranscriptional reduction in IFNγ levels previously described in exhausted CD8⁺ T cells (126, 127). Decreased AKT activity also causes reduced phosphorylation (and enhanced nuclear retention) of the transcription factor FOXO1, which represses antiviral molecules such as granzyme B and T-BET while inducing expression of PD1, EOMES, and the survival molecule BCL2 (123). Of note, FOXO3, which is also inactivated downstream of mTOR signaling, promotes apoptosis (128), thereby supporting a contrasting role for FOXO1 and FOXO3 in exhausted CD8⁺ T cells.

In addition, mTOR signaling uses hypoxia-inducible factors (HIFs) to drive glycolysis-related genes (129). During chronic LCMV infection, enforcement of HIF accumulation by deleting the von Hippel–Lindau (VHL) tumor suppressor (a negative regulator of HIFs) promotes glycolysis and the expression of effector molecules (e.g., granzyme B), which leads to pathology and death in VHL-null mice (130). mTOR signaling also reduces autophagy (131), which is required by LCMV-specific T cells to form memory, and T cells lacking autophagy proteins during chronic LCMV infection expand normally but do not persist, causing delayed viral control (132). These results suggest that defects in a variety of metabolic processes (perhaps triggered by nutrient and/or oxygen restrictions) might underlie T cell exhaustion but may be necessary to prevent lethal immunopathology.

Epigenetic Regulation of CD8⁺ T Cell Exhaustion

Interestingly, when transgenic virus-specific CD8⁺ T cells isolated early after LCMV Cl13 infection are transferred into a similarly chronic infectious environment in the absence of their cognate antigen, their function and memory formation are restored (74, 99, 100). In contrast, transfer of exhausted CD8⁺ T cells later during infection into naive animals allows only partial restoration of function, and these cells maintain PD1 expression (82, 133). These results suggest antigen-driven, progressive, heritable genomic changes in exhausted CD8⁺ T cells. This is consistent with decreased DNA methylation of the PD1 locus in CD8⁺ T cells from both chronic LCMV and HIV infections (134, 135). Furthermore, Zhang et al. (136) recently showed reduced diacetylated histone 3 in exhausted LCMV-specific CD8⁺ T cells, and treatment with a histone deacetylase inhibitor increased IFNγ production and memory differentiation during chronic infection. Interestingly, many DNA/histone methylation marks promote HIV latency via long terminal repeat silencing; consequently, the histone deacetylase inhibitor SAHA (vorinostat) is proceeding to clinical trials as potential treatment to reduce the latent HIV reservoir (137). It will be interesting to evaluate whether this inhibitor also restores T cell function.

Although global epigenetic maps in exhausted CD8⁺ T cells have not yet been published, the aforementioned studies indicate that epigenetic modifications help maintain this cellular state. Future studies might allow therapeutic reprogramming of T cell responsiveness.

CD8⁺ T Cell Exhaustion Can Be Therapeutically Reversed

Characterization of exhausted CD8⁺ T cells has opened numerous therapeutic windows, many of which are related to modulation of γC cytokine pathways, namely the IL-2, IL-7, IL-15, and IL-21 pathways (138). Treatment with IL-7, IL-2, or IL-21 in established chronic LCMV infection is successful in amplifying the T cell response and reducing the viral titer (91, 139–142). In contrast, multiple phase III clinical trials of IL-2 treatment in HIV-positive patients concluded with no difference in outcome (143). IL-7 is currently in phase II trials, and IL-15 and IL-21 did not lower viremia in SIV-infected macaques despite increasing CD8⁺ T cell numbers (144–146).

In recent studies, therapeutic targeting of inhibitory surface receptors or cytokines induced strong restoration of T cell function, in particular the PD1/PDL1 and IL-10/IL-10R pathways (96, 147–150). Targeting other pathways [CTLA-4, TIM-3, 4-1BB, or TGFβ (151, 152)] alone is not effective to reduce viral loads. However, coblockade of α-PDL1 with α-TIM-3 (153) or α-IL-10 (147); α-PDL1 combined with costimulation with 4-1BB (154) or OX40 (155); or administration of low-dose IL-2 (156) results in synergistic enhancement of T cell numbers and function during LCMV Cl13 infection to reduce viral titer. Anti-PD1 clinical trials in HIV-infected patients are now in progress (157). These results support the idea that further understanding of the complex mechanisms underlying CD8⁺ T cell exhaustion may illuminate and refine effective therapeutic interventions that simultaneously enhance immune function, diminish viral load, and minimize immunopathology.

