Spontaneous resolution of hepatitis C virus (HCV) infection in humans usually affords long-term immunity to persistent viremia and associated liver diseases. Here, we report that memory CD4+ Tcells are essential for this protection. Antibody-mediated depletion of CD4+ Tcells before reinfection of two immune chimpanzees resulted in persistent, low-level viremia despite functional intra-hepatic memory CD8+ Tcell responses. Incomplete control of HCV replication by memory CD8+ Tcells in the absence of adequate CD4+ Tcell help was associated with emergence of viral escape mutations in class I major histocompatibility complex-restricted epitopes and failure to resolve HCV infection.

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Signalling activated by Toll-like receptors (TLRs) can result in the production of tumour necrosis factor alpha (TNF-α) which is implicated in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infection. No study has examined or compared hepatic expression of TLRs in both HCV and HCV/HIV. Liver and peripheral blood mononuclear cells (PBMCs) were obtained from HCV & HCV/HIV-infected patients and PBMCs from HIV-infected patients. Liver RNA was analysed by microarray and reverse transcription quantitative PCR (RT-qPCR). PBMCs were analysed by flow cytometry. Associations with hepatic histology and infection type were sought. Forty-six HCV, 20 HIV and 27 HCV/HIV-infected patients were recruited. Increasing Metavir inflammatory activity score was associated with increased hepatic TLR mRNA by RT-qPCR: TLR2 (P ≤ 0.001), TLR4 (P = 0.008) and TNF-α (P ≤ 0.001). A high degree of correlation was seen between hepatic mRNA expression of TNF-αvs TLR2 (r(2) = 0.66, P < 0.0001) and TLR4 (r(2) = 0.60, P < 0.0001). No differences in TLR gene or protein expression was observed between HCV, HCV/HIV- or HIV-infected groups. Hepatic TLR2, TLR4 and TNF-α mRNA are associated with hepatic inflammation in both HCV and HCV/HIV infection. High correlation between TNF-α and TLR2/TLR4 suggests a role for the innate immune response in TNF-α production. Activation of the innate immune response appears to be independent of infection type.

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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.

GB virus B (GBV-B) is a hepatotropic virus that is closely related to hepatitis C virus (HCV). GBV-B causes acute hepatitis in infected marmosets and tamarins and is therefore a useful small-animal model for the study of HCV. We investigated virus-specific T-cell responses in marmosets infected with GBV-B. Gamma interferon (IFN-␥) enzyme-linked immunospot (ELISPOT) assay responses in the peripheral blood of two marmosetswere assessed throughout the course of GBV-B infection. These T-cell responses were directed against the GBV-B nonstructural proteins 3 (NS3), 4A (NS4A), and 5B (NS5B), and their appearance was temporally associated with clearance of viremia. These marmosets were then rechallenged with GBV-B at least 3 months after clearance of the primary infection to determine if the animals were protected from reinfection. There was no detectable viremia following reinfection, although a sharp increase in T-cell responses against GBV-B proteins was observed. Epitope mapping of T-cell responses to GBV-B was performed with liver and blood samples from both marmosets after rechallenge with GBV-B. Three shared, immunodominant T-cell epitopes within NS3 were identified in animals with multiple common major histocompatibility complex class I alleles. IFN-␥ ELISPOT responses were also detected in the livers of two marmosets that had resolved a primary GBV-B infection. These responses were high in frequency and were directed against epitopes within GBV-B NS3, NS4A, and NS5B proteins. These results indicate that virus-specific T-cell responses are detectable in the liver and blood of GBV-B-infected marmosets and that the clearance of GBV-B is associated with the appearance of these responses.A major impediment in the development of antiviral agents and vaccines against hepatitis C virus (HCV) has been the lack of a convenient small-animal model to test their efficacy. The chimpanzee is the only animal model that supports virus replication and has been used in studies examining vaccine efficacy (12, 26), but due to the high cost involved in their enrollment and care and their endangered status, these animals are beyond the reach of most research groups. Consequently, protective or immunosuppressive regions of the HCV polyprotein that should be incorporated into or excluded from vaccine design are still unknown. A potential small-animal model for studying HCV immunity is the common marmoset (Callithrix jacchus). These animals are relatively inexpensive, are not endangered, and are easily kept in captivity. Furthermore, marmosets are often relatively inbred, which facilitates the study of major histocompatibility complex (MHC)-restricted T-cell immunity. Marmosets develop acute hepatitis following infection with GB virus B (GBV-B) (4, 19), but there have been reported cases of persistent infection with similar pathological and virologic changes to those seen with persistent HCV infection (21). Infection of marmosets with GBV-B produces a peak viremia ranging from 10 7 to 10 9 genomic equivalents/ml (GE/ml) for a period of...

Resolution of hepatitis C virus (HCV) infection is associated with strong and sustained virus-specific CD4؉ T cell responses. In this study, we investigated the evolution of functional T cell responses during acute infection of a chimpanzee and the longevity of these lymphocytes in blood and liver after resolution of infection. Viremia increased through the first 3 weeks of infection and then remained stable until the onset of T cell responses at weeks 6 and 8 postinfection. CD4؉ T cells targeting nonstructural HCV proteins were detected in proliferation assays by week 6 postinfection, but they failed to produce interferon ␥ (IFN-␥). HCV-specific CD4؉ and CD8؉ T cells with the ability to produce IFN-␥ appeared at week 8 when a rapid 10-fold reduction in plasma viremia was first observed. This cytokine response persisted through to week 24 when infection apparently resolved. T cell lines targeting 3 CD4؉ T cell epitopes and 1 CD8؉ T cell epitope were derived from liver and their Patr major histocompatibility complex (MHC) restriction elements were identified. In retrospective studies performed on cryopreserved peripheral blood mononuclear cells (PBMCs) collected at various timepoints after infection, the onset of an IFN-␥ response measured against the class II restricted epitopes correlated with viral clearance. In conclusion, the characterization of the HCV epitopes and MHC class II restriction elements described here will facilitate a detailed comparison of CD4؉ T cell function in animals with resolved and persistent infections. (HEPATOLOGY 2003;38:1297-1306

These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer.