Divergent contributions of regulatory T cells to the pathogenesis of chronic hepatitis C

Abstract

Hepatitis C virus, a small single-stranded RNA virus, is a major cause of chronic liver disease. Resolution of primary hepatitis C virus infections depends upon the vigorous responses of CD4⁺ and CD8⁺ T cells to multiple viral epitopes. Although such broad CD4⁺ and CD8⁺ T-cell responses are readily detected early during the course of infection regardless of clinical outcome, they are not maintained in individuals who develop chronic disease. Purportedly, a variety of factors contribute to the diminished T-cell responses observed in chronic, virus-infected patients including the induction of and biological suppression by CD4⁺FoxP3⁺ regulatory T cells. Indeed, a wealth of evidence suggests that regulatory T cells play diverse roles in the pathogenesis of chronic hepatitis C, impairing the effector T-cell response and viral clearance early during the course of infection and suppressing liver injury as the disease progresses. The factors that affect the generation and biological response of regulatory T cells in chronic, hepatitis C virus-infected patients is discussed.

Keywords: :

• dendritic cells
• hepatitis C
• immune tolerance
• liver disease
• regulatory T cells

Introduction

Hepatitis C virus (HCV, a small single-stranded RNA virus) is the cause of hepatitis C, the most common blood-borne infection in the world; an estimated 180 million people globally, ~3 million in the US, are chronically infected.^1,2 The RNA genome consists of a large open reading frame that encodes an approximate 3,000 amino acid polyprotein precursor that is cleaved by viral and cellular proteases to yield three structural proteins [core, envelope 1 (E1) and E2] and seven non-structural proteins, which are involved in viral synthesis.³ HCV is primarily transmitted via contact with infected blood or bodily fluids. The risk of contracting hepatitis C from contaminated blood or blood products in the US is negligible due to mandatory and reliable blood screening.⁴ Despite a safe blood supply, however, isolated nosocomial/iatrogenic infections continue to occur emphasizing the need for enforcement of universal precautions and proper sterilization of medical devices.⁴ Individuals at highest risk of infection in the US today are injection drug users.⁵ In contrast, a large percentage of the population in many developing countries is infected owing to substandard infrastructure and technology required to provide a safe blood supply.⁶

The majority of HCV-infected patients develops chronic disease; only an approximate 20% resolves infection spontaneously.² A variety of factors such as age, sex, ethnicity, human immunodeficiency virus or hepatitis B co-infection, and host genetics (e.g., IL28B gene variants and HLA haplotype) affect spontaneous clearance of HCV.^7-10 Standard treatment of unresolved infections, consisting of the combined administration of PEGylated interferon (IFN) and ribavirin over an extended period of 24 to 48 weeks, is often accompanied by significant side effects. Moreover, only 45–50% of treated patients infected with HCV genotype 1 (the primary etiologic agent of hepatitis C in the US) demonstrate a sustained virologic response following treatment withdrawal.¹¹ Additional treatment approaches that combine protease inhibitors, e.g., boceprevir and telaprevir, with ribavirin and PEGylated IFN result in sustained virologic responses in up to 80% of patients infected with HCV genotype 1, though the severity of accompanying side effects can also increase dramatically.^12-14

The vigorous responses of HLA class I- (CD8⁺) and class II-restricted (CD4⁺) T cells to numerous viral epitopes derived from both structural and nonstructural proteins are required for the resolution of primary HCV infections.¹⁵ Such broad responses are readily detected during the early course of infection despite clinical outcome, but subside in patients who later develop chronic disease.¹⁶ While patients who spontaneously clear infection continue to exhibit sustained, proliferative responses to a broad array of class I- and class II-restricted epitopes, chronically infected individuals respond to only a limited number.¹⁷ Purportedly, multiple factors contribute to the reduction in T-cell responses found in patients with chronic infections, notably, the induction of and biological suppression by CD4⁺FoxP3⁺ regulatory T_(reg) cells.^18-22

