Combined OX40 stimulation and PD-L1 blockade reinforce HBV-specific CD4 T cell responses

Chronic hepatitis B virus (cHBV) infection is a common cause of progressive liver disease that eventually leads to hepatocellular carcinoma. Antiviral therapeutic options are still limited and cannot achieve the elimination of persistent HBV infection. HBV-specific T cells are crucial for HBV control and act in a protective manner after discontinuation of antiviral therapy but appear functionally impaired during cHBV infection. Thus, T-cell based immunotherapy appears to be a promising therapeutic approach.

Aim of the study was to characterize the ex vivo phenotype of HBV-specific CD4 T cells, the identification of new HBV-specific CD4 T cell epitopes and to analyze whether the functionality of CD4 T cells can be enhancement by targeting different immunological pathways.

The expression of relevant costimulatory and coinhibitory molecules (e.g. CD127, OX40 and PD-1) on HBV- and Influenza (Flu)-specific CD4 T cells was analyzed using MHC class II-tetramers in 11 cHBV patients and 11 healthy volunteers, respectively. 66 patients (cHBV, genotype D) were screened for HBV-specific CD4 T cell responses after stimulation with overlapping peptides (OLPs) spanning the entire HBV-polyprotein. Control responses (Flu, Epstein-Barr virus (EBV) and tetanus toxoid (TT)) were analyzed in 59 healthy donors (HD). Stimulation with recombinant IL-7, an agonistic OX40-antibody or blockade of PD-L1 was performed in antigen-specific in vitro cultures. Cytokine secretion and expression of transcription factors were analyzed by flow cytometry.

HBV-specific CD4 T cells showed significantly higher expression of OX40 and PD-1 compared to Flu-specific CD4 T cells ex vivo and predominantly targeted the HBV polymerase and core proteins whereas responses to the surface protein were not detectable in cHBV patients. Consistent with the elevated ex vivo surface expression combined OX40 stimulation and PD-L1 blockade resulted in an increased frequency of IL-21 and IFN-g secreting CD4 T cells after antigen-specific in vitro culture. In addition, Th1- and Tfh-associated transcription factors T-bet and Bcl6 were strongly expressed in cytokine producing cells. In line with a lower expression of OX40 and PD-1 on Flu-specific CD4 T cells ex vivo, the augmentation of cytokine production was observed to a lesser extent for viral control responses to Flu and also EBV epitopes in HD.

Collectively, our observations demonstrate that synergistic effects of combined OX40 stimulation and PD-L1 blockade augment the secretion of Th1 and Tfh signature cytokines IFN-g and IL-21, suggesting that these pathways are promising candidates for immunotherapeutic interventions for cHBV infection.