Heterologous T cells can help restore function in dysfunctional hepatitis C virus nonstructural 3/4A-specific T cells during therapeutic vaccination

The hepatitis C virus (HCV)-specific T cell response in patients with chronic HCV is dysfunctional. In this study, we aimed at restoring immunological function through therapeutic vaccination in a transgenic mouse model with impaired HCV-specific T cell responses due to a persistent presence of hepatic HCV nonstructural (NS)3/4A Ags. The HCV-specific T cells have an actively maintained dysfunction reflected in reduced frequency, impaired cytokine production, and impaired effector function in vivo, which can be partially restored by blocking regulatory T cells or programmed cell death ligand 1. We hypothesized that the impairment could be corrected by including sequences that created a normal priming environment by recruiting "healthy" heterologous T cells and by activating innate signaling. Endogenously expressed hepatitis B core Ag (HBcAg) can recruit heterologous T cells and activate TLR (TLR7) signaling. Hence, by combining HCV NS3/4A with different forms of HBcAg we found that heterologous sequences somewhat improved activation and expansion of NS3/4A-specific T cells in a wild-type host. Importantly, the signals provided by HBcAg effectively restored the activation of HCV-specific T cells in a tolerant NS3/4A-transgenic mouse model. The adjuvant effect could also be transferred to the priming of dysfunctional HLA-A2-restricted NS3-specific T cells in vivo. Thus, recruiting healthy heterologous T cells to the site of priming may also help restore HCV-specific responses present in a chronically infected host.     

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.     

Two Distinct Functional Patterns of Hepatitis C Virus (HCV)-Specific T Cell Responses in Seronegative,Aviremic Patients

In hepatitis C Virus (HCV) high-risk groups, HCV-specific T cell responses have been detected in seronegative, aviremic persons who have no evidence of HCV infection. Herein, we investigated functional profiles of HCV-specific T-cell responses in seronegative, aviremic patients of a HCV high-risk group. Seventy seven hemodialysis patients with chronic renal disease were analyzed by IFN-γ ELISpot assays, and eight of 71 (11.3%) seronegative, aviremic patients displayed HCV-specific T-cell responses. Their HCV-specific memory T cells were characterized by assessing cytokine polyfunctionality, known to provide antiviral protection. By intracellular staining of IFN-γ, TNF-α, IL-2 and MIP-1β, we identified two distinct populations in the seronegative, aviremic patients: polyfunctional responders and TNF-α-predominant responders. In further analysis, occult HCV infection was excluded as a cause of the HCV-specific T cell response via secondary nested RT-PCR of HCV RNA in peripheral blood mononuclear cell samples. HCV-specific T cells targeted multiple epitopes including non-structural proteins in a single patient, implying that their T cells might have been primed by HCV proteins synthesized within the host. We conclude that HCV-specific memory T cells of seronegative, aviremic patients arise from authentic HCV replication in the host, but not from current occult HCV infection. By functional pattern of HCV-specific T cells, there are two distinct populations in these patients: polyfunctional responders and TNF-α-predominant responders.     

Cellular immune responses persist and humoral responses decrease two decades after recovery from a single-source outbreak of hepatitis C

As acute hepatitis C virus (HCV) infection is clinically inapparent in most cases, the immunologic correlates of recovery are not well defined. The cellular immune response is thought to contribute to the elimination of HCV-infected cells and a strong HCV-specific T-helper-cell (Th) response is associated with recovery from acute hepatitis C (ref. 2). However, diagnosis of resolved hepatitis C is based at present on the detection of HCV-specific antibodies and the absence of detectable HCV RNA, and detailed comparison of the humoral and cellular immune response has been hampered by the fact that patient cohorts as well as HCV strains are usually heterogeneous and that clinical data from acute-phase and long-term follow-up after infection generally are not available. We studied a cohort of women accidentally exposed to the same HCV strain of known sequence and found that circulating HCV-specific antibodies were undetectable in many patients 18-20 years after recovery, whereas HCV-specific helper and cytotoxic T-cell responses with an interferon (IFN)-gamma-producing (Tc1) phenotype persisted. The data indicate these HCV-specific CD4 + and CD8+ T cells are biomarkers for a prior HCV exposure and recovery. Because of undetectable antibodies against HCV, the incidence of self-limited HCV infections and recovery may be underestimated in the general population.     