Regulatory T Cells

Background

CD4⁺ regulatory T_(reg) cells constitute one of the major mechanisms underlying immunological homeostasis and self-tolerance.²³ T_reg cells are characterized by the constitutive expression of the transcription factor forkhead box P3 (FoxP3) and interleukin (IL)-2 receptor α chain (CD25) on the cell surface. FoxP3 mutation and T_reg cell dysfunction result in fatal immunopathology and autoimmune disease in both mice and men.^24-26 T_reg cells also moderate effector immune responses to infectious diseases that, if sustained at elevated levels, can lead to serious host tissue and organ damage.²⁷ Although a key factor in the maintenance of immune homeostasis, T_reg cells can also suppress effective immune responses to autologous tumors (e.g., hematological malignancies and metastatic melanoma) and promote persistent infections by a wide variety of microbial pathogens.^27-29

T_reg cell phenotype. Two distinct T_reg cell subsets, classically distinguished by site of origin, are described in the literature. Natural (n)T_reg cells are generated by high-avidity selection in the thymus; inducible (i)T_reg cells, on the other hand, derive from conventional (CD4⁺CD25^-FoxP3^-) T cells in the periphery following stimulation.^30-32 nT_reg cells can induce “infectious tolerance” by converting conventional T cells into iT_reg cells via two primary methods: cytokine (IL-10, IL-35 or TGF-β)-dependent and dendritic cell (DC)-mediated, cytokine-independent mechanisms.^33,34 Purportedly, nT_reg and iT_reg cells possess complementary immune functions: prevention of autoimmunity and maintenance of a non-inflammatory environment, respectively.³¹

Notably, no specific marker defines T_reg cells or differentiates nT_reg and iT_reg cell subsets. While FoxP3 expression is a common attribute of both subsets, conventional human T cells lacking immunosuppressive capacity can also express FoxP3 transiently following activation.³² Moreover, despite the near exclusive expression of CD25 by nT_reg cells in naïve mice, CD25 is expressed by a much more heterogeneous T-cell population in humans.³² Recent studies report the high level expression of neuropilin-1 on the surface of nT_reg, but not iT_reg, cells in mice enabling differentiation and separation of these two subsets.^35,36 Activated human FoxP3⁺ T_reg cells that express high suppressive activity are also distinguished by presence of glycoprotein A repetitions predominant (GARP, or LRRC32), a cell surface transmembrane protein that contains leucine-rich repeats.^37-40 GARP mRNA is specifically expressed by CD4⁺CD25^hi T_reg cells, and is rapidly upregulated following T-cell receptor engagement.^37,38 GARP anchors transforming growth factor (TGF)-β to the cell surface conferring increased suppressive activity and the ability to induce infectious tolerance.³⁹ Lastly, cell surface expression of ectonucleotidase, CD39, distinguishes activated, effector memory T_reg cells capable of abrogating DC maturation and T cell-dependent cytotoxicity.⁴¹

T_reg Cell Function

Contact-independent mechanisms

Activated T_reg cells are able to suppress the activity of a variety of immune cell types, i.e., both CD8⁺ and CD4⁺ T cells, NK cells, NKT cells, B cells, macrophages and DCs.^42-46 Multiple mechanisms contribute to this suppressive activity although it is widely believed that nT_reg cell-mediated suppression is dependent upon direct, cell–cell contact.⁴⁶ The synthesis of inhibitory cytokines constitutes a principal contact-independent mechanism by which T_reg cells in general suppress T_eff cell activity (Fig. 1). Both the soluble and membrane-bound forms of TGF-β, for example, play key roles in inducing and/or maintaining iT_reg and nT_reg cells, and in suppressing conventional effector T_(eff) cell activation.^45,47,48 Similarly, IL-10 plays a critical role in suppressing CD4⁺ T_eff cell responses to a variety of pathogens used in animal models, as well as those that contribute to human disease.²⁷

Figure 1. Increases in both the number and function of T_reg cells have been implicated in the pathogenesis of chronic hepatitis C. Virus-associated regulatory T cell epitopes, homologous to peptide sequences found in the human plasma proteome, induce nT_reg cell activation, conversion of T_eff to iT_reg cells and infectious tolerance (A). Viral epitopes lacking human homology, which are presented by immature DCs, elicit additional HCV-specific iT_reg cells (B). T_reg cells inhibit T_eff cell function by direct, contact-dependent and -independent mechanisms and by indirect mechanisms that affect DC maturation and/or immunostimulatory activity (C).

