T cells with a CD4+CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection

Chronic hepatitis C virus (HCV) infection is associated with impaired proliferative, cytokine, and cytotoxic effector functions of HCV-specific CD8(+) T cells that probably contribute significantly to viral persistence. Here, we investigated the potential role of T cells with a CD4(+)CD25(+) regulatory phenotype in suppressing virus-specific CD8(+) T-cell proliferation during chronic HCV infection. In vitro depletion studies and coculture experiments revealed that peptide specific proliferation as well as gamma interferon production of HCV-specific CD8(+) T cells were inhibited by CD4(+)CD25(+) T cells. This inhibition was dose dependent, required direct cell-cell contact, and was independent of interleukin-10 and transforming growth factor beta. Interestingly, the T-cell-mediated suppression in chronically HCV-infected patients was not restricted to HCV-specific CD8(+) T cells but also to influenza virus-specific CD8(+) T cells. Importantly, CD4(+)CD25(+) T cells from persons recovered from HCV infection and from healthy blood donors exhibited significantly less suppressor activity. Thus, the inhibition of virus-specific CD8(+) T-cell proliferation was enhanced in chronically HCV-infected patients. This was associated with a higher frequency of circulating CD4(+)CD25(+) cells observed in this patient group. Taken together, our results suggest that chronic HCV infection leads to the expansion of CD4(+)CD25(+) T cells that are able to suppress CD8(+) T-cell responses to different viral antigens. Our results further suggest that CD4(+)CD25(+) T cells may contribute to viral persistence in chronically HCV-infected patients and may be a target for immunotherapy of chronic hepatitis C.     

Cellular immune responses associated with occult hepatitis C virus infection of the liver

Occult hepatitis C virus (HCV) infection is a type of recently identified chronic infection that is evidenced only by detection of HCV RNA in liver; patients consistently test negative for antibodies to HCV and HCV RNA in serum. Using ex vivo and in vitro measures of T-cell responses, we have identified functional virus-specific memory CD4(+) and CD8(+) T cells in the peripheral blood of patients with occult HCV infection. The features of the virus-specific T cells were consistent with immune surveillance functions, supporting previous exposure to HCV. In addition, the magnitudes of CD4(+) and CD8(+) T-cell responses were in parallel and correlated inversely with the extent of liver HCV infection. The detection of HCV-specific T cells in individuals in whom HCV RNA can persist in the liver despite the absence of viremia and antibodies indicates that HCV replication is prolonged in the face of virus-specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate that HCV-specific cellular immune responses are markers not only of previous exposure to and recovery from HCV but also of ongoing occult HCV infection.     

Bone marrow of persistently hepatitis C virus-infected individuals accumulates memory CD8+ T cells specific for current and historical viral antigens: a study in patients with benign hematological disorders

The role of virus-specific T cells in hepatitis C virus (HCV) pathogenesis is not clear. Existing knowledge on the frequency, phenotype, and behavior of these cells comes from analyses of blood and liver, but other lymphoid compartments that may be important sites for functionally mature T cells have not yet been analyzed. We studied HCV-specific T cells from bone marrow, in comparison to those from peripheral blood and liver biopsy tissue, from 20 persistently HCV-infected patients with benign hematological disorders. Bone marrow contained a sizeable pool of CD8(+) T cells specific for epitopes from structural and nonstructural HCV proteins. These cells displayed the same effector memory phenotype as liver-derived equivalents and the same proliferative potential as blood-derived equivalents but had greater antiviral effector functions such as Ag-specific cytotoxicity and IFN-gamma production. These features were not shared by influenza virus-specific CD8(+) T cells in the same bone marrow samples. Despite their highly differentiated phenotype and activated status, some bone marrow-resident HCV-specific CD8(+) T cells were not directed against the infecting virus but, instead, against historical HCV Ags (i.e., viral species of a previous infection or minor viral species of the current infection). These findings provide a snapshot view of the distribution, differentiation, and functioning of virus-specific memory T cells in patients with persistent HCV infection.     

