Regulatory T cells and the liver: a new piece of the puzzle.

Tcells with immune suppressive properties were originally described in the 70's.1 However, the inability to elucidate the mechanism of suppression resulted in serious skepticism about their existence during the 1980s.2 With the demonstration of CD4-mediated immunological tolerance in the 1990s,3, 4 the suppressor T cells are now back with a vengeance under a less threatening name of regulatory T cells.2 With the increasing appreciation for their global immune regulatory role, this editorial attempts to provide an overview about regulatory T cells while highlighting several recent studies related to liver including 2 papers on hepatitis B virus (HBV) infection and on hepatocellular carcinoma (HCC) in this issue. HBV, hepatitis B virus; HCC, hepatocellular carcinoma; DC, dendritic cell; IBD, inflammatory bowel disease. Regulatory T cells are a heterogeneous group of naturally occurring and/or inducible T cells. In addition to the CD4+CD25+ T cells (also called CD25+ Tregs) discussed primarily in this review, they include peripherally induced antigen-specific CD4 or CD8 T cells that secrete immunoregulatory cytokine IL10 (Tr1 cells) or TGFβ (Th3 cells) as well as NKT cells and γδ cells. There are also CD4+CD25-negative regulatory T cells and naïve T cells that become tolerant in certain settings (‘infectious tolerance’).5 Particular attention has focused on the naturally occurring thymic-derived CD4 T cell subset expressing the IL-2 receptor α-chain (i.e., CD25). These CD4+CD25+ T cells (CD25+ Tregs) represent 5%-10% of peripheral CD4 T cells and mediate self-tolerance/autoimmunity as well as immune response to tumor, grafts and pathogens.2 Similarly suppressive and hyporesponsive CD4+CD25+ T cells are detected in humans.5 CD4+CD25+ Tregs express various memory or activation markers detectable by flowcytometry including CD45RO (or CD45 RB-low in mice), CTLA4 (cytotoxic T lymphocyte associated antigen-4), GITR (glucocorticoid-induced TNF receptor-related protein), CD62L, HLA-DR, CD38 and IL2Rβ, among others. Generally, a combined panel of markers (e.g., CD4, CD25, CD62L, CD45RO, CTLA4) is used to detect CD25+ Tregs. However, none of these markers are specific for Tregs. For example, activated T cells upregulate their CD25, CD45RO, CTLA4 and GITR expression and cannot be distiguished from CD25+ Tregs by surface phenotype, particularly in inflammatory conditions. They tend to secrete TGFβ and/or IL10 with low levels of IFNγ or IL4 but not IL2 in vitro.2, 6 The most specific marker of CD25+ Treg is the forkhead transcription factor Foxp3 or scurfin. Foxp3 is a key factor in murine CD25+ Treg development and function in vivo, conferring suppressive phenotype in naive CD25-cells upon forced expression.7 Genetic defects in Foxp3 result in severe autoimmune and inflammatory syndromes in mice (Scurfy) and humans (immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome or IPEX). A hallmark of the CD4+CD25+ Tregs is their functional hyporesponsiveness, low IL2 production and cell–cell contact–dependent suppression of CD4 and CD8 T cell function in vitro. Tregs require initial activation through the T cell receptor before exerting their suppressive function in an antigen-nonspecific manner. Neither the antigen specificity nor trafficking of the circulating CD25+ Tregs is well defined. The findings on the mechanism of their suppression are somewhat conflicting, implicating (and refuting) CTLA4, IL10 or TGFβ in various in vivo and in vitro systems.2 T cell suppression in-vitro tends to be independent of IL4, IL10 or TGFβ and overcome by exogenous IL2 or CD28 costimulation. By contrast, immunoregulatory cytokines are involved in vivo in various animal models.8 Interestingly, dendritic cell (DC) stimulation via the toll-like receptors may render the effector T cells resistant to Treg-mediated suppression in part through IL6-mediated pathway.9, 10 Further, T cells deficient in B7 expression resist suppression by CD25+ Tregs,11 thus providing multiple levels of immune regulation and counter-regulation. The central role of CD25+ Tregs in immune tolerance has been elegantly demonstrated in vivo in mouse models (e.g., day 3 thymectomized animals) where adoptively transferred CD4+CD25+ T cells prevent autoimmune diseases including autoimmune gastritis, thyroiditis, oophoritis and inflammatory bowel disease (IBD) while their depletion precipitates autoimmune diseases.2 Thus, CD25+ Tregs control the balance between tolerance and rejection of self, non-self (pathogens, transplant) or altered-self (cancer). In acute murine HSV infection, CD25+ Treg depletion enhanced the primary HSV-specific CD8 T cell response with a rapid viral clearance while adoptive transfer of CD25+ Tregs suppressed HSV-specific CD8 T cell response and delayed viral clearance.12 Similarly, CD25+ Tregs control Leishmania major persistence and immunity.13 Furthermore, Friend murine retroviral infection persists with increased number of CD4+ Tregs that suppress virus-specific CD8 T cells in vivo.14, 15 Relevant for tumor-immunity, suppressive CD4+CD25+ T cells are present in large amounts in tumor tissues of cancer patients.16, 17 Interestingly, antigen-specific CD25+ Tregs may abrogate CD8 T cell-mediated tumor rejection by suppressing their cytolytic activity in a TGFβ-dependent mechanism.18 Along this line, CD25-depletion with and without