The constitutive, high-level expression of CD25 (IL-2 receptor α chain) constitutes an additional contact-independent mechanism underlying T_reg cell-mediated suppression. T_reg cells produce relatively low levels of IL-2 and, as such, require an exogenous source of IL-2 in order to proliferate and survive.⁴⁹ As a consequence of the rapid consumption of IL-2 by T_reg cells, T_eff cell populations are deprived of the cytokine necessary for activation.⁴⁹ The cell surface expression of CD39 and CD73 ectonucleotidases constitutes another mechanism by which T_reg cells disrupt the metabolic activity of T_eff cells.⁵⁰ The activity expressed by these molecules abrogates the proinflammatory response of T_eff cells by rapidly degrading extracellular ATP released by neighboring, activated or damaged cells.⁵⁰ Additionally, adenosine generated as a byproduct of ATP degradation further suppresses T_eff cell function by binding A2A receptors expressed on the cell surface and inducing T cell anergy.^50-52

Contact-dependent mechanisms

A number of contact-dependent mechanisms also facilitate the ability of T_reg cells to suppress T_eff cell function. For example, T_reg cells can exhibit cytotoxic activity and induce T_eff cell apoptosis dependent upon the production of granzyme A, granzyme B and perforin.^48,53 In addition, cell-surface galectin-1 appears to contribute to the immunosuppressive activity of T_reg cells.⁵⁴ A member of a highly conserved family of β-galactosidase-binding proteins, galectin-1 inhibits proliferation and promotes apoptosis of activated T_eff cells.⁵⁴

Apart from regulating T_eff cell function directly, T_reg cells can inhibit the maturation and immunostimulatory activity of DC and, consequently, suppress the activity of conventional T cells indirectly.⁴⁸ T_reg cells can render DCs tolerogenic and limit their capacity to sensitize naïve T cells by a process termed trans-endocytosis whereby cytotoxic T lymphocyte-4 (CTLA-4) molecules expressed by T_reg cells capture, internalize, and degrade co-stimulatory molecules (i.e., CD80 and CD86) expressed on the surface of DCs.⁵⁵ Lymphocyte activation gene-3 (LAG-3 of CD223), a CD4 homolog capable of binding MHC class II molecules, further suppresses DC maturation and the expression of co-stimulatory molecules.^56,57 Lastly, neuropilin-1 expressed by nT_reg cells inhibits the sensitization of naïve CD4⁺ T cells by promoting extended interaction between T_reg cells and immature DCs.⁵⁸ Undoubtedly, the suppressive functions of T_reg cells depend upon a combination of the mechanisms described.

T_reg Cells in Hepatitis C

The increased numbers of T_reg cells present in the liver and peripheral blood of chronic HCV-infected patients relative to patients who spontaneously clear infection suggests that T_reg cells play a significant role in the pathogenesis of chronic hepatitis C.^{18,19,27,59-63} In this regard, CD4⁺CD25⁺ T cells obtained from the peripheral blood of patients infected with HCV suppressed virus-specific proliferation and IFN-γ production by CD4⁺ and CD8⁺ T cell; depletion of CD25⁺ cells including T_regs from the total peripheral blood mononuclear cell (PBMC) population enhanced the proliferation of cells that remained.^18,63 Whether the increase number of T_reg cells found in the livers of chronically-infected patients represents an virus-specific T_reg cell response or arises as a non-specific consequence of chronic inflammation and disease is not entirely clear.

HCV encodes T_reg cell epitopes

Researchers have identified T_reg cell epitopes present in both the structural and non-structural proteins synthesized by HCV.^{20,59,60,64-66} Using HCV peptide-loaded MHC class II tetramers to label and quantify the cells, Ebimuna and coworkers reported HCV epitope-specific recognition by FoxP3⁺ T_reg cells present in the peripheral blood of chronically infected patients.⁶⁴ Moreover, a number of studies now report an increase in FoxP3⁺CD25^high cells that resemble nT_reg cells and express an anergic cytokine profile following stimulation with HCV derived epitopes.^59,61,64 Comparing FACS-sorted CD4⁺CD25^high T cells (nT_reg phenotype) derived from uninfected and HCV-infected individuals, however, one study found only minimal differences in global gene expression.⁶⁴ In contrast, other investigators reported an increase in HCV epitope-specific T_reg cells that resembled iT_reg cells phenotypically and exhibited both IL-10- and TGF-β-dependent suppressive activity.^60,65 Regardless, a general consensus supports the heterogeneous nature of the expanded T_reg cell population found in chronic, HCV-infected patients (i.e., composed of both nT_reg and iT_reg cell subsets). The factors that contribute to this expansion are neither well understood nor characterized. Conceivably, the initial response of nT_reg cells to infection promotes the conversion of conventional T cells to iT_reg cells and subsequent suppression (i.e., infectious tolerance).³⁴