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.     

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.     

Defining target antigens for CD25+ FOXP3 + IFN-gamma- regulatory T cells in chronic hepatitis C virus infection

The mechanism behind the apparent lack of effective antiviral immune responses in chronic hepatitis C virus (HCV) patients is poorly understood. It remains unclear if natural regulatory T cells (Treg) contribute to the induction and maintenance of HCV persistence. We herein report for the first time that CD25(high)IFN-gamma(-)FOXP3(high) Tregs can be rapidly induced by culturing peripheral blood mononuclear cells (PBMCs) of HCV-positive patients with HCV protein-derived peptides. The HCV-specific Tregs, generally CD4(+)CD45RO(+), did not proliferate in response to HCV peptide and failed to produce interferon (IFN)-gamma, in distinct contrast to antiviral effector cells. Stimulation of healthy donor PBMCs with HCV peptides did not result in CD25 and FOXP3 upregulation above non-antigen background. To further investigate the antigen specificity of these potentially disease-associated natural Tregs, CD25(+) cells were isolated from PBMCs, labeled with carboxyfluorescein diacetate succinimidylester and added back to CD25-depleted PBMCs, and the co-cultures were then stimulated with individual peptides derived from the HCV core protein. We found that the actual peptide that can stimulate Treg varied between patients, but within any given subject only a small number of the peptides were able to stimulate Treg, suggesting the existence of dominant Treg epitopes. Although functional experiments for these peptides are ongoing in our laboratory, data presented here suggests that HCV-specific natural Tregs are abundant in infected individuals, in contrast to the extremely low frequency of anti-HCV effector T cells, supporting the view that natural Treg may be implicated in host immune tolerance during HCV infection.     

Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection

The cellular immune response contributes to clearance of hepatitis C virus (HCV) and persists for decades after recovery from infection. The immunological basis for the inefficiency of the cellular immune response in chronically infected persons is not known. Here, we used four HLA-A2 tetramers, specific for two HCV core and two HCV NS3 epitopes, to investigate at the single-cell level effector function and phenotype of HCV-specific CD8+ T cells in 20 chronically infected and 12 long-term recovered patients. Overall, HCV-specific, tetramer+ T cells were more frequently found in PBMCs of chronically infected patients than in those of recovered patients. However, when compared with HCV-tetramer+ T cells of recovered patients, they displayed an impaired proliferative capacity. As a result of the impaired proliferative capacity, HCV-specific T cell lines derived from chronically infected patients displayed less peptide-specific cytotoxicity than those from recovered patients. In addition, proliferation and ex vivo IFN-gamma production of HCV-tetramer+ cells, but not influenza-virus-specific T cells, were defective in chronically infected patients and could not be restored by in vitro stimulation with peptide and IL-2. At least three distinct phenotypes of HCV-specific CD8+ T cells were identified and associated with certain functional characteristics. In addition, impairment of proliferative, cytokine, and cytotoxic effector functions of tetramer+ T cells in viremic patients was associated with weak ex vivo HCV-specific CD4+ T cell responses. Thus, the defective functions of HCV-specific CD8+ T cells might contribute to viral persistence in chronically infected patients, and knowledge on their reversibility may facilitate the development of immunotherapeutic vaccines.     

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.     

HCV-specific interleukin-21+CD4+ T cells responses associated with viral control through the modulation of HCV-specific CD8+ T cells function in chronic hepatitis C patients

Interleukin-21 (IL-21)+CD4+ T cells are involved in the immune response against hepatitis B virus (HBV) by secreting IL-21. However, the role of IL-21+CD4+ T cells in the immune response against chronic hepatitis C (CHC) virus infection is poorly understood. This study aimed to investigate the role of IL-21+CD4+ T cells in CHC patients and the potential mechanisms. The study subjects included nineteen CHC patients who were grouped by viral load (low, < 10⁶ RNA copies/ml, n = 8; high, > 10⁶ RNA copies/ml, n = 11). The peripheral frequency of HCV-specific IL-21+CD4+ T cells was higher in the low viral load group and was negatively correlated with the serum HCV RNA viral load in all CHC patients. Meanwhile, IL-21+ cells accumulated in the liver in the low viral load group. In vitro, IL-21 treatment increased the expression of proliferation markers and cytolytic molecules on HCV-specific CD8+ T cells. In summary, these findings suggest that HCV-specific IL-21+CD4+ T cells might contribute to HCV control by rescuing HCV-specific CD8+ T cells in CHC patients.     