CTLA-4 blockade enhanced the effectiveness of a tumor vaccine in mice, suggesting that tumor immunotherapy may be more effective with concurrent Treg inhibition.19 The potential role of Tregs in human liver disease is just beginning to be defined. Tregs may limit liver injury by controlling inflammation (e.g., fulminant or autoimmune hepatitis) or promote transplant tolerance. Alternatively, Tregs may promote persistent infection by suppressing immune response (e.g., in HBV, HCV) or control tumor growth (e.g., liver cancer). The following section reviews some of the first studies on liver cancer and viral hepatitis B and C with many more to come.1 Potential role for regulatory T cells in liver disease pathogenesis. Regulatory T cells are a diverse group of cells including the CD4+CD25+ (Foxp3+) Tregs, IL10+ Tr1, TGFβ+ Th3 and NK T cells among others. The balance between the regulatory and effector T cells may contribute to the pathogenesis of various immune-mediated liver diseases. For example, overregulation/suppression of antiviral or antitumor immune response could result in chronic HBV/HCV infection and liver cancer. By contrast, inadequate regulation could result in autoimmune hepatitis, rejection and fulminant hepatitis. Liver cancer usually develops in the setting of chronic hepatitis and cirrhosis. The study by Unitt et al. in this issue provides the first report of increased CD4+CD25+ T cell frequency within the tumor tissue compared to nontumor tissue in HCC+ patients.20 The HCC-infiltrating CD4+CD25+ T cells were suppressive, hypoproliferative, Foxp3+ and TGFβ+, consistent with the expected Treg phenotype. Although their circulating frequency was similar between HCC+ and healthy subjects, CD25+ T cells from HCC+ patients had significantly greater expression of membranous TGFβ1, similar to the tumor-infiltrating CD25+ Tregs. These findings suggest that tumor-specific T cells are suppressed on-site by the CD25+ Tregs via immunosuppressive cytokine TGFβ. In another study, Stoop and colleagues21 examined the potential role of CD25+ Tregs in HBV infection. In this study, the frequency of CD25+CD45RO+CTLA4+ Treg subset among the circulating CD4 T cells was significantly greater in chronically HBV-infected than in uninfected subjects (particularly HBeAg+). Increased CD25+ Treg frequency in HBV-infected patients was accompanied with increased Foxp3 mRNA expression in peripheral lymphocytes. As for their suppressive function, HBV-specific T cell proliferation and IFNγ secretion in peripheral lymphocytes was enhanced by CD25-depletion and directly suppressed by CD25+ Tregs in a dose-dependent manner. These results suggested that CD25+CD45RO+CTLA4+ T cells in HBV-infected patients represent true CD25+Tregs, rather than activated T cells. The authors concluded that these Tregs may promote inadequate HBV-specific immune response leading to persistent infection. In another compelling study by Franzese et al.,22 immune modulating effect of CD25+ Treg was also demonstrated but without a frequency difference between HBV-infected and uninfected subjects. While this discrepancy in frequency may reflect different criteria for Treg detection, CD25+ Tregs suppressed HBV-specific T cells in both HBV-infected and resolved individuals, consistent with a regulatory (if not a pathogenetic) role for CD25+ Tregs in HBV infection. While further studies are needed, these 2 studies are the first to highlight the potential impact of CD25+ Tregs in HBV infection and immunity. In HCV-infected patients, virus-specific T cells with IL10 or TGFβ production have been reported for some time.23-25 HCV-specific CD8+ Tr1 cells were detected in the liver of HCV-infected patients, demonstrating potential role in controlling hepatic effector T cells.26 As for CD25+ Tregs, HCV persistence was associated with increased circulating CD25+Tregs that directly suppressed HCV-specific CD8 T cell IFNγ response ex –vivo.27 In this study by Sugimoto et al., HCV-specific CD8 T cell response was augmented by CD4-depletion in HCV-infected but not in HCV-recovered subjects, suggesting that CD4 T cells (presumably CD25+ Tregs) were suppressing HCV-specific CD8 T cells during chronic infection. Subsequently, increased frequency and suppressive function of CD25+ Tregs in HCV-infected patients was confirmed in a detailed study by Cabrera et al., further demonstrating Treg secretion of TGFβ and IL10 and inhibition of Treg suppression by TGFβ1 blockade.28 CD25+ Tregs from HCV-infected patients also displayed HCV-specific IL10 secretion and their frequency correlated with HCV titer and HAI but not ALT or liver fibrosis. Although further studies are needed to confirm and extend these initial findings, these studies suggest that Tregs could influence immune regulation and the outcome of HCV. Collectively, these early studies on potential impact of Tregs in liver disease combined with the evolving knowledge about regulatory T cells define a new direction in studying the pathogenetic mechanisms for various liver diseases, including viral hepatitis, liver cancer, autoimmune liver disease, transplant tolerance, fulminant hepatitis, alcoholic liver disease and HIV/HCV coinfection. They also provide potential immunotherapeutic opportunities taking the knowledge gained at the bench to the bedside. I would like to acknowledge Dr. Antonio Bertoletti for generously sharing his preprint for Journal of Virology.