Regulatory T cells suppress in vitro proliferation of virus-specific CD8+ T cells during persistent hepatitis C virus infection

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8(+) T cells. The role of CD4(+)CD25(+) T regulatory (T(reg)) cells in priming and expanding virus-specific CD8(+) T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8(+) T-cell proliferation and gamma interferon (IFN-gamma) frequency were analyzed with/without depletion of T(reg) cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4(+)CD25(+) T(reg) cells inhibited anti-CD3/CD28 CD8(+) T-cell proliferation and perforin expression. Depletion of CD4(+)CD25(+) T(reg) cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-gamma-expressing CD8(+) T cells. Although stimulated CD8(+) T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4(+)CD25(+) regulatory T cells on CD8(+) T-cell proliferation. In conclusion, marked CD4(+)CD25(+) regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8(+) T-cell responses and viral persistence.     

The clearance of hepatitis C virus infection in chimpanzees may not necessarily correlate with the appearance of acquired immunity

Clearance of hepatitis C virus (HCV) infection in humans and chimpanzees is thought to be associated with the induction of strong T-cell responses. We studied four chimpanzees infected with HCV derived from an infectious full-length HCV genotype 1b cDNA. Two of the chimpanzees cleared the infection to undetectable levels for more than 12 months of follow-up; the other two became persistently infected. Detailed analyses of HCV-specific immune responses were performed during the courses of infection in these chimpanzees. Only weak and transient T helper responses were detected during the acute phase in all four chimpanzees. A comparison of the frequency of gamma interferon (IFN-gamma)-producing CD4(+) and CD8(+) T cells in peripheral blood by ELISpot assay did not reveal any correlation between viral clearance and T-cell responses. In addition, analyses of IFN-gamma, IFN-alpha, and interleukin-4 mRNA levels in liver biopsies, presumably indicative of intrahepatic T-cell responses, revealed no distinct pattern in these chimpanzees with respect to infection outcome. The present study suggests that the outcome of HCV infection in chimpanzees is not necessarily attributable to HCV sequence variation and that chimpanzees may recover from HCV infection by mechanisms other than the induction of readily detectable HCV-specific T-cell responses.     

Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression

The majority of people infected with hepatitis C virus (HCV) fail to generate or maintain a T-cell response effective for viral clearance. Evidence from murine chronic viral infections shows that expression of the coinhibitory molecule PD-1 predicts CD8+ antiviral T-cell exhaustion and may contribute to inadequate pathogen control. To investigate whether human CD8+ T cells express PD-1 and demonstrate a dysfunctional phenotype during chronic HCV infection, peripheral and intrahepatic HCV-specific CD8+ T cells were examined. We found that in chronic HCV infection, peripheral HCV-specific T cells express high levels of PD-1 and that blockade of the PD-1/PD-L1 interaction led to an enhanced proliferative capacity. Importantly, intrahepatic HCV-specific T cells, in contrast to those in the periphery, express not only high levels of PD-1 but also decreased interleukin-7 receptor alpha (CD127), an exhausted phenotype that was HCV antigen specific and compartmentalized to the liver, the site of viral replication.     

Identification and in vitro expansion of functional antigen-specific CD25+ FoxP3+ regulatory T cells in hepatitis C virus infection