CD4⁺ T CELLS: DISTINCT FUNCTIONAL SPECIALIZATION DURING CHRONIC VIRAL INFECTIONS

Following vaccination and acute viral infections, CD4⁺ T cells have the capacity to differentiate into a number of subsets that sustain and enhance both CD8⁺ T cell– and antibody-mediated immunity. In HIV-1, HBV, and HCV infections, enhanced virus-specific CD4⁺ T cell responses are associated with improved viral control (158–161). Consistently, major histocompatibility complex class II human leukocyte antigen haplotypes correlate with disease prognosis in HIV-positive individuals (162, 163). Furthermore, CD4⁺ T cell–depleted chimpanzees fail to clear HBV or HCV and develop severe liver disease (164, 165). Similarly, CD4⁺ T cell–depleted mice fail to control persistent LCMV infection (63, 166), whereas the adoptive transfer of virus-specific CD4⁺ T cells into LCMV Cl13–infected mice helps restore CD8⁺ T cell exhaustion (167) and can provide B cell help (168).

Altogether, these studies indicate that CD4⁺ T cells are essential in the immune responses to chronic viral infections. However, excessive numbers of CD4⁺ T cells, caused by NK cell depletion or by vaccines that solely elicit an antiviral CD4⁺ T cell response, can result in pronounced inflammation and mortality during chronic LCMV infection, suggesting that enhanced CD4⁺ T cell responses should be sought carefully (169, 170). In the following sections we discuss key features of CD4⁺ T cell functional specialization and heterogeneity during chronic viral infections (Figure 2).

Reduced T Helper 1 Features and Enhanced Interleukin-21 Production

The prototypic antiviral CD4⁺ T cell response is characterized by the differentiation of T helper 1 (Th1) cells and their secretion of IFNγ, TNFα, and IL-2. Like CD8⁺ T cells (discussed above), CD4⁺ T cells also exhibit altered function during chronic infection that has historically been considered a form of functional exhaustion. Although less is known about the precise mechanisms behind this process, this CD4⁺ T cell state is characterized by reduced IL-2, TNFα, and IFNγ production (168, 171, 172). Similar to CD8⁺ T cells, virus-specific CD4⁺ T cells from LCMV-, HCV-, and HIV-infected individuals also express higher levels of inhibitory receptors (173–177), and blockade of PD1/PDL1 is sufficient to restore the function of HIV- and HCV-specific CD4⁺ T cells in vitro (174, 176). Chronic viral infections also increase the expression of the inhibitory cytokine IL-10 by CD4⁺ T cells (reviewed in 178), and loss of IL-10 signaling during chronic LCMV infection improves CD4⁺ T cell functionality and viral control (95, 96, 150, 179).

Despite reduced expression of prototypic Th1 cytokines, CD4⁺ T cells show enhanced production of IL-21 during chronic versus acute LCMV infection (142, 180–182). IL-21 is critical for the proliferation and survival of germinal center B cells (183). However, IL-21 not only promotes higher antiviral antibody levels but also is essential for the maintenance of virus-specific CD8⁺ T cells, suppression of regulatory T cell (Treg) expansion, and subsequent viral control during chronic LCMV infection (142, 180–182). Elevated IL-21 levels and increased frequencies of IL-21⁺CD4⁺ T cells are also associated with improved viral control in HIV, HBV, and HCV infections (184–190). Although the signals that promote IL-21 production in CD4⁺ T cells during human chronic infections have yet to be investigated, we demonstrated that, during chronic LCMV infection, cell-intrinsic signaling via the shared IL-6 family cytokine coreceptor, gp130, is essential for IL-21 production by virus-specific CD4⁺ T cells (191). Overall, the aforementioned studies are consistent with a model in which, rather than being exhausted, virus-specific CD4⁺ T cells favor distinct functional specialization (e.g., IL-21 production) during chronic viral infections.

