TP20: Impact of interferon-stimulated genes on virus-specific CD8+ T cells and NK cells in viral hepatitisThe overall aim of this project is to identify the role of Interferon-stimulated genes (ISGs) in T-cell and NK-cell differentiation and function in order to determine the impact on the failure of the cytotoxic immune response in chronic viral hepatitis. For this, we will determine ISG signatures and the responsiveness towards interferons of different virus-specific CD8+ T-cell and NK-cell subsets in viral hepatitis by RNAseq, ATACseq and high-dimensional FACS analyses complemented by in vitro assays following gain- and loss-of-function experiments.
A. Alfei F, Kanev K, Hofmann M, Wu M, Ghoneim HE, Roelli P, Utzschneider DT, Hösslin M, Cullen J, Fan Y, Eisenberg V, Wohlleber D, Steiger K, Merkler D, Delorenzi M, Knolle PA, Cohen C, Thimme R*, Youngblood B*, Zehn D*. 2019. Tox reinforces the phenotype and longevity of dysfunctional T cell populations during chronic viral infection. Nature. 571(7764):265-269 * co-corresponding authors B. Karimzadeh H*, Kiraithe MM*, Oberhardt V*, Salimi Alizei E, Bockmann J, Schulze Zur Wiesch J, Bude-us B, Hoffmann D, Wedemeyer H, Cornberg M, Krawczyk A, Rashidi-Alavijeh J, Rodríguez-Frías F, Casillas R, Buti M, Smedile A, Alavian SM, Heinold A, Emmerich F, Panning M, Gostick E, Price DA, Timm J, Hofmann M, Raziorrouh B, Thimme R, Protzer U, Roggendorf M, Neumann-Haefelin C. 2019. Mutations in Hepatitis D Virus Allow It to Escape Detection by CD8+ T Cells and Evolve at the Popula-tion Level. Gastroenterology. 156(6):1820-1833 *equal contributionC. Jacobi FJ, Wild K, Smits M, Zoldan K, Csernalabics B, Flecken T, Lang J, Ehrenmann P, Emmerich F, Hofmann M, Thimme R, Neumann-Haefelin C, Boettler T. 2019. OX40 stimulation and PD-L1 blockade synergistically augment HBV-specific CD4 T cells in patients with HBeAg-negative infection. J Hepatol. 70(6):1103-1113D. Schuch A, Salimi Alizei E*, Heim K*, Wieland D, Kiraithe MM, Kemming J, Llewellyn-Lacey S, Sogukpi-nar Ö, Ni Y, Urban S, Zimmermann P, Nassal M, Emmerich F, Price DA, Bengsch B, Luxenburger H, Neumann-Haefelin C, Hofmann M*, Thimme R*. 2019. Phenotypic and functional differences of HBVcore- versus HBVpolymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load. GUT. 68(5) *equal senior authorshipE. Schuch A*, Zecher BF*, Müller PA, Correia MP, Daul F, Rennert C, Tauber C, Schlitt K, Böttler T, Neumann-Haefelin C, Hengel H, Pircher H, Cerwenka A, Thimme R, Hofmann M. 2019. NK-cell respons-es are based towards CD16-mediated effector functions in chronic hepatitis B virus infection. J He-patol. 70(3):351-360 *equal contributionF. Provine NM*, Binder B*, FitzPatrick MEB, Schuch A, Garner LC, Williamson KD, van Wilgenburg B, Thimme R, Klenerman P, Hofmann M. 2018. Unique and Common Features of Innate-Like Human Vδ2+ γδT Cells and Mucosal-Associated Invariant T Cells. Front Immunol. 23;9:756 *equal contributionG. Karimzadeh H, Kiraithe MM, Kosinska AD, Glaser M, Fiedler M, Oberhardt V, Salimi Alizei E, Hofmann M, Mok JY, Nguyen M, van Esch WJE, Budeus B, Grabowski J, Homs M, Olivero A, Keyvani H, Rodríguez-Frías F, Tabernero D, Buti M, Heinold A, Alavian SM, Bauer T, Schulze Zur Wiesch J, Razior-rouh B, Hoffmann D, Smedile A, Rizzetto M, Wedemeyer H, Timm J, Antes I, Neumann-Haefelin C, Protzer U, Roggendorf M. 2018. Amino acid substitutions within HLA-B*27-restricted T cell epitopes prevent recognition by hepatitis delta virus-specific CD8+ T cells. J Virol. 92(13). pii: e01891-17H. Utzschneider DT, Delpoux A, Wieland D, Huang X, Lai CY, Hofmann M, Thimme R, Hedrick SM. 2018. Active Maintenance of T Cell Memory in Acute and Chronic Viral Infection Depends on Continuous Ex-pression of FOXO1. Cell Rep. 22(13):3454-3467I. Wieland D, Kemming J, Schuch A, Emmerich F, Knolle P, Neumann-Haefelin C, Held W, Zehn D, Hof-mann M*, Thimme R*. 2017. TCF1+ hepatitis C virus-specific CD8+ T cells are maintained after cessa-tion of chronic antigen stimulation. Nat Commun. 8:15050 *equal senior authorshipJ. Utzschneider DT, Charmoy M, Chennupati V, Pousse L, Ferreira DP, Calderon-Copete S, Danilo M, Alfei F, Hofmann M, Wieland D, Pradervand S, Thimme R, Zehn D, Held W. 2016. T Cell Factor 1-Expressing Memory-like CD8(+) T Cells Sustain the Immune Response to Chronic Viral Infections. Im-munity. 45(2):415-2