HCV T_reg cell epitopes and human homology

The expressed function of nT_reg cells is to suppress autoimmunity and the response to self antigens.^23-26 Activation of T_reg cells subsequent to HCV infection suggests the existence of viral epitopes that exhibit homology to self and the ability to simulate these nT_reg cells and viral persistence.^59,67-70 Indeed, Kanduc and colleagues reported that 3003 unique pentapeptides comprised the HCV polyprotein; of these, 2760 pentapeptides occurred a total of 46,731 times (including matches/repeats) in 20,269 proteins that comprised the human proteome.^68,70 Furthermore, a BLAST search and JanusMatrix analysis (a new computer algorithm, which identifies epitopes that are highly cross-reactive on the T cell receptor aspect) revealed extensive homology between a number of published T_reg cell epitopes derived from HCV structural and non-structural proteins and sequences encoded by the human genome (Moise et al., manuscript submitted) (Losikoff et al., manuscript in preparation).⁷¹ These epitopes induced the response of T_reg cells present in the peripheral blood of chronic HCV-infected patients, but not in blood obtained from HCV non-infected individuals.^22,59,71 Taken together, these findings suggest that viral epitopes, homologous to peptide sequences constituting the human proteome, influence the pathogenesis of chronic hepatitis C by inducing the expansion of the iT_reg cell population and infectious tolerance.^62,72

An evolving body of research demonstrates the import of the human microbiome in shaping the immune response to viral pathogens.⁷³ Recent studies found the microbiome can induce the differentiation and expansion of iT_reg cells that exhibit a unique T cell receptor repertoire specific for commensal bacteria.^74,75 Conceivably, homology between viral epitopes and T_reg cell epitopes associated with the human microbiome represents an additional mechanism underlying the expansion of T_reg cells in chronic HCV.

T_reg Cells Impair Viral Clearance

Several cross-sectional studies demonstrated a strong correlation between chronic HCV infection and increased T_reg cell frequencies.^18,63,64 The specific contribution of T_reg cells to viral persistence, however, could not be elucidated due to the occurrence of infection at some previous, undetermined date. Furthermore, two longitudinal studies failed to demonstrate a significant difference in the frequency or function of T_reg cells in the blood of patients acutely infected with HCV regardless of clinical outcome (i.e., whether the virus was eventually cleared or chronic disease developed).^61,76 In one of these studies, T_reg cells obtained from patients who subsequently developed chronic disease demonstrated significantly more suppressive activity than did T_reg cells obtained from patients who spontaneously cleared infection.⁶¹ As a consequence, the authors concluded that maintenance of an elevated T_reg cell population promoted the development of chronic hepatitis C.

Recently, Cusick and coworkers described the emergence of a variant of an immunodominant HCV epitope during the course of chronic HCV infection.⁶⁶ This variant elicited a T_reg cell population that suppressed a T_eff cell response to the cognate, but not an unrelated, peptide. This finding suggests that variant, MHC class II-restricted viral epitopes arise as a consequence of immune pressure during the course of chronic HCV infection. Rather than induce the activity of conventional CD4⁺ T cells, these variant epitopes elicit an epitope-specific T_reg cells response that diminishes the CD4⁺ T cell help required for viral clearance. Thus, in cases of chronic disease, T_reg cells impair T_eff cell function and promote viral persistence by inhibiting virus-specific helper T cell activity.

