Viral Hepatitis is a major health challenge that in many cases leads to progression to liver cirrhosis, fibrosis and hepatocellular carcinoma. Different hepatitis viruses impose changes in the host cells and create dis-tinct immune environments. The complexity of the innate immune response mediated by Natural Killer (NK) cells and other tissue-resident Innate Lymphoid Cell (ILC) subsets producing type 1, 2, or 3 cytokines against different hepatitis viruses remains incompletely understood. Here, we postulate that not only stress-induced changes in the infected cells, but also changed immune environments shape NK/ILC reactivity de-pendent on the subtype of the infecting hepatitis virus. In acutely infected HCV patients, for instance, an early and strong NK cell activation is associated with HCV clearance. In this context, it is still unclear, how HCV-infected cells directly and/or indirectly modulate NK function. Moreover, the role of cytokine producing ILCs in the liver during HCV infection is elusive. During the first funding period, we investigated mechanisms of NK cell reactivity to hepatitis-infected hepatoma cells with a focus on HCV. In collaboration with Barten-schlager, Lohmann, and Thimme, we demonstrated that in co-cultures of HCV replicon-carrying or HCV-infected hepatocytes with peripheral blood mononuclear cells (PBMCs), a subset of NK cells increased expression of the high affinity IL-2 receptor αchain (CD25), proliferated rapidly and produced IFN-ɣ. This involved the interaction with myeloid cells and the co-stimulatory molecule OX40 and its ligand OX40L (Pollmann et al., 2018; Pollmann et al. 2017). We performed transcriptome analysis of the expanded NK subset and observed many differentially expressed genes including an upregulation of the integrin CD49a and chemokine receptors on NK cells upon HCV infection. Future studies will address the functional rele-vance of these candidates for the NK reactivity and liver homing in response to HCV. Since NK cells isolat-ed from peripheral blood substantially differ from NK in human liver, we will determine the reactivity of NK cells isolated from human liver in HCV-replicon co-cultures. Moreover, tissue-resident ILC subsets isolated from liver will be co-cultured with HCV infected hepatoma cells. The liver is known to be a hypoxic organ and we will investigate effects of low oxygen levels and other factors of the liver microenvironment in our co-cultures.
During the first funding period, in collaboration with Hofmann and Thimme we detected expansion of the memory-like NK cell subset in chronically HBV-infected patient cohorts (Schuch et al. 2019). In in vitro co-cultures with HBV-infected hepatoma cells, however, we did not observe major changes in NK cell reac-tivity. In contrast, we noted strong NK cell activation in co-cultures upon HDV infection (collaboration with Urban). HDV depends on the envelope proteins of HBV and it occurs as co-infection or superinfection of HBV-infected individuals, increasing the risk of a more severe form of hepatitis, progression of liver cirrho-sis and cancer. So far, we observed an upregulation of NK cell activating ligands and classical/non-classical MHC class I molecules on HDV-infected hepatocytes and an elevated expression of activation markers and cytolytic mediators on co-cultured NK cells. Based on these results, we will extend our focus to study NK/ILC activation/dysfunction/exhaustion in response to HDV infection or HBV/HDV co-infection (in collaboration with Urban). We will i) functionally evaluate changes of activating and inhibitory receptors on HDV-reactive NK cells, ii) dissect cytokine production and the mechanistic underpinnings of NK cell-mediated killing of HDV-infected cells and iii) determine the heterogeneity of NK cell reactivity to HDV by single cell sequencing of sorted NK cells (collaboration with Z03N). Moreover, we will examine the interaction of NK/ILCs with T cells and different types of myeloid cells, as well as the involvement of type I (IFN-/βand type III (IFN-λ) interferons that are produced by HDV-infected hepatoma cells. Together with Neumann-Haefelin, we will validate candidate molecules that are modulated in our in vitro co-cultures in HDV/HBV co-infected patient cohorts.
Results of our study on the regulation of reactivity of blood or liver NK/ILCs to different hepatitis vires will not only facilitate the development of novel treatment options e.g. for HBV/HDV infection, but will also pro-vide novel insight into general anti-viral mechanisms of NK cells and ILC subsets and their tissue specificity.

Milestones of the first funding period:

- Explore pathways of human NK cell responses to HCV- or HBV-infected cells and their modulation by the microenvironment:
- Dissect the direct or indirect impact of IFN-λ/α on NK cell interaction with other immune cells and HCV/HBV-infected cells
- Characterize NK receptors/NK ligands in livers/blood and sera from HCV- or HBV-infected individuals