We found a link between the expression of CTLA-4 and the proapoptotic mediator Bim in HBV-specific CD8. Longitudinal study of a cohort of CHB patients commencing antiviral therapy showed that viral load reduction did not reduce CTLA-4 or Bim levels in antiviral T cells. We therefore explored the potential to manipulate this coinhibitory pathway in vitro to restore expansion of HBV-specific CD8

T cells. ALT, alanine transaminase; CHB, chronic hepatitis B virus infection; CTLA-4, cytotoxic T lymphocyte antigen-4; HBV, hepatitis B virus; OLP, overlapping peptides. The study was approved by the local Ethical Committees selleck kinase inhibitor and written informed consent was obtained from all patients. A total of 86 patients with CHB, three patients with resolved HBV infection, and 23 healthy volunteers participated in the study; there were no significant differences in their demographics (Table 1). All participants were HCV and HIV seronegative and cytomegalovirus (CMV) seropositive. Patients with CHB were stratified by HBV DNA levels above or below 2,000 IU/mL (determined by real-time polymerase chain reaction [PCR]), according to European Association for the Study of the Liver (EASL) guidelines.13 All CHB patients were treatment-naïve at recruitment; a subgroup of seven patients was followed

longitudinally after commencing lamivudine and adefovir (Table 2). Hepatitis B s-antigen (HBsAg) was quantitated with the Architect assay. Paired peripheral blood and liver biopsy Cell Cycle inhibitor specimens (surplus to diagnostic requirements) were obtained from eight patients with CHB (Table 1). PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation and cultured on anti-CD3 monoclonal antibody (mAb)-coated plates (1 μg/mL) or in medium alone for 16 hours before analysis of CTLA-4 in total CD8 T cells. For detection of intracellular CTLA-4 on virus-specific CD8 T cells ex vivo, cells were stained with human leukocyte antigen A2 (HLA-A2)/c18-27, HLA-A2/e183-191, HLA-A2/e335-343,

and HLA-A2/e348-357 HBV dextramers (Immudex) before stimulation with HBV-specific peptides for 4 hours in the presence of Brefeldin A. CMV-specific CD8 T cells were detected by HLA-A2/NLVPMVAYV pentamers (Proimmune). For functional detection of virus-specific CD8 T cells, cells were stimulated with HBV or control viral peptide and cultured for 10 (-)-p-Bromotetramisole Oxalate days, supplemented with 20 U/mL IL-2 at 0 and 4 days, restimulated with 1 μM peptide for 16 hours in the presence of 1 μg/mL Brefeldin A (Sigma-Aldrich), and identified by intracellular staining for IFN-γ. To examine the effect of blocking inhibitory pathways, purified, NA/LE monoclonal antibodies against CTLA-4 (BD Biosciences), PD-L1, PD-L2 (eBioscience), or control IgG (BD-Biosciences) were added at 5 μg/mL with peptide at onset of culture. Responses were analyzed as described above. Liver sections from biopsies were homogenized and filtered.