Cytotoxic capacity of hepatitis C virus (HCV)--specific lymphocytes after in vitro immunization with HCV-derived lipopeptides.

BACKGROUND: Hepatitis C virus (HCV)-derived lipopeptides can induce epitope-specific immune responses in lymphocytes from HCV-naive individuals. We analyzed whether such T cells generated by in vitro immunization with HCV core-derived lipopeptides exert HCV-specific cytolytic activity. METHODS: Using a sensitive flow cytometric cytotoxicity assay we characterized HCV-specific cytotoxicity in T cells generated in vitro with HCV core-derived 25-mer lipopeptides. In addition, we studied expressions of Fas ligand and perforin and interferon-gamma (IFN-gamma) secretion in HLA-A2-HCV(core_35-44) tetramer-positive T cells generated with lipopeptide amino acid 20-44 (LP20-44). RESULTS: CD8+ T cells induced in vitro with HCV core-derived lipopeptides only infrequently exerted HCV-specific cytotoxicity, irrespective of whether antigen-coated T2 cells or autologous B lymphoblasts were used as targets. Detailed analysis of HLA-A2-HCV(core_35-44) tetramer-positive T cells generated with LP20-44 revealed that in vitro immunization resulted in T cells that secreted IFN-gamma after antigen-specific restimulation and that upregulated expression of Fas ligand but not of perforin. CONCLUSIONS: Our data confirm at the functional level that HCV lipopeptides induce antigen-specific T lymphocytes that produce IFN-gamma but exert significant cytotoxicity in only a minority of experiments, probably because expression of cytolytic effector molecules is not enhanced in their granules.     

Intact Dendritic Cell Pathogen-Recognition Receptor Functions Associate with Chronic Hepatitis C Treatment-Induced Viral Clearance

Although studies have addressed the exhaustion of the host''s immune response to HCV and its role in treatment, there is little information about the possible contribution of innate immunity to treatment-induced clearance. We hypothesized that because intact myeloid dendritic cell (MDC) pathogen sensing functions are associated with improved HCV-specific CD8⁺ T cell functionality in some chronically infected patients, it might enhance HCV clearance rate under standard interferon therapy. To investigate this hypothesis, TLR-induced MDC activation and HCV-specific CD8⁺ T cell response quality were monitored longitudinally at the single-cell level using polychromatic flow cytometry in chronically infected patients undergoing interferon therapy. We correlated the immunological, biochemical and virological data with response to treatment. We demonstrate that the clinical efficacy of interferon-induced viral clearance is influenced by the extent to which HCV inhibits MDC functions before treatment, rather than solely on a breakdown of the extrinsic T cell immunosuppressive environment. Thus, viral inhibition of MDC functions before treatment emerges as a co-determining factor in the clinical efficacy of interferon therapy during chronic HCV infection.     

CD127 Expression,Exhaustion Status and Antigen Specific Proliferation Predict Sustained Virologic Response to IFN in HCV/HIV Co-Infected Individuals

Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality in the HIV co-infected population. Interferon-alpha (IFN-α) remains a major component of anti-HCV therapy despite its deleterious effects on the immune system. Furthermore, IFN-α was recently shown to diminish the size of the latent HIV reservoir. The objectives of this study were to monitor the impact of IFN-α on T cell phenotype and proliferation of HIV and HCV-specific T cells during IFN therapy, and to identify immune markers that can predict the response to IFN in HICV/HIV co-infected patients. We performed longitudinal analyses of T cell numbers, phenotype and function in co-infected patients undergoing IFN-α therapy with different outcomes including IFN-α non-responders (NR) (n = 9) and patients who achieved sustained virologic response (SVR) (n = 19). We examined the expression of activation (CD38, HLA-DR), functional (CD127) and exhaustion markers (PD1, Tim-3, CD160 and CD244) on total CD4 and CD8 T cells before, during and after therapy. In addition, we examined the HIV- and HCV-specific proliferative responses against HIV-p24 and HCV-NS3 proteins. Frequencies of CD127⁺ CD4 T cells were higher in SVR than in NR patients at baseline. An increase in CD127 expression on CD8 T cells was observed after IFN-α therapy in all patients. In addition, CD8 T cells from NR patients expressed a higher exhaustion status at baseline. Finally, SVR patients exhibited higher proliferative response against both HIV and HCV antigens at baseline. Altogether, SVR correlated with higher expression of CD127, lower T cell exhaustion status and better HIV and HCV proliferative responses at baseline. Such factors might be used as non-invasive methods to predict the success of IFN–based therapies in co-infected individuals.     

