A number of emerging molecules and pathways have been implicated in mediating the T-cell exhaustion characteristic of chronic viral infection. Not all dysfunctional T cells express PD-1, nor are they all rescued by blockade of the PD-1/PD-1 ligand pathway. In this study, we characterize the expression of T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) in chronic hepatitis C infection. For the first time, we found that Tim-3 expression is increased on CD4 ؉ and CD8 ؉ T cells in chronic hepatitis C virus (HCV) infection. The proportion of dually PD-1/Tim-3-expressing cells is greatest in liver-resident T cells, significantly more so in HCV-specific than in cytomegalovirus-specific cytotoxic T lymphocytes. Tim-3 expression correlates with a dysfunctional and senescent phenotype (CD127 low CD57 high ), a central rather than effector memory profile (CD45RA negative CCR7 high ), and reduced Th1/Tc1 cytokine production. We also demonstrate the ability to enhance T-cell proliferation and gamma interferon production in response to HCV-specific antigens by blocking the Tim-3-Tim-3 ligand interaction. These findings have implications for the development of novel immunotherapeutic approaches to this common viral infection.Hepatitis C virus (HCV) is a major causative agent of chronic hepatitis, affecting approximately 200 million people throughout the world; the majority of individuals exposed to HCV become persistently infected (19). A broad array of functional impairments of virus-specific T cells from early to chronic stages of infection, including exhaustion (decreased antiviral cytokine production, cytotoxicity, and proliferative capacity) (8, 24) and arrested stages of differentiation (1, 13), is supported by considerable evidence. Recently, upregulation of programmed death 1 (PD-1) and downmodulation of CD127 (interleukin-7 [IL-7] receptor) have been linked to functional exhaustion of T cells in chronic HCV infection (5-7, 15, 21, 22). However, not all exhausted T cells express these phenotypic changes, and blockade of the PD-1/PD-L1 signaling pathway does not always reconstitute Th1/Tc1 cytokine production (4, 5), indicating that other molecules may contribute to the exhaustion typically associated with chronic viral infections (9). One such molecule is Tim-3 (T-cell immunoglobulin and mucin domain-containing molecule 3), a membrane protein initially identified on terminally differentiated Th1 but not Th2 cells in mice (9). A recent analysis of human immunodeficiency virus (HIV) infection demonstrates that Tim-3 is upregulated on both CD4 ϩ and CD8 ϩ T cells from patients with chronic infection relative to uninfected individuals and that virus-specific cells expressing high levels of Tim-3 secrete less IFN-␥ than do Tim-3-negative cells (10). In light of these findings, for the first time, this study assessed the expression of Tim-3 in chronic HCV infection. We found a higher frequency of Tim3-expressing CD4 ϩ and CD8 ϩ T cells in chronic HCV infection, with the highest on HCV-specific cytotoxic T...

Infection with hepatitis C virus (HCV) is associated with persistence in the majority of individuals. We demonstrate here that the inhibitory molecule programmed death-1 (PD-1) is significantly upregulated on total and HCV-specific CD8 ؉ cytotoxic T lymphocytes (CTLs) in the peripheral blood and livers of patients with chronic infection compared to subjects with spontaneous HCV resolution, patients with nonviral liver disease, and normal controls. PD-1 expression on cytomegalovirus-specific CTLs also varies according to HCV status and is highest in patients with chronic infection. HCV-specific CTLs that are PD-1 high express higher levels of the senescence marker CD57 than PD-1 low CTLs, and CD57 expression is greater in chronic than in resolved infection. In vitro blockade of PD-1 by monoclonal antibodies specific to its ligands (PD-L1 and PD-L2) results in restoration of functional competence (proliferation and gamma interferon and interleukin-2 secretion) of HCV-specific CTLs, including those residing in the liver. This reversal of CTL exhaustion is evident even in individuals who lack HCV-specific CD4 ؉ T-cell help. Our data indicate that the PD-1/PD-L pathway is critical in persistent HCV infection in humans and represents a potential novel target for restoring function of exhausted HCV-specific CTLs. Hepatitis C virus (HCV) is the major causative agent of chronic hepatitis and has an estimated global prevalence of 3% (31). It is not precisely understood why the majority of individuals exposed to HCV develop viral persistence and only a minority experience spontaneous resolution. Moreover, antiviral therapy is effective in only about half of chronically infected patients, and those who fail antiviral therapy are at increased risk of disease progression, including development of cirrhosis and end-stage liver disease (17). Chronic HCV infection is manifested by cytotoxic T lymphocytes (CTLs) that are functionally impaired or exhausted (decreased antiviral cytokine production, cytotoxicity, and proliferative capacity) (15,30) and may exhibit phenotypic features of early stages of differentiation (1, 18). Recent reports indicate that PD-1 is markedly upregulated on surface of exhausted virus-specific CD8 ϩ T cells in mice with lymphocytic choriomeningitis virus (3) and in humans with human immunodeficiency virus (HIV) infection (8,24,28), and emerging data indicate a significant role for this immunoreceptor in HCV infection (23,25,29). In this regard, hepatic expression of PD-1 mRNA recently was shown to be increased in four acutely infected chimpanzees that subsequently developed persistence in contrast to lower levels in the two animals who spontaneously resolved HCV (26).In the present study, we report the expression of PD-1 on bulk and HCV-specific CTLs from patients with chronic infection and subjects with spontaneous recovery, as well as the consequences of manipulating PD-1/PD-L pathway on proliferation and effector cytokine production by these cells. We find that PD-1 is markedly upregulated in the peri...

Interleukin-7 (IL-7) is required for the establishment and maintenance of memory CD4 ؉ and CD8 ؉ T lymphocytes, and cells lacking IL-7R␣ (CD127) demonstrate impaired IL-2 secretion and have a short life-span. Chronic HCV is characterized by T cells that are functionally impaired and exhibit an immature phenotype. To investigate the potential role of IL-7/IL-7R␣ in the outcome of HCV infection, we used multiparameter flow cytometry to characterize patients with acute infection (n ‫؍‬ 24), long-term chronic infection (12) and normal subjects (13). HCV infection per se resulted in downregulation of CD127 on total CD4 ؉ and CD8 ؉ T lymphocytes as compared to normal controls. Total expression was lowest in those patients who subsequently developed persistence and intermediate in those patients with acute-resolving infection. This reduction affected both naïve and effector/memory T cells. CD127 correlated phenotypically with upregulation of chemokine receptors CCR7 and CXCR4, expression of the anti-apoptotic molecule B cell leukemia/lymphoma 2 (Bcl-2), and enhanced IL-2 production. In six HLA A2-positive patients, we longitudinally tracked tetramer responses to HCV and CMV epitopes; at baseline, reflecting the expression of CD127 on whole T cell populations, viral-specific CTLs in patients who became chronic demonstrated lower CD127. In conclusion, CD127 is a useful marker of functional CD4 ؉ and CD8 ؉ T cells and its expression correlates with virologic outcome of acute HCV. These data provide a mechanistic basis for the observation that CTLs generated in early infection rapidly decline as chronicity is established; CD127 expression should be considered in the design of novel immunotherapeutic approaches. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/ 0270-9139/suppmat/index.html).