Hepatitis C virus (HCV) dysregulates innate immune responses and induces persistent viral infection. We previously demonstrated that HCV core protein impairs IL-12 expression by monocytes/macrophages (M/MΦs) through interaction with a complement receptor gC1qR. Because HCV core-mediated lymphocyte dysregulation occurs through the negative immunomodulators programmed death-1 (PD-1) and suppressor of cytokine signaling-1 (SOCS-1), the aim of this study was to examine their role in HCV core-mediated IL-12 suppression in M/MΦs. We analyzed TLR-stimulated, primary CD14+ M/MΦs from chronically HCV-infected and healthy subjects or the THP-1 cell line for PD-1, SOCS-1, and IL-12 expression following HCV core treatment. M/MΦs from HCV-infected subjects at baseline exhibited comparatively increased PD-1 expression that significantly correlated with the degree of IL-12 inhibition. M/MΦs isolated from healthy and HCV-infected individuals and treated with HCV core protein displayed increased PD-1 and SOCS-1 expression and decreased IL-12 expression, an effect that was also observed in cells treated with gC1qR’s ligand, C1q. Blocking gC1qR rescued HCV core-induced PD-1 upregulation and IL-12 suppression, whereas blocking PD-1 signaling enhanced IL-12 production and decreased the expression of SOCS-1 induced by HCV core. Conversely, silencing SOCS-1 expression using small interfering RNAs increased IL-12 expression and inhibited PD-1 upregulation. PD-1 and SOCS-1 were found to associate by coimmunoprecipitation studies, and blocking PD-1 or silencing SOCS-1 in M/MΦ led to activation of STAT-1 during TLR-stimulated IL-12 production. These data suggested that HCV core/gC1qR engagement on M/MΦs triggers the expression of PD-1 and SOCS-1, which can associate to deliver negative signaling to TLR-mediated pathways controlling expression of IL-12, a key cytokine linking innate and adaptive immunity.

Vaccination for hepatitis B virus (HBV) in the setting of hepatitis C virus (HCV) infection is recommended, but responses to vaccination are blunted when compared to uninfected populations. The mechanism for this failure of immune response in HCV-infected subjects remains unknown but is thought to be a result of lymphocyte dysfunction during chronic viral infection. We have recently demonstrated that PD-1, a novel negative immunomodulator for T cell receptor (TCR) signaling, is involved in T and B lymphocyte dysregulation during chronic HCV infection. In this report, we further investigated the role of the PD-1 pathway in regulation of CD4+ T cell responses to HBV vaccination in HCV-infected individuals. In a prospective HCV infected cohort, a poor response rate to HBV vaccination as assayed by seroconversion was observed in HCV-infected subjects (53%), while a high response rate was observed in healthy or spontaneously HCV-resolved individuals (94%). CD4+ T cell responses to ex vivo stimulations of anti-CD3/CD28 antibodies or hepatitis B surface antigen (HBsAg) were found to be lower in HBV vaccine non-responders compared to those responders in HCV-infected individuals who had received a series of HBV immunizations. PD-1 expression on CD4+ T cells were detected at relatively higher levels in these HBV vaccine non-responders than those who responded, and this was inversely associated with the cell activation status. Importantly, blocking the PD-1 pathway improved T cell activation and proliferation in response to ex vivo HBsAg or anti-CD3/CD28 stimulation in HBV vaccine non-responders. These results suggest that PD-1 signaling may be involved in impairing CD4+ T cell responses to HBV vaccination in subjects with HCV infection, and raise the possibility that blocking this negative signaling pathway might improve success rates of immunization in the setting of chronic viral infection.

T regulatory (TR) cells suppress T cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death-1 (PD-1) also plays a pivotal role in regulation of T cell functions during chronic viral infection. To examine the role of PD-1 pathway in regulating TR cell functions that inhibit T cell responses during virus-associated malignancy, TR cells were investigated in the setting of hepatitis C virus-associated lymphoma (HCV-L), non-HCV-associated lymphoma (non-HCV-L), HCV infection alone, and healthy subjects (HS). Relatively high numbers of CD4+CD25+ and CD8+CD25+ TR cells as well as high levels of PD-1 expressions on these TR cells were found in the peripheral blood of subjects with HCV-L compared to those from non-HCV-L or HCV alone or HS. TR cells from the HCV-L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of TR cells in PBMC from HCV-L improved T cell proliferation. Additionally, the suppressed T cell activation and proliferation in HCV-L was partially restored by blocking the PD-1 pathway ex vivo, resulting in both a reduction in TR cell number and the ability of TR to suppress the activity of effector T cells. This study suggests that the PD-1 pathway is involved in regulating TR cells that suppress T cell functions in the setting of HCV-associated B cell lymphoma.

Chronic hepatitis C virus (HCV) infection is associated with T-cell exhaustion that is mediated through upregulation of the PD-1 negative regulatory pathway. PD-1 expression is induced by HCV core protein, which also induces upregulation of SOCS-1, a key modulator that controls the Jak/STAT pathway regulating cytokine expression. To determine whether these two negative regulatory pathways are linked during T-cell signaling, SOCS-1 expression was examined by blocking the PD-1 pathway in T cells stimulated with anti-CD3/CD28 in the presence of HCV core protein. T cells isolated from healthy subjects or HCV-infected individuals were treated with anti-PD-1 or anti-PDL-1 antibodies in the presence or absence of HCV core protein, and SOCS-1 gene expression was detected by RT-PCR or immunoblotting, while T-cell functions were assayed by flow cytometric analyses. Both PD-1 and SOCS-1 gene expression were upregulated in healthy T cells exposed to HCV core protein, and blocking the PD-1 pathway downregulated SOCS-1 gene expression in these cells. Additionally, T cells isolated from chronically HCV-infected subjects exhibited increased PD-1 and SOCS-1 expression compared to healthy subjects, and SOCS-1 expression in T cells isolated from HCV-infected subjects was also inhibited by blocking PD-1 signaling; this in turn enhanced the phosphorylation of STAT-1, and improved the impaired T-cell proliferation observed in the setting of HCV infection. These data demonstrate that PD-1 and SOCS-1 are linked in dysregulating T-cell signaling during HCV infection, and their cross-talk may coordinately inhibit T-cell signaling pathways that lead to T-cell exhaustion during chronic viral infection.

HCV infection is associated with immune dysregulation and B cell Non-Hodgkins lymphoma (HCV-NHL). We have previously shown in vitro that HCV core protein differentially regulates T and B cell functions through two negative signaling pathways, programmed death-1 (PD-1) and suppressor of cytokine signaling-1 (SOCS-1). In this report, we performed a detailed immunologic analysis of T and B cell functions in the setting of HCV-NHL. We observed that T cells isolated from patients with HCV-NHL exhibited an exhausted phenotype including decreased expression of viral-specific and non-specific activation markers; whereas B cells exhibited an activated phenotype including over-expression of cell activation markers and immunoglobulins compared to healthy subjects. Individuals with HCV alone or NHL alone exhibited abnormal T and B cell phenotypes, but to a lesser extent compared to HCV-NHL. This differential activation of T and B lymphocytes was inversely associated with the expression of PD-1 and SOCS-1. Interestingly, blocking PD-1 during TCR activation inhibited SOCS-1 gene expression, suggesting that these regulatory pathways are linked in T cells. Importantly, blocking PD-1 also restored the impaired T cell functions observed in the setting of HCV-NHL. These results support a coordinated mechanism by which HCV might cause immune dysregulation that is associated to HCV-NHL.