Induction of Intrahepatic HCV NS4B,NS5A and NS5B-Specific Cellular Immune Responses following Peripheral Immunization

Numerous studies have suggested that an effective Hepatitis C Virus (HCV) vaccine must induce strong cytotoxic and IFN-γ+ T cell responses targeting the non-structural region of the virus. Most importantly, these responses must be able to migrate into and remain functional within the liver, an organ known to cause T cell tolerance. Using three novel HCV DNA vaccines encoding non-structural proteins NS4B, NS5A and NS5B, we assessed the ability of peripheral immunization to induce functional intrahepatic immunity both in the presence and absence of cognate HCV antigen expression within the liver. We have shown that these constructs induced potent HCV-specific CD4+ and CD8+ T cell responses in the spleen of C57BL/6 mice and that these responses were detected within the liver following peripheral immunization. Additionally, using a transfection method to express HCV antigen within the liver, we showed that intrahepatic HCV-specific T cells remained highly functional within the liver and retained the ability to become highly activated as evidenced by upregulation of IFN-γ and clearance of HCV protein expressing hepatocytes. Taken together, these findings suggest that peripheral immunization can induce potent HCV-specific T cell responses able to traffic to and function within the tolerant environment of the liver.     

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.     

Comparison of Immune Restoration in Early versus Late Alpha Interferon Therapy against Hepatitis C Virus

Early alpha interferon (IFN-α) therapy against hepatitis C virus (HCV) rescues polyfunctional, virus-specific memory CD8⁺ T cells, but whether immune restoration is possible during late therapy remains controversial. We compared immune restoration of HCV-specific memory T cells in patients who cleared HCV infection spontaneously and following early or late IFN therapy. Multifunctional CD4⁺ and CD8⁺ memory T cells were detected in spontaneous resolvers and in individuals treated early following an acute infection. In contrast, limited responses were detected in patients treated during chronic infection, and the phenotype of HCV-specific cells was influenced by autologous viral sequences. Our data suggest that irreversible damage to the HCV-specific memory T-cell response is associated with chronic HCV infection.The majority of acute hepatitis C virus (HCV) infections become chronic, with persistent viremia and serious liver complications (12). Alpha interferon (IFN-α)-based therapy is the only approved treatment for chronic HCV; its success rate ranges from 40 to 90% depending on the infecting genotype (9, 18). The success of therapy is characterized by a sustained virological response (SVR), defined as undetectable HCV RNA in plasma at 6 months after termination of therapy. SVR rates are greatly enhanced if therapy is started between 3 and 6 months following acute HCV infection, but the underlying mechanisms are not well understood (27, 28). We have demonstrated that early interferon therapy for HCV can rescue and select for long-lived polyfunctional CD8⁺ memory T cells (1). Treatment-induced memory T cells were similar in phenotype and function to natural memory T cells generated following spontaneously resolved infection. They expressed high levels of CD127 and Bcl-2 (CD127^hi, Bcl-2^hi) and low levels of PD1 (PD1^lo) and were polyfunctional in nature (1). However, restoration of HCV-specific memory CD4⁺ T cells has not been examined. Furthermore, whether immune restoration is possible following the late initiation of therapy during the chronic phase remains controversial. Kamal et al. demonstrated that SVR is associated with a recovery in HCV-specific CD4⁺ T-cell responses (13). In contrast, Barnes et al. and Rahman et al. demonstrated that the induction of HCV-specific immunity during therapy does not correlate with outcomes (2, 21).     

