In vitro use of autologous dendritic cells improves detection of T cell responses to hepatitis B virus (HBV) antigens.

T lymphocyte responses to hepatitis B virus (HBV) core antigen (HBcAg) are vigorous and easily detectable in vitro during recovery from acute hepatitis B but significantly weaker in patients with chronic HBV infection. In contrast, T cell responses to hepatitis B surface antigen (HBsAg) are almost undetectable during infection and even in a substantial fraction of subjects receiving vaccination with HBsAg. The aim of this study was to investigate whether the use of dendritic cells (DCs) in an in vitro assay could increase the detection of HBV-specific T cells in these conditions. Autologous monocyte-derived DCs, compared to direct HBsAg addition to the cultures, increased the stimulation of HBs- specific T cells. These were detected in 73% of healthy subjects who had recently received hepatitis B vaccine and in 43% of patients recovering from acute hepatitis B. Likewise, proliferation in response to DC-presented HBcAg was detected in both CD4(+) and CD8(+) T cells from the majority of chronic hepatitis B patients. A longitudinal evaluation of HBc-specific T cell responses during and after a 1-year treatment with pegylated interferon (IFN)-alpha showed that HBc-specific CD4(+) T cell responses had no correlation with sustained virus suppression whereas CD8(+) T cell responses were more frequently detected in patients able to control HBV replication after therapy interruption. The use of autologous DCs as antigen-presenting cells appears applicable to clinically relevant in vitro evaluation of T cell responses, particularly in those conditions characterized by low frequency of circulating antigen-specific cells and suboptimal in vivo activation.

Induction of regulatory T-cells and interleukin-10-producing cells in non-responders to pegylated interferon-alpha therapy for chronic hepatitis B.

BACKGROUND: Treatment with interferon-alpha (IFN-alpha) leads to a response in only a minority of patients with chronic hepatitis B virus (HBV) infection, but the reasons for this are poorly understood. It was recently shown that in patients with chronic HBV infection, CD4+CD25+ regulatory T-cells (Treg) can suppress the HBV-specific immune response. We aimed to investigate whether in non-responders to IFN-alpha therapy Treg contribute to treatment failure by downregulating the HBV-specific T-cell responses. PATIENTS AND METHODS: Fourteen patients positive for hepatitis B e antigen received pegylated IFN-alpha monotherapy for 52 weeks and were followed for 26 weeks. RESULTS: Compared with non-responders, responders displayed an increased HBV-specific T-helper cell proliferation. At the start of treatment there was no difference in the frequencies of CD4+CD25+ Treg between responders and non-responders. During therapy, the frequency of CD4+CD25+ Treg increased in non-responders, but not in responders. In contrast to the responders, the non-responders showed a significant increase in the frequency of interleukin-10-producing cells. Treg depletion resulted in increased proliferation capacity, but did not affect the frequency of interleukin-10-producing cells measured during the course of the treatment. CONCLUSION: This study indicates that Treg might have an important role in HBV persistence during and after pegylated IFN-alpha therapy.

Reciprocal changes of naïve and effector/memory CD8+ T lymphocytes in chronic hepatitis B virus infection.

Persistent viruses, such as cytomegalovirus or human immunodeficiency virus, cause major perturbations of CD8+ T-lymphocyte subpopulations. To test whether chronic infection with hepatitis B virus (HBV) could also be responsible for such modifications, we analyzed the expression of CD27, CD28, CCR7, and perforin in blood CD8+ T lymphocytes. In comparison to healthy subjects and patients recovering from acute hepatitis B, chronic hepatitis B patients showed higher percentages of naïve CD8+ T lymphocytes (CD45RA+CD27+CD28+), and lower percentages of intermediately-differentiated CD27+CD28⁻CD8+ T cells. The late differentiated CD45RA+CD27⁻CD28⁻ subset was also present in a large percentage in some patients, but no statistically significant difference with healthy controls was observed. Removal from the circulation of intermediately-differentiated CD8+ T lymphocytes may occur during chronic HBV infection, favoring the recruitment of naïve cells. This may result in impairment of the generation of functionally-competent memory cells, and an inability to achieve control of HBV replication.

Antiviral treatment with alpha interferon up-regulates CD14 on liver macrophages and its soluble form in patients with chronic hepatitis B.

To investigate whether therapy with alpha interferon (IFN-alpha) induces changes in intrahepatic antigen-presenting cells (APCs), we obtained liver biopsy specimens before, during, and after therapy with IFN-alpha from chronic hepatitis B patients whose viral load had already been reduced by at least 8 weeks of treatment with lamivudine. HLA-DR, CD1a, and CD83 were not modified by the therapy. The intralobular expression of CD68 on Kupffer cells remained stable, denoting no changes in the number of resident macrophages during IFN-alpha treatment. In contrast, CD14 was weakly expressed in the absence of IFN-alpha and was significantly up-regulated during therapy. At the same time, the levels of soluble CD14 and interleukin-10 in plasma increased significantly. In vitro, monocytes maintained in the presence of IFN-alpha differentiated into macrophages or dendritic cells with higher levels of expression of CD14 than that for the control cultures. During therapy with IFN-alpha, T-cell infiltration in the portal spaces was reduced, mainly due to a significant decrease in the number of CD8(+) T cells. These findings show that IFN-alpha is biologically active on APCs in vivo and in vitro and suggest that this newly described regulatory function, together with the already known inhibitory effects on lymphocytes, may cooperate to reduce inflammation and consequent tissue damage in patients with chronic viral hepatitis.