Negative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+ T cells.

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.

Prospective analysis of effector and regulatory CD4+ T cells in chronic HCV patients undergoing combination antiviral therapy.

BACKGROUND/AIMS: The role of HCV-specific CD4(+) T cells and regulatory T cells in influencing the outcome of antiviral therapy is incompletely defined. METHODS: CD4(+) IFN-gamma ELISPOT assays (n=58) and flow cytometric analysis of FoxP3-expressing T regulatory cells (n=62) were performed on patients from the Virahep-C study at baseline, during and after cessation of antiviral therapy. RESULTS: Total HCV-specific IFN-gamma CD4(+) T cell ELISPOT responses did not increase with therapy, but rather decreased by 8 weeks and remained below baseline 24 weeks after cessation of therapy. There were no statistically significant differences with respect to viral kinetics, race and virologic outcome. In contrast, viral relapse after treatment was associated with a three-fold increase in HCV-specific responses. The frequency and phenotype of regulatory T cells during therapy were not significantly different in terms of race, viral kinetic groups or virologic outcome. CONCLUSIONS: A contraction of HCV-specific CD4(+) T cell responses was found during treatment with recovery of responses in patients experiencing virologic relapse after treatment. The levels of FoxP3-expressing regulatory T cells did not vary by race and were not predictive of virologic outcome. Work is ongoing to explore the contribution of mechanisms independent of CD4(+) T cells in therapy-induced viral clearance.

Upregulation of PD-1 expression on circulating and intrahepatic hepatitis C virus-specific CD8+ T cells associated with reversible immune dysfunction.

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.

Phenotypic and functional changes of cytotoxic CD56pos natural T cells determine outcome of acute hepatitis C virus infection.

Innate CD56(pos) natural killer (NK) and natural T (NT) cells comprise important hepatic antiviral effector lymphocytes whose activity is fine-tuned through surface NK receptors (NKRs). Dysregulation of NKRs in patients with long-standing hepatitis C virus (HCV) infection has been shown, but little is known regarding NKRs in acute infection. Treatment-naïve patients with acute HCV (n = 22), including 10 with spontaneous recovery, were prospectively studied. CD56(pos) NT levels were reduced early in acute HCV infection and did not fluctuate over time. In resolving HCV infection, NT cells with a more activated phenotype (lower CD158A and higher natural cytotoxicity receptor expression) at baseline predated spontaneous recovery. Moreover, NKG2A expression on CD56(+) NT cells correlated directly with circulating HCV RNA levels. Deficient interleukin-13 (IL-13) production by NT cells and reduced IL-2-activated killing (LAK) at baseline were associated with the ultimate development of persistence. These results indicate a previously unappreciated role for NT cells in acute HCV infection and identify a potential target for pharmacologic manipulation.

Loss of IL-7 receptor alpha-chain (CD127) expression in acute HCV infection associated with viral persistence.

Interleukin-7 (IL-7) is required for the establishment and maintenance of memory CD4(+) and CD8(+) T lymphocytes, and cells lacking IL-7Ralpha (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-7Ralpha 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.