Cutting edge: programmed death-1 expression is increased on immunocytes in chronic hepatitis C virus and predicts failure of response to antiviral therapy: race-dependent differences.

Up-regulation of programmed death-1 (PD-1) identifies exhausted T cells in various mouse and human viral models including chronic hepatitis C virus (HCV) infection, which is characterized by impaired CTL function. A large proportion of patients fail to eradicate HCV with current IFN-based antiviral therapy; in particular, African Americans are less likely to respond, but the mechanisms for these differences are not fully elucidated. In this study, in 72 treatment-naive patients with persistent HCV we found that PD-1 was significantly up-regulated on CD4(+) and CD8(+) T cells, HCV-specific CTLs, and NK cells. Increased PD-1 on HCV-specific CTLs was significantly associated with failed early and sustained virologic response to therapy in African American but not Caucasian American patients. Patients with sustained virologic response showed decreases in PD-1 on total CD4(+) T cells, HCV-specific CTLs, and the CD56(bright) NK subset after therapy completion. Collectively, these data indicate that PD-1 is critical in persistent HCV and successful therapy results in global down-regulation of its expression.     

Hepatitis C virus structural proteins impair dendritic cell maturation and inhibit in vivo induction of cellular immune responses

Hepatitis C virus (HCV) chronic infection is characterized by low or undetectable cellular immune responses against HCV antigens. Some studies have suggested that HCV proteins manipulate the immune system by suppressing the specific antiviral T-cell immunity. We have previously reported that the expression of HCV core and E1 proteins (CE1) in dendritic cells (DC) impairs their ability to prime T cells in vitro. We show here that immunization of mice with immature DC transduced with an adenovirus encoding HCV core and E1 antigens (AdCE1) induced lower CD4(+)- and CD8(+)-T-cell responses than immunization with DC transduced with an adenovirus encoding NS3 (AdNS3). However, no differences in the strength of the immune response were detected when animals were immunized with mature DC subsequently transduced with AdCE1 or AdNS3. According to these findings, we observed that the expression of CE1 in DC inhibited the maturation caused by tumor necrosis factor alpha or CD40L but not that induced by lipopolysaccharide. Blockade of DC maturation by CE1 was manifested by a lower expression of maturation surface markers and was associated with a reduced ability of AdCE1-transduced DC to activate CD4(+)- and CD8(+)-T-cell responses in vivo. Our results suggest that HCV CE1 proteins modulate T-cell responses by decreasing the stimulatory ability of DC in vivo via inhibition of their physiological maturation pathways. These findings are relevant for the design of therapeutic vaccination strategies in HCV-infected patients.     

Galectin-9 and IL-21 Mediate Cross-regulation between Th17 and Treg Cells during Acute Hepatitis C

Loss of CD4 T cell help correlates with virus persistence during acute hepatitis C virus (HCV) infection, but the underlying mechanism(s) remain unknown. We developed a combined proliferation/intracellular cytokine staining assay to monitor expansion of HCV-specific CD4 T cells and helper cytokines expression patterns during acute infections with different outcomes. We demonstrate that acute resolving HCV is characterized by strong Th1/Th17 responses with specific expansion of IL-21-producing CD4 T cells and increased IL-21 levels in plasma. In contrast, viral persistence was associated with lower frequencies of IL-21-producing CD4 T cells, reduced proliferation and increased expression of the inhibitory receptors T cell immunoglobulin and mucin-domain-containing-molecule-3 (Tim-3), programmed death 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) on HCV-specific CD8 T cells. Progression to persistent infection was accompanied by increased plasma levels of the Tim-3 ligand Galectin-9 (Gal-9) and expansion of Gal-9 expressing regulatory T cells (Tregs). In vitro supplementation of Tim-3^high HCV-specific CD8 T cells with IL-21 enhanced their proliferation and prevented Gal-9 induced apoptosis. siRNA-mediated knockdown of Gal-9 in Treg cells rescued IL-21 production by HCV-specific CD4 T cells. We propose that failure of CD4 T cell help during acute HCV is partially due to an imbalance between Th17 and Treg cells whereby exhaustion of both CD4 and CD8 T cells through the Tim-3/Gal-9 pathway may be limited by IL-21 producing Th17 cells or enhanced by Gal-9 producing Tregs.