T_reg Cells Suppress Chronic Hepatitis C-Associated Liver Injury

In addition to inhibiting effective immune responses and promoting persistent infections by a wide variety of pathogenic microorganisms, T_reg cells moderate T_eff cell responses that could otherwise lead to serious host tissue and organ damage.^{18,21,27,28,77-79} This is evidenced in patients with chronic hepatitis C by an inverse correlation between the level of T_reg cell activity found in the peripheral blood and the extent of liver injury.^18,78,80 Both the serum alanine aminotransferase concentrations and the histopathologic scores of liver biopsies were higher in chronic HCV-infected patients who exhibited a relative reduction in T_reg cell activity suggesting that the expanded T_reg cell population ameliorated excessive pathology and tissue damage. The balance between T_reg cells and CD8⁺ T_eff cells appears to be a crucial factor. Sturm and colleagues reported a strong inverse correlation between the severity of liver fibrosis and the accumulation of CD4⁺FoxP3⁺ T_reg cells in close contact with CD8⁺ T cells in necro-inflammatory sites in patients with chronic hepatitis C.²¹ The ratio of FoxP3⁺ to CD8⁺ T cells was elevated in the early stages of fibrosis, and reduced substantially in cirrhotic livers. The authors also reported a link between IL-10 and TGF-β message expression in the liver and presence of FoxP3⁺ and CD8⁺ T cells suggesting that these anti-inflammatory cytokines served both to maintain the T_reg cell pool and inhibit the production of pro-inflammatory cytokine (e.g., TNF-α and IFN-γ) by CD8 T_eff cells.²¹

Thus, an equilibrium between T_reg cells and T_eff cells that permits both viral clearance and protection from HCV-related hepatopathology is critical for an optimal response to HCV infection.^21,77 In cases of chronic disease, however, the elevated presence of T_reg cells appears to benefit both the pathogen (i.e., viral persistence) and the host (i.e., prevention of immunopathogenesis).^21,79 In this regard, it is Interesting to note that increased numbers of CD25⁺FoxP3⁺ T_reg cells were found in the livers of patients who underwent successful chemotherapy, suggesting that the continued presence of T_reg cells moderated immunopathology despite viral clearance. These divergent roles of T_reg cells during the development of chronic hepatitis C are illustrated in Figure 2.

Figure 2. Contrasting contributions of T_reg cells to the pathogenesis of chronic hepatitis C. An increased ratio of T_reg to T_eff cells impairs spontaneous viral clearance and suppresses liver injury and the pathogenesis of chronic hepatitis C as the disease progresses.

Impaired DC Function Promotes the Generation of T_reg Cells

Whether HCV infection elicits the response of T_reg cells directly or indirectly (dependent upon the intermediary role of another cell type) remains to be determined. Accumulating evidence suggests, however, that DCs play a key role in the expansion of T_reg cells during the pathogenesis of chronic disease. DCs are professional antigen presenting cells characterized by the potent capacity to elicit primary T cell responses.⁸¹ While there is no general consensus regarding the effects of HCV infection on DC function, it is often agreed that chronically infected patients have significantly fewer DCs circulating in the peripheral blood, which correlates inversely with the severity of liver disease.^82-86 Conversely, increased numbers of DCs are found in the liver of patients chronically infected with HCV.^85,87-89 Regardless of their physiologic distribution (i.e., peripheral blood vs. liver), it is frequently suggested that the DCs are functionally impaired in patients with chronic hepatitis C.^83,89-98 These impairments include lowered expression of HLA-DR and costimulatory molecules (e.g., CD86), decreased allostimulatory activity and the ability to secrete IFN-α and IL-12 and increased IL-10 production and the ability to prime T_reg cells.^{83-85,89,90,92-96,99-103} Additionally, it was reported that DCs derived from chronically infected patients remain phenotypically immature and lack the capacity to upregulate the expression of maturation or costimulatory marker following stimulation.^91,97

Mechanisms that underlie impaired DC function

The specific mechanisms underlying DC dysfunction in patients chronically infected with HCV remain to be clarified. Recent studies suggest, on the one hand, that DCs are susceptible to HCV infection.^104,105 The interaction between envelope glycoproteins E1 and E2 and DC-SIGN, a C-type lectin expressed by DCs, mediates the ingestion of HCV viral particles.¹⁰⁵ This finding supports the speculation that, even in the absence of a productive infection, E1 and E2 bound to DC-SIGN transmit a signal that promotes T_reg cell formation and tolerance. Other structural and non-structural viral proteins further impair DC function by decreasing costimulatory molecule and HLA expression, reducing cytokine production, preventing TLR signaling and diminishing allostimulatory activity.¹⁰⁶ As described above, T_reg cells can also inhibit DC maturation and immunostimulatory capacity by a variety of mechanisms and, thereby, suppress the response of conventional T cells.⁴⁸ A growing body of evidence supports the critical role of DCs in the induction and maintenance of T_reg cells during chronic HCV infection, regardless of the specific mechanisms that contribute to a diminution in DC function.