CD4(+)CD25(+) regulatory T cells (CD25(+) Tregs) play a key role in immune regulation. Since hepatitis C virus (HCV) persists with increased circulating CD4(+)CD25(+) T cells and virus-specific effector T-cell dysfunction, we asked if CD4(+)CD25(+) T cells in HCV-infected individuals are similar to natural Tregs in uninfected individuals and if they include HCV-specific Tregs using the specific Treg marker FoxP3 at the single-cell level. We report that HCV-infected patients display increased circulating FoxP3(+) Tregs that are phenotypically and functionally indistinguishable from FoxP3(+) Tregs in uninfected subjects. Furthermore, HCV-specific FoxP3(+) Tregs were detected in HCV-seropositive persons with antigen-specific expansion, major histocompatibility complex class II/peptide tetramer binding affinity, and preferential suppression of HCV-specific CD8 T cells. Transforming growth factor beta contributed to antigen-specific Treg expansion in vitro, suggesting that it may contribute to antigen-specific Treg expansion in vivo. Interestingly, FoxP3 expression was also detected in influenza virus-specific CD4 T cells. In conclusion, functionally active and virus-specific FoxP3(+) Tregs are induced in HCV infection, thus providing targeted immune regulation in vivo. Detection of FoxP3 expression in non-HCV-specific CD4 T cells suggests that immune regulation through antigen-specific Treg induction extends beyond HCV.     

Analysis of CD127 and KLRG1 expression on hepatitis C virus-specific CD8+ T cells reveals the existence of different memory T-cell subsets in the peripheral blood and liver

The differentiation and functional status of virus-specific CD8+ T cells is significantly influenced by specific and ongoing antigen recognition. Importantly, the expression profiles of the interleukin-7 receptor alpha chain (CD127) and the killer cell lectin-like receptor G1 (KLRG1) have been shown to be differentially influenced by repetitive T-cell receptor interactions. Indeed, antigen-specific CD8+ T cells targeting persistent viruses (e.g., human immunodeficiency virus and Epstein-Barr virus) have been shown to have low CD127 and high KLRG1 expressions, while CD8+ T cells targeting resolved viral antigens (e.g., FLU) typically display high CD127 and low KLRG1 expressions. Here, we analyzed the surface phenotype and function of hepatitis C virus (HCV)-specific CD8+ T cells. Surprisingly, despite viral persistence, we found that a large fraction of peripheral HCV-specific CD8+ T cells were CD127+ and KLRG1- and had good proliferative capacities, thus resembling memory cells that usually develop following acute resolving infection. Intrahepatic virus-specific CD8+ T cells displayed significantly reduced levels of CD127 expression but similar levels of KLRG1 expression compared to the peripheral blood. These results extend previous studies that demonstrated central memory (CCR7+) and early-differentiated phenotypes of HCV-specific CD8+ T cells and suggest that insufficient stimulation of virus-specific CD8+ T cells by viral antigen may be responsible for this alteration in HCV-specific CD8+ T-cell differentiation during chronic HCV infection.     

Impaired hepatitis C virus (HCV)-specific effector CD8+ T cells undergo massive apoptosis in the peripheral blood during acute HCV infection and in the liver during the chronic phase of infection

A majority of patients infected with hepatitis C virus (HCV) do not sustain an effective T-cell response, and viremia persists. The mechanism leading to failure of the HCV-specific CD8⁺ T-cell response in patients developing chronic infection is unclear. We investigated apoptosis susceptibility of HCV-specific CD8⁺ T cells during the acute and chronic stages of infection. Although HCV-specific CD8⁺ T cells in the blood during the acute phase of infection and in the liver during the chronic phase were highly activated and expressed an effector phenotype, the majority was undergoing apoptosis. In contrast, peripheral blood HCV-specific CD8⁺ T cells during the chronic phase expressed a resting memory phenotype. Apoptosis susceptibility of HCV-specific CD8⁺ T cells was associated with very high levels of programmed death-1 (PD-1) and low CD127 expression and with significant functional T-cell deficits. Further evaluation of the “death phase” of HCV-specific CD8⁺ T cells during acute HCV infection showed that the majority of cells were dying by a process of cytokine withdrawal, mediated by activated caspase 9. Contraction during the acute phase occurred rapidly via this process despite the persistence of the virus. Remarkably, in the chronic phase of HCV infection, at the site of infection in the liver, a substantial frequency of caspase 9-mediated T-cell death was also present. This study highlights the importance of cytokine deprivation-mediated apoptosis with consequent down-modulation of the immune response to HCV during acute and chronic infections.     

