| This study is mainly comprised of three sections. Firstly, we investigated the expression of B7-H1 and PD-1 on myeloid dendritic cells (mDCs) and T cells and evaluated the correlation between their expression and the state of disease progression. Second, we analyzed the effect of B7-H1/PD-1-mediated pathway on immune function of T cells in patients with chronic hepatitis B virus (HBV) infection, particularly on the function of HBV-specific T cells. Finally, we longitudinally explored the dynamic expression of B7-H1 on mDCs and T cells in chronic hepatitis B (CHB) patients undergoing interferon-αtherapy.Peripheral blood sample from 35 CHB patients, 10 healthy control and 10 autoimmune hepatitis patients were investigated. Flow cytometry was applied to study the expression of B7-H1 and PD-1 on mDCs and T cells. In contrast to healthy subjects, B7-H1 was significantly upregulated on T cells and mDCs in CHB patients and autoimmune hepatitis patients. In addition, the expression of B7-H1 or PD-1 was positively correlated with serum ALT level and HBV DNA load in these CHB patients, suggesting that the expression of B7-H1 and PD-1 in CHB patients is closely associated with the state of disease. We also found that inflammatory cytokines including IFN-αand IFN-γcould upregulate B7-H1 on mDCs and T cells in vitro, while HBV core antigen hardly affected the expression of B7-H1. Thus, the upregulation of B7-H1 in CHB patients and autoimmune hepatitis patients may, partly if not all, result from chronic inflammation.Based on the above results, we further investigated the effect of B7-H1/PD-1 signal pathway on T-cell immune function. Since mDCs have a pivotal role in presenting antigen and interacting with effector T cells, functional deficit in mDC-T cell interaction has been proposed to be responsible for HBV persistence. Herein, we focused on the effect of mDC-associated B7-H1 upregulation on T-cell function. To address this issue, firstly, we respectively cultured mDCs from CHB patients and healthy individual with T cells from a third healthy individual at different ratio of mDCs and T cells. T-cell proliferation was assessed by the incorporation of [~3H] thymidine. The results showed that mDCs of CHB patients had a significantly reduced capacity to stimulate allogeneic CD4~+ and CD8~+ T cells at all ratios tested compared to healthy controls, while blockade B7-H1/PD-1 pathway could significantly reverse the capacity. Meanwhile, IFN-γ, IL-2, IL-12 and IL-10 in the co-cultured supernatants were detected by enzyme-linked immunosorbent assay (ELISA). MDCs of CHB patients showed a significantly reduced capacity to produce IL-12 and IL-10 and to stimulate allogeneic CD4~+ and CD8~+ T cells to produce IFN-γand IL-2 at the 1:10 ratio compared to healthy controls, whereas blockade B7-H1/PD-1 pathway significantly reversed the capacity to produce these cytokines.Moreover, we further sought to demonstrate the effect of mDC-associated B7-H1 upregulation on HBV-specific T-cell responses. HBcAg-pulsed MoDCs were co-cultured with autologous T cells isolated from the CHB patients in the presence or absence of anti-B7-Hl. HBV-specific T-cell responses were analyzed by proliferation and cytokine measuring. We observed blockade of MoDC-associated B7-H1 signals could enhance HBV-specific T-cell proliferation and type 1 cytokine production. This recovery following antibody treatment was also closely associated with increased IL-12 and decreased IL-10 production by MoDCs. In addition, IFN-γ-producing T cells were assayed using ELISPOT and intracellular cytokine staining. We found that blocking B7-H1 on mDCs could result in an increased numbers of IFN-γ-producing HBV-specific T cells compared to a group treated with control IgG. This result was confirmed by intracellular cytokine staining. These data suggested that B7-H1 up-regulation on mDCs may be responsible for the defective HBV-specific T-cell function in chronic HBV infection.Since both B7-H1 and PD-1 are significantly upregulated on T cells from HBV chronic infected patients, we preliminarily explored the effect of T-cell associated B7-H1 on immune function of T cells. Proliferation of T cells was detected by CFSE staining. We found that blocking T-cell associated B7-H1 could enhance specific and nonspecific proliferation of T cells from patients, as well as the frequency of HBV-specific IFN-γ-producing effector T cells, so B7-H1/PD-1 pathway between T-T cells also influences effective function of T cells in CHB patients.Finally, we longitudinally studied the dynamic changes of B7-H1 expression on mDCs and T cells in CHB patients undergoing interferon-αtherapy, and analyzed the association with the efficiency of interferon-αtherapy. We found that the expression of B7-H1 on mDCs, CD4~+ T cell and CD8~+ T cells was all significantly upregulated at 4-week after the initial of interferon-αtherapy. After the timepoint, B7-H1 expression persistently decreased in responders, while non-responders maintained high level of B7-H1 expressions. In addition, we found the frequency of HBV-specific IFN-γ-producing T cells significantly increased in responders, but significantly decreased in non-responders. Blocking B7-H1/PD-1 signal pathway could enhance the frequency of HBV-specific IFN-γ-producing T cells in both respondes and non-responders.Overall, our study indicates the expression of B7-H1 and PD-1 on mDCs and T cells was significantly increased in CHB patients, which may subsequently mediate virus-specific T-cell exhaustion. Blocking the B7-H1/PD-1 pathway can revitalize the exhausted virus-specific T-cell repertoire. In addition, we found that the B7-H1 upregulation in CHB patients with interferon-αtherapy was closely associated the responsiveness to interferon-γtherapy. In conclusion, our findings begin to shed light on the understanding of immune pathogenesis during chronic HBV infection and further support the notion that blockade of B7-H1-mediated inhibitory pathway may represent a potential therapeutic strategy against chronic hepatitis B. |
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