Regulatory T Cells Suppress Antiviral Responses and Limit Pathology

Foxp3⁺ Tregs maintain immune system quiescence and prevent reactivity that would otherwise promote immunopathology (reviewed in 192). Chronic HIV, HCV, LCMV, and Friend virus (FV) infections all result in the expansion of Tregs (182, 193–197). In vitro, these Tregs have a potent capacity to suppress virus-specific CD8⁺ T cell function (194, 197–199), and depletion of Tregs during murine LCMV and FV infections has established the same role in vivo (200, 201). Of note, Treg depletion enhances CD8⁺ T cell responses and improves viral control during FV infection, but it has no effect on viral control during LCMV Cl13 infection [despite increased CD8⁺ T cell responses (182, 200, 201)]. This failure in viral control was associated with increased expression of PDL1, and a combination of Treg ablation and PDL1 blockade resulted in a greater reduction in viral titers than did PDL1 blockade alone (201). Tregs therefore appear to negatively impact the ability of the host to resolve an actively replicating chronic viral infection. There is evidence, however, that Tregs may play an important role in limiting pathology and morbidity during chronic LCMV infection (182). During HIV infection, reduced frequency and functionality of Tregs are associated with the onset of immune activation, increased viral load, and overall HIV disease progression (202, 203). Similar observations of higher Treg frequency associated with reduced pathology have also been made in HCV and SIV infections (204, 205). Thus, Tregs may be part of a disease tolerance mechanism (206) that minimizes the tissue damage caused by antiviral immune responses and thereby reduces the host’s fitness costs associated with a chronic viral infection.

T Follicular Helper Cell Escalation and Antibody-Mediated Immunity

T follicular helper (T_FH) cells are a CD4⁺ T cell subset specialized to provide B cell help and promote antibody-mediated immunity (reviewed in 207, 208). Among other molecular features, they are primarily characterized by high expression of the chemokine receptor CXCR5 (which promotes their migration to the B cell zone of secondary lymphoid organs), the transcriptional repressor BCL6 (which is necessary for T_FH differentiation), and the cytokine IL-21.

Recent studies indicate that T_FH cell differentiation is favored during chronic viral infections. Chronic LCMV infection leads to the pronounced accumulation of T_FH cells that migrate into the B cell zone, enhance the quantity and quality of germinal center B cell and virus-specific antibodies, and promote LCMV Cl13 control (209, 210). Increased frequencies of circulating T_FH-like or lymph node T_FH cells are also observed in patients infected with HIV, HBV, or HCV and in macaques infected with SIV and correlate with the levels of germinal centers, plasma cells, and/or virus-specific antibodies (211–214). Moreover, higher frequencies of circulating T_FH cells correlate with the appearance of HIV-specific broadly neutralizing antibodies, and improved T_FH function is associated with better vaccination outcomes in HIV-positive patients (215, 216). However, circulating T_FH-like and lymph node T_FH cells from HIV-infected patients exhibit reduced B cell helper capacity (217, 218), which can be restored by blockade of PD1/PDL1 on HIV-specific lymph node T_FH cells (217). Importantly, T_FH cell frequencies do not correlate with viral control during SIV or HIV infections (213–215). This is different from the role of T_FH cells in the control of persistent LCMV discussed above and could be related to the efficacy of antibody-mediated immunity in containing distinct persistent viruses. Indeed, permanent control of LCMV requires the presence of B cells (219, 220) and the production of virus-specific antibodies (221, 222). However, the presence of antibodies does not prevent reinfection with similar strains of HCV in either humans or chimpanzees (223, 224), nor does it prevent progression of HIV to AIDS (225), possibly due to antibody escape mutants (226). The late appearance of neutralizing or broadly neutralizing antibodies during chronic infection or vaccination could also contribute to their limited efficacy (reviewed in 227). This view is consistent with the observation that, when administered before HIV infection or even during HCV infection, broadly neutralizing antibodies are capable of promoting viral containment (228, 229). One confounding factor that may influence the capacity of T_FH cells to aid in the control of HIV-1 or SIV infections is that they are preferentially infected by both HIV-1 and SIV (213, 214, 230–233). This could result in the presence of a viral reservoir, the localization of which provides the virus with protection from clearance by CD8⁺ T cells (230, 231). Indeed, it was recently shown that T_FH cells act as a viral sanctuary in SIV-infected rhesus macaques (234).

The accumulation of T_FH cells during the late stages of LCMV infection is, in part, driven by increased IL-6 signaling, which also promotes optimal levels of functional molecules (e.g., ICOS) in virus-specific T_FH cells (210). Consistently, increased IL-6 signaling in T_FH cells is also observed during the chronic stage of SIV infection (210, 214). Another IL-6 family member, IL-27, promotes survival of virus-specific CD4⁺ T cells during chronic LCMV infection and is essential for viral control (191). Another factor promoting T_FH cell accumulation is the presence of chronic TCR stimulation; mice receiving either a chronic viral infection or recurrent antigen administration have increased T_FH cell frequencies compared with mice receiving acute inflammatory stimuli (209, 235, 236). Moreover, whereas T_FH cell development during acute infection or vaccination requires cognate B cell interactions (207, 208), B cells are not required in the context of chronic antigen (209, 236). Finally, the miR-17~92 family of microRNAs is essential for T_FH cell formation and viral control during LCMV Cl13 infection (237).