Hepatitis C virus (HCV)-specific CD8+ cells produce transforming growth factor beta that can suppress HCV-specific T-cell responses

Hepatitis C virus (HCV)-specific T-cell responses are rarely detected in peripheral blood, especially in the presence of human immunodeficiency virus (HIV) coinfection. Based on recent evidence that T-regulatory cells may be increased in chronic HCV, we hypothesized that functional blockade of regulatory cells could raise HCV-specific responses and might be differentially regulated in the setting of HIV coinfection. Three groups of subjects were studied: HCV monoinfected, HCV-HIV coinfected, and healthy controls. Frequencies of peripheral T cells specific for peptides derived from HCV core, HIV type 1 p24, and recall antigens were analyzed by gamma interferon (IFN-gamma) enzyme-linked immuno-spot assay. HCV-specific T-cell responses were very weak in groups with HCV and HCV-HIV infections. Addition of blocking antibodies against transforming growth factor beta1 (TGF-beta1), -2, and -3 and interleukin-10 specifically increased the HCV-specific T-cell responses in both infected groups; however, this increase was attenuated in the group with HCV-HIV coinfection compared to HCV infection alone. No increase in recall antigen- or HIV-specific responses was observed. Flow cytometric sorter analysis demonstrated that regulatory-associated cytokines were produced by HCV-specific CD3(+)CD8(+)CD25(-) cells. Enhancement of the IFN-gamma effect was observed for both CD4 and CD8 T cells and was mediated primarily by TGF-beta1, -2, and -3 neutralization. In conclusion, blockade of TGF-beta secretion could enhance peripheral HCV-specific T-cell responses even in the presence of HIV coinfection.     

Hepatitis C virus-specific T-cell gamma interferon and proliferative responses are more common in perihepatic lymph nodes than in peripheral blood or liver

The activation state, differentiation state, and functions of liver lymphocytes and perihepatic lymph nodes during chronic hepatitis C virus (HCV) infection are not well understood. Here, we performed phenotypic and functional analyses of freshly prepared lymphocytes isolated from the livers, perihepatic lymph nodes, and peripheral blood compartments of chronic HCV-infected and disease control subjects with end-stage liver disease undergoing liver transplantation. We measured lymphocyte subset frequency and memory T-cell gamma interferon (IFN-γ) and proliferative responses to HCV peptide and control viral antigens in direct ex vivo assays. We found higher frequencies of CD4 cells in the lymph node compartment than in the other compartments for both HCV-infected and disease control subjects. Lymph node CD4 and CD8 cells less commonly expressed the terminal differentiation marker CD57, a finding consistent with an earlier differentiation state. In HCV-infected subjects, HCV-specific IFN-γ-producing and proliferative responses were commonly observed in the lymph node fraction, while they were uncommonly observed in the peripheral blood or liver fractions. In contrast, control viral CD4 protein antigen and CD8 peptide antigen-specific IFN-γ responses were commonly observed in the periphery and uncommonly observed in the lymph nodes of these same subjects. These findings are consistent with a selective defect in HCV-specific T-cell effector function or distribution in patients with advanced chronic HCV infection. The high frequency of HCV-reactive T cells in perihepatic lymph nodes indicates that a failure to generate or sustain T-lymphocyte HCV reactivity is not responsible for the paucity of functional cells even in end-stage liver disease.     

High-potency human immunodeficiency virus vaccination leads to delayed and reduced CD8+ T-cell expansion but improved virus control

CD8(+) T lymphocytes are thought to play an important role in the control of acute and chronic human immunodeficiency virus infections. However, there is a significant delay between infection and the first observed increase in virus-specific CD8(+) T-cell numbers. Prior to this time, viral kinetics are not significantly different between controls and vaccinees. Surprisingly, higher initial virus-specific CD8(+) T-cell numbers lead to a longer delay prior to initial CD8(+) T-cell expansion, and slower CD8(+) T-cell increases. Nevertheless, higher initial CD8(+) T-cell numbers were associated with reduced peak and chronic viral loads and reduced CD4(+) T-cell depletion.     