In chronic viral hepatitis, the cellular cytotoxic immune response mediated by virus-specific CD8+ T cells and NK cells is altered resulting in insufficient viral control. The functional capacity of virus-specific CD8+ T cells and NK cells, including cytotoxicity, cytokine/chemokine secretion and proliferation, and thus their potential to control viral infection is linked to their differentiation and heavily influenced by the inflammatory microenvironment. The inflammatory microenvironment of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) is, however, quite different. In particular, HBV does not induce interferons (IFNs; (“stealth virus”) that represent important inflammatory mediators while HCV infection is accompanied by a strong IFN re-sponse. It is well established that IFNs have an impact on the differentiation and function of virus-specific CD8+ T cells and NK cells in a time- and dose-dependent manner. Thus, the IFN response links inflamma-tion to the differentiation and function within the cellular cytotoxic immune response. With respect to chronic HBV and HCV infection, however, the underlying molecular mechanisms of how the differences in the IFN response affect the differentiation and thus the functional alterations of virus-specific CD8+ T cells and NK cells are insufficiently understood. In the first funding period of TP01 we observed that in chronic HCV in-fection, HCV-specific CD8+ T-cell subsets that are most severely impaired in their effector function and that are associated with a terminally exhausted differentiation stage exhibited a dominant signature of IFN-stimulated genes (ISG) on a gene expression level. Furthermore, co-regulatory networks of ISGs were large-ly diverse in dysfunctional HCV-specific CD8+ T cells from chronically HCV-infected patients compared to protective HCV-specific CD8+ T cells from patients, who spontaneously resolved HCV. Given the amount of work inherent to further investigate the impact of the different ISG signatures on the functional capacity of T cells, we split TP01 into two complementary projects: First, TP01 continues to define fate and function of a distinct HCV-specific CD8+ T-cell subset, namely memory-like cells, in chronic HCV infection. Second, the present project TP20N aims to determine the role of ISGs in the failure of the cellular cytotoxic immune re-sponse in chronic viral hepatitis. Since failure of the cellular cytotoxic immune response is linked to the differentiation of virus-specific CD8+ T cells and NK cells we will in particular address the link of ISGs with the altered CD8+ T-cell and NK-cell differentiation observed in chronic viral hepatitis. To achieve this, we will first extend our initial analysis and comprehensively assess ISG signatures on epigenetic (low input ATACseq), mRNA (low input/single cell RNAseq) and protein (high-dimensional FACS) levels comparing functionally distinct virus-specific CD8+ T-cell and NK-cell subsets (collaboration with Thimme, Neumann-Haefelin, Boettler, Cerwenka, Bartenschlager, Lohmann, DaoThi/Boulant, Binder/Pichlmair, Z02 and Z03N). Subsequently, we plan to associate specific ISGs, or ISG patterns, with relevant transcription factors and metabolic pathways (collaboration with Bengsch) in CD8+ T-cell and NK-cell differentiation by network anal-yses (collaboration with Z03N) and by in vitro analyses after gene knock-down and over-expression (collab-oration with Thimme and Cerwenka). Comparisons of HBV- and HCV-specific CD8+ T cells and NK cells from HBV- and HCV-infected patients enable to distinguish between a potentially intrinsic and an IFN-induced ISG signature. Moreover, we will test the type I IFN responsiveness of heterogeneous virus-specific CD8+ T-cell (collaboration with Thimme, Neumann-Haefelin, Boettler Bartenschlager, Lohmann, Knolle) and NK-cell subsets (collaboration with Cerwenka) by performing signalling and functional studies in vitro and ex vivo using banked samples from PEG-IFN treated viral hepatitis patients. With this proposed work program we will gain molecular insights into the impact of IFNs/ISGs on virus-specific CD8+ T-cell and NK-cell re-sponses that are key determinants in clearance versus persistence of hepatitis virus infections. In addition, this will also have relevance with respect to potentially adjuvant and also adverse effects of IFNs in thera-peutic and preventive strategies in general.
Virus-specific CD8+ T cell responses in hepatitis B virus/ hepatitis D virus co-infection: mechanisms of failure and strategies for restoration
Role of platelets in control and clearance of hepatitis B virus infection via modulation of liver tissue environment and metabolism
Identification of systems properties determining the dynamics of the hepatitis B virus infection cycle and cross-talk to host cell responses
Viral determinants and innate immunity in persistent hepatitis B virus and hepatitis D virus infections
Role of DNA repair, SUMOylation and nuclear bodies in the formation of the hepatitis B virus cccDNA persistence reservoir
Contribution of innate immune responses to persistence versus clearance of hepatitis C virus and hepatitis A virus infections
Combinatorial knock-down/knock-out strategies to reconstitute antiviral im-munity and eliminate persisting hepatitis B virus cccDNA
Impact of the enteric stage of hepatitis A virus and hepatitis E virus on the outcome of liver infection