Accumulation of exhausted virus-specific CD8+ T cells (TEX) with impaired antiviral function is a major fea-ture of unsuccessful immune responses in patients with chronic hepatitis B and C virus (HBV and HCV) in-fection. We previously identified a major role for the control of cellular metabolism downstream of inhibitory receptor signalling (i.e. via PD-1) in the development of TEX in murine models of chronic infection that in-volved regulation of glucose metabolism, induction of mitochondrial depolarization and ROS production (Bengsch et al 2016). Importantly, reversion of mitochondrial dysfunction resulted in increased T cell effec-tor function. Understanding the metabolic regulation of HCV- and HBV-specific TEX will be important for a better understanding of T cell dysfunction and should reveal novel targets for immune-based therapy ap-proaches. In preliminary work, we have profiled metabolic hallmarks of virus-specific T cells ex vivo in pa-tients with chronic HBV and HCV infection. These results indicate that unlike in mice, glucose uptake is maintained in HBV- and HCV-specific TEX, while mitochondrial dysfunction occurs in both settings. However, our data also indicate that the metabolic profile differs significantly between HBV and HCV. We hypothesize that the differential metabolic programming of virus-specific T cells determines the severity of exhaustion and is linked to differences in antigen recognition and inflammation.
We therefore plan to perform a detailed characterization of the metabolism of HBV- and HCV-specific CD8 T cells to understand the metabolic determinants of functional vs. exhausted hepatitis virus-specific T cells in different settings of viral hepatitis and viral control. We will use a comprehensive approach with high-parametric metabolic single-cell profiling using flow cytometry and mass cytometry, analysis of metabolic pathways on the transcriptional level using low-input RNA-Seq (with help from Z02 and Z03N) and metabolic flux studies. The comparison of different hepatitis virus infections (focus on HBV and HCV infection, exten-sion in collaboration to HDV, HEV) and animal models of hepatitis virus infections is possible due to the collaborations with Thimme, Neumann-Haefelin, Boettler, Hofmann, Urban, Heikenwälder and Wohlleber and will allow us to dissect the effects of different conditions of successful and dysfunctional immune respons-es (e.g., acute infection, spontaneous clearance vs. chronic infection). A high-dimensional exhaustion-directed phenotypic and functional assessment using mass cytometry will be used with help of Z02 to de-termine the differences in the degree and differentiation of TEX subsets linked to different metabolic states and clinical outcomes. In selected samples, we will attempt to correlate cytometry of peripheral blood phe-notypes to liver histology together with Z02. We will then seek to understand the impact of metabolic altera-tions and manipulations on the function of the antiviral T cell response by antagonizing immunomodulatory pathways regulating TEX (e.g., PD-1 or CD39) and promote metabolic pathways identified as deficient (such as via stimulation of metabolic regulators or provision of anaplerotic nutrient sources). Moreover, to under-stand how antigen and therapy impact on TEX metabolism, we will test samples from patients with chronic HCV infection undergoing direct acting antiviral (DAA) therapy and compare them to patients that underwent interferon-based therapy. Similarly, we will test patients with chronic HBV and HDV infection undergoing antiviral therapy with standard therapy (nucleos(t)ide analogues, interferon treatment) or the novel entry in-hibitor MyrcludexB. These ex vivo analyses will be combined with in vitro assays testing the effects of inter-ferons on T cell metabolism and inhibition of viral replication in T cell – hepatocyte co-cultures in collabora-tion with Bartenschlager, Heikenwälder and Lohmann. In collaboration, we will test specific results, such as candidate metabolic checkpoints in the glycolytic pathway, in murine models of viral hepatitis in collabora-tion with Wohlleber. Together, the planned experiments will shed light on the metabolic underpinnings of the dysfunctional antiviral T cell response in chronic viral hepatitis and reveal novel metabolism-based targets that could be harnessed to enhance T cell function.