It is well established that CD4 T cells are central players in orchestrating antiviral humoral and cellular immun-ity. Despite this important role, little is known about the composition and fate of the virus-specific CD4 T cell compartment in viral hepatitis. In the first funding period, we therefore focussed on the longitudinal analysis and functional modification of hepatitis B virus (HBV)- and hepatitis C virus (HCV)-specific CD4 T cells during chronic infection. Specifically, we identified the costimulatory receptor OX40 in combination with the inhibitory receptor PD-1 as potential targets for immune interventions that aim at improving HBV-specific CD4 T cell function (Jacobi/Wild et al., 2019). In addition, we observed dynamic changes of the HCV-specific CD4 T cell phenotype in patients undergoing antiviral therapy with a shift from a predominant Th1 phenotype in the chronic phase of the infection to a Tfh biased phenotype after viral elimination (Smits et al., 2019). However, in contrast to observations in mouse models where virus-specific Th1 cells can sharply be separated from virus-specific Tfh cells, we observed a strong phenotypic overlap between Th1 and Tfh signatures in HCV-specific CD4 T cells. The implications on Th1 and Tfh cell function and the mech-anisms responsible for this phenotypic overlap remain to be identified. Thus, for the second funding period, we propose to continue our studies on antiviral CD4 T cell responses in viral hepatitis, specifically HBV, HCV and hepatitis E virus (HEV) infection. One focus will be on the identification of the lineage commitment of antiviral CD4 T cells in different phases of viral hepatitis in order to identify whether the lack of a distinct lineage commitment is specific for chronic infection or can also be observed in acute and resolved infection (with Thimme, Hofmann, Bengsch, Z03N). Our preliminary results outlined below also suggest that HCV-specific CD4 T cells in chronic infection have an impaired plastic potential to re-differentiate into different lineages. This lack of plasticity could be a consequence of continuous antigen and interferon (IFN) expo-sure in chronic infection as well as metabolic reprogramming. Indeed, in own previous work, we observed that type I IFNs have the potential to potently impact CD4 T cell function. We found that type I IFNs pro-foundly enhance the activation and function of Tfh cells, suggesting that the strong IFN signature in chronic HCV infection might contribute to the expansion of intrahepatic Tfh cells we have previously observed. Therefore, during the next funding period, we will address the role of type I IFNs in modulating CD4 T helper cell responses, especially follicular T helper (Tfh) cell responses (with Hofmann, Knolle and Klingmül-ler/Timmer). By using newly established HCV-specific CD4 T cell clones and primary HCV-specific CD4 T cells, we plan to identify the pathways that are involved in IFN-mediated enhancement of Tfh responses (with Bartenschlager). Specifically, we aim to identify IFN stimulated genes (ISGs) in T cells after culture in the presence of IFN and in patients with chronic viral hepatitis with the goal to analyse the impact of ISGs on Tfh cell activation, metabolism and function (with Bengsch). In addition, we will analyse CD4 T helper cell responses in HEV infection (with Thimme and Neumann-Haefelin). HEV infection is an emerging disease that serves as an excellent model for the analysis of successful immune responses leading to spontaneous viral clearance in immunocompetent hosts. However, spontaneous clearance of HEV does not necessarily pre-vent from re-infection, suggesting a lack of long-lasting, potent neutralizing antibody (nAb) responses. The failure to establish potent, long-lasting humoral immunity has recently been associated with impaired Tfh cell help, particularly help by Tfh cells with a co-existent Th1 signature. Thus, we aim to analyse the T cell re-sponses elicited by HEV infection (with Z03N and Neumann-Haefelin) to analyse whether the breadth and quality of the Tfh response is associated with the nAb responses against HEV (with Dao Thi/Boulant). More-over, we aim to identify MHC class II restricted epitopes from HEV in order to precisely define the viral pro-teins that are targeted and to generate MHC class II tetramers for the analysis of HEV-specific CD4 T cell responses. These analyses will not only give important insights into the role of CD4 T cell responses in HEV infection, but may also be of clinical relevance for vaccine design. Collectively, we aim to identify the ideal Tfh signature that provides the most potent B cell help and to identify factors that influence Tfh differentia-tion and maintenance.

Milestones of the first funding period:
- Identification and comprehensive profiling of Tfh responses following HBV-vaccination and in acute HBV infection in humans
- Analysis of Tfh responses in a HBV mouse model
- Role of Tfh cells in chronic HBV infection