TP20: Impact of interferon-stimulated genes on virus-specific CD8+ T cells and NK cells in viral hepatitis

The 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.

Prof. Dr. Maike Hofmann

Winkler F, Hipp AV, Ramirez C, Martin B, Villa M, Neuwirt E, Gorka O, Aerssens J, Johansson SE, Rana N, Llewellyn-Lacey S, Price DA, Panning M, Gross O, Pearce EL, Herrmann CM, Schumann K, Hannibal L, Neumann-Haefelin C, Boettler T, Knolle P, Hofmann M, Wohlleber D, Thimme R, Bengsch B. Enolase represents a metabolic checkpoint controlling the differential exhaustion programs of hepatitis virus-specific CD8+ T cells. Gut accepted (TP01, TP02, TP04, TP05, TP13, TP20, TP21, Z03)

Kotsiliti E, ..., Hofmann M, Rippe K, Zitzlesberger H, Weber A, Malek N, Lüdde T, Vucur M, Augustin HG, Flavell R, Parnas O, Rad R, Pabst O, Henderson NC, Huber S, Macpherson A, Knolle P, Claasen M, Geier A, Trautwein C, Unger K, Elinav E, Waisman A, Abdullah Z, Haller D, Tacke F, Anstee QM, Heikenwalder M. (2023) Intestinal B-cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling. J Hepatol. 2023 May 9:S0168-8278(23)00325-2. doi: 10.1016/j.jhep.2023.04.037.

Luxenburger H, Reeg D, Lang-Meli J, Reinscheid M, Eisner M, Bettinger D, Oberhardt V, Alizei ES, Wild K,Graeser A, Karl V, Sagar, Emmerich F, Klein F, Panning M, Huzly D, Bengsch B, Boettler T, Elling R, Thimme R, Hofmann M, Neumann-Haefelin C. (2023) Boosting compromised SARS-CoV-2-specific immunity with mRNA vaccination in liver transplant recipients. J Hepatol. 78(5):1017-1027. doi: 10.1016/j.jhep.2023.02.007.

Reinscheid M, Luxenburger H, Karl V, Graeser A, Giese S, Ciminski K, Reeg DB, Oberhardt V, Roehlen N, Lang-Meli J, Heim K, Gross N, Baum C, Rieg S, Speer C, Emmerich F, Breisinger S, Steinmann D