Thus, the escalation of T_FH cells during chronic viral infections is induced by a combination of signals, including cytokines and continuous TCR stimulation. Concomitant with attenuated Th1 functions and expanded numbers of Tregs, biased T_FH cell differentiation may be part of a common host mechanism to drive a less pathogenic response mediated by antiviral antibodies.

Distinctive CD4⁺ T Cell Transcriptional Signature and Heterogeneity

A recent microarray analysis of virus-specific CD4⁺ T cells from LCMV Cl13–infected mice revealed a transcriptional signature that is distinct from the prototypic CD4⁺ T cell fates (e.g., Th1, Th2, Th17, induced Treg) (238). This study highlighted the expression of several key transcription factors in CD4⁺ T cells from chronically infected mice, including low expression of Tbx21 (T-BET) and increased expression of Prdm1 (BLIMP1), Ikzf2 (HELIOS), and Eomes (EOMES). As mentioned above, in virus-specific CD8⁺ T cells, T-BET is critical for maintaining function and high BLIMP1 expression is associated with increased inhibitory receptor expression and exhaustion, and it is conceivable that these transcription factors would play similar roles in CD4⁺ T cells (112, 114, 238). Neither HELIOS nor EOMES has been previously implicated in T cell dysfunction during chronic viral infection; however, the Ikaros family of transcription factors, which includes HELIOS, is associated with cytokine production by CD4⁺ T cells (239). By contrast, CD4⁺ T cell expression of EOMES has recently been found to drive a distinct subset of cytotoxic CD4⁺ T cells in melanomas (240). Interestingly, Crawford et al. (238) found that high expression of BLIMP1 and EOMES was restricted to distinct populations of CD4⁺ T cells during chronic LCMV infection.

These studies highlight the presence of CD4⁺ T cell heterogeneity during chronic viral infection and the potentially distinct differentiation and/or abundance of CD4⁺ T cell subsets with respect to vaccinations or acute infections.

CONCLUDING REMARKS

Given the hyporesponsiveness of innate and adaptive immune cells during chronic viral infections, the term exhaustion could be applied to almost all aspects of immunity discussed in this review (e.g., pDCs and T cells). In all cases, however, an argument could be made to switch this terminology to adaptation or recalibration of immune cells, as has recently been proposed for CD8⁺ T cells (241). Adaptation or recalibration (rather than exhaustion) emphasizes reprogramming of innate and adaptive immune cells to establish an equilibrium with the new environment while remaining partially effective during chronic viral infections. This involves multiple layers of cell-intrinsic transcriptional, epigenetic, and posttranscriptional processes that respond to cell-extrinsic changes, including sustained stimulation via TCRs, B cell receptors, and/or PRRs; a distinct inflammatory milieu; altered nutrient and oxygen levels; and, likely, increased damage-associated molecular patterns and tissue repair factors. Notably, the molecular mechanisms underlying immune adaptation appear to be conserved in great part during chronic infections with distinct viruses in a range of host species. It is important to emphasize, however, that the ultimate effectiveness of particular immune mediators (e.g., IFN-I, T_FH cells, antibodies) in promoting viral control depends on the specific life cycle and immune-evasion strategies of each infectious agent (e.g., tropism, mutation rate, susceptibility to ISGs, etc.).

Technological advances will continue to allow greater understanding of innate and adaptive immune regulation during chronic viral infections. For instance, advances in single-cell sequencing in combination with multiparameter flow cytometry, including mass cytometry, should provide clarification on the extent of heterogeneity in different immune cell compartments during chronic versus acute viral infections. Similarly, high-throughput approaches to epigenetic, posttranscriptional, and metabolomic processes should provide greater clarity about their roles in immunity to chronic viral infections. Additionally, the increasing evidence for cross talk between the host’s immune system and microbiome should also prove an intriguing avenue of discovery.

In conclusion, the molecular networks underlying immune cell adaptation likely evolved as a safety rheostat to counteract immune responses that, although well tolerated for a limited time in an acute infection, have the capacity to cause considerable pathology in the presence of persistent pathogens. Sterilizing therapeutics will likely benefit from a combination of drugs or gene therapies that boost different arms of the immune system and target key steps in the virus life cycle. Further understanding of the unique and sophisticated adaptation of immune cells to a chronic infectious environment will move us closer to this goal.