HCV Specific IL-21 Producing T Cells but Not IL-17A Producing T Cells Are Associated with HCV Viral Control in HIV/HCV Coinfection

BackgroundDecreased hepatitis C virus (HCV) clearance, faster cirrhosis progression and higher HCV RNA levels are associated with Human Immunodeficiency virus (HIV) coinfection. The CD4⁺ T helper cytokines interleukin (IL)-21 and IL-17A are associated with virus control and inflammation, respectively, both important in HCV and HIV disease progression. Here, we examined how antigen-specific production of these cytokines during HCV mono and HIV/HCV coinfection was associated with HCV virus control.MethodsWe measured HCV-specific IL-21 and IL-17A production by transwell cytokine secretion assay in PBMCs from monoinfected and coinfected individuals. Viral control was determined by plasma HCV RNA levels.ResultsIn acutely infected individuals, those able to establish transient/complete HCV viral control tended to have stronger HCV-specific IL-21-production than non-controllers. HCV-specific IL-21 production also correlated with HCV viral decline in acute infection. Significantly stronger HCV-specific IL-21 production was detected in HAART-treated coinfected individuals. HCV-specific IL-17A production was not associated with lower plasma HCV RNA levels in acute or chronic HCV infection and responses were stronger in HIV coinfection. HCV-specific IL-21/ IL-17A responses did not correlate with microbial translocation or fibrosis. Exogenous IL-21 treatment of HCV-specific CD8⁺ T cells from monoinfected individuals enhanced their function although CD8⁺ T cells from coinfected individuals were somewhat refractory to the effects of IL-21.ConclusionsThese data show that HCV-specific IL-21 and IL-17A-producing T cells are induced in HIV/HCV coinfection. In early HIV/HCV coinfection, IL-21 may contribute to viral control, and may represent a novel tool to enhance acute HCV clearance in HIV/HCV coinfected individuals.     

High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome

We monitored expression of PD-1 (a mediator of T-cell exhaustion and viral persistence) on hepatitis C virus (HCV)-specific CD8⁺ and CD4⁺ T cells from blood and liver during acute and chronic infections and after the resolved infection stage. PD-1 expression on HCV-specific T cells was high early in acute infection irrespective of clinical outcome, and most cells continued to express PD-1 in resolved and chronic stages of infection; intrahepatic expression levels were especially high. Our results suggest that an analysis of PD-1 expression alone is not sufficient to predict infection outcome or to determine T-cell functionality in HCV infection.     

Differences in hepatitis C virus (HCV)-specific CD8 T-cell phenotype during pegylated alpha interferon and ribavirin treatment are related to response to antiviral therapy in patients chronically infected with HCV

CD8 T cells play a major role in antiviral immune responses. Their importance for progression to chronic hepatitis C and response to treatment are still unclear. To address these issues, hepatitis C virus (HCV)-specific CD8 T-cell responses were monitored, at the single-cell level, using HLA class I pentamers specific for HCV core and HCV NS3 epitopes, in 23 chronically infected patients during treatment with pegylated alpha interferon and ribavirin. Patients who presented a sustained-response to therapy had stronger HCV-specific CD8 T-cell responses at all time points studied. Moreover, there were clear differences in the phenotypes of these cells during therapy: in responder patients, terminally differentiated effector cells increased more rapidly, and their frequency was always higher than in nonresponder patients. Sustained-responder patients also showed a higher frequency of HCV-specific CD8 T cells producing cytotoxic factors. Overall, a late and inefficient differentiation process of HCV-specific CD8 T cells might be associated with lack of response to treatment. A better knowledge of the mechanisms underlying this impairment may be important for the development of new therapeutic strategies to maintain, restore, or increase CD8 T-cell effectiveness in chronic HCV infection.     