DCs and immunological tolerance

DCs play a key role in establishing and maintaining immunological tolerance to both foreign and self antigens.^107,108 Interactions between T_reg cells and DCs in the prototypic, tolerogenic environment maintained in the liver foster the development of chronic infectious diseases such as viral hepatitis.¹⁰⁹ While it is generally agreed that immature DCs promote T_reg cell function, the underlying mechanisms remain to be fully delineated.^107-111 Notably, DCs obtained from human liver principally exhibit an immature phenotype characterized by low or negligible expression of cell-surface co-stimulatory molecule (e.g., CD40, CD80, CD83 and CD86).¹⁰⁹ Relative to DCs purified from the peripheral blood, DCs derived from liver also produce lower levels of proinflammatory cytokine (i.e., TNF-α, IL-1β and IL-6) and significantly higher levels of IL-10 following stimulation. Moreover, the IL-10-dependent production of CD4⁺CD25⁺FoxP3⁺ T_reg cells was greater in co-cultures that contained naïve, allogeneic CD4⁺ T cells and liver DCs, compared with co-cultures that contained DCs derived from blood.¹¹⁰ Similar results were obtained when allogeneic T cells and DCs obtained from chronic, HCV-infected patients were co-cultured, i.e., greater expansion of the T_reg cell population than observed in co-cultures that contained DCs obtained from healthy individuals.¹⁰³

Summary and Clinical Implications

Eighty to 85% of the patients in the US infected with HCV develop chronic disease.² Persistent viral infection correlates with increased T_reg cell number and function in the tissues of HCV-infected patients. While triple drug therapy (e.g., boceprevir or telapervir administered in conjunction with interferon and ribavirin) increases the sustained virologic response, the adverse side effects are often severe, the costs are extraordinarily high, and a significant portion of treated patients remains infected.^12-14 Additional approaches, e.g., therapeutic vaccination, are urgently needed. To date, four vaccine strategies have demonstrated varied and only limited success in clinical trials: recombinant protein, peptide, genetic or DNA-based and vector-mediated.^112,113 Recent evidence suggests that viral epitopes may activate nT_reg cells and, subsequently, induce expansion of the iT_reg cell population and immunosuppression via the direct effects of T_reg cells on T_eff cell activity or by indirect effects dependent upon intervening DCs. DCs that exhibit an immature phenotype, in turn, can promote the expansion and activity of T_reg cells and, thus, play a key role in exacerbating the pathogenesis of chronic hepatitis C. As a consequence, future efforts to develop a therapeutic vaccine must employ strategies to avoid incorporating epitopes that promote T_reg cell activity.

In the absence of a therapeutic vaccine, a full understanding of the response of T_reg cells to HCV infection could lead to the development of treatments capable of sustaining T_eff cell-mediated immunity while limiting tissue damage.¹¹⁴ In this regard, continued research efforts are needed to determine the most effective means of manipulating the T_reg cell response in a clinical setting.¹⁸ Progress in understanding the role of T_reg cells and the factors that influence protective immunity to hepatitis C are hampered, however, by the lack of a good rodent model and the ability of HCV to infect humans and chimpanzees only.¹¹⁵

References

Abbreviations:
DCs	=	dendritic cells
FoxP3	=	transcription factor forkhead box P3
HCV	=	hepatitis C virus
IFN	=	interferon
IL	=	interleukin
iTreg cell	=	inducible regulatory T cell
nTreg cell	=	natural regulatory T cell
Teff cells	=	effector T cells
TGF-β	=	transforming growth factor-beta

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Support was provided by National Institutes of Health Research Grant: U19 AI082642.