The early kinetics of cytomegalovirus-specific CD8+ T-cell responses are not affected by antigen load or the absence of perforin or gamma interferon

Both innate and adaptive immune responses participate in the control of murine cytomegalovirus (mCMV) infection. In some mouse strains, like BALB/c, the control of infection relies on the activities of CD8(+) T cells. mCMV-specific CD8(+) T-cell responses are unusual in that, even after mCMV has been controlled in the periphery, the numbers of circulating virus-specific CD8(+) T cells remain high compared to those observed in other viral infections. To better understand the generation and maintenance of mCMV-specific CD8(+) T-cell responses, we evaluated how antigen load and effector molecules, such as perforin (Prf) and gamma interferon (IFN-gamma), influence these responses during acute infection in vivo. Viral burden affected the magnitude, but not the early kinetics, of antigen-specific CD8(+) T-cell responses. Similarly, the magnitude of virus-specific CD8(+) T-cell expansion was affected by Prf and IFN-gamma, but contraction of antigen-specific responses occurred normally in both Prf- and IFN-gamma-deficient mice. These data indicate that control of mCMV-specific CD8(+) T-cell expansion and contraction is more complex than anticipated and, despite the role of Prf or IFN-gamma in controlling viral replication, a full program of T-cell expansion and contraction can occur in their absence.     

Diminished primary and secondary influenza virus-specific CD8(+) T-cell responses in CD4-depleted Ig(-/-) mice

Optimal expansion of influenza virus nucleoprotein (D(b)NP(366))-specific CD8(+) T cells following respiratory challenge of naive Ig(-/-) microMT mice was found to require CD4(+) T-cell help, and this effect was also observed in primed animals. Absence of the CD4(+) population was consistently correlated with diminished recruitment of virus-specific CD8(+) T cells to the infected lung, delayed virus clearance, and increased morbidity. The splenic CD8(+) set generated during the recall response in Ig(-/-) mice primed at least 6 months previously showed a normal profile of gamma interferon production subsequent to short-term, in vitro stimulation with viral peptide, irrespective of a concurrent CD4(+) T-cell response. Both the magnitude and the localization profiles of virus-specific CD8(+) T cells, though perhaps not their functional characteristics, are thus modified in mice lacking CD4(+) T cells.     

Early interferon therapy for hepatitis C virus infection rescues polyfunctional, long-lived CD8+ memory T cells

The majority of acute hepatitis C virus (HCV) infections progress to chronicity and progressive liver damage. Alpha interferon (IFN-alpha) antiviral therapy achieves the highest rate of success when IFN-alpha is administered early during the acute phase, but the underlying mechanisms are unknown. We used a panel of major histocompatibility complex class I tetramers to monitor the phenotypic and functional signatures of HCV-specific T cells during acute HCV infection with different infection outcomes and during early IFN therapy. We demonstrate that spontaneous resolution correlates with the early development of polyfunctional (IFN-gamma- and IL-2-producing and CD107a(+)) virus-specific CD8(+) T cells. These polyfunctional T cells are distinguished by the expression of CD127 and Bcl-2 and represent a transitional memory T-cell subset that exhibits the phenotypic and functional signatures of both central and effector memory T cells. In contrast, HCV-specific CD8(+) T cells in acute infections evolving to chronicity expressed low levels of CD127 and Bcl-2, exhibited diminished proliferation and cytokine production, and eventually disappeared from the periphery. Early therapeutic intervention with pegylated IFN-alpha rescued polyfunctional memory T cells expressing high levels of CD127 and Bcl-2. These cells were detectable for up to 1 year following discontinuation of therapy. Our results suggest that the polyfunctionality of HCV-specific T cells can be predictive of the outcome of acute HCV infection and that early therapeutic intervention can reconstitute the pool of long-lived polyfunctional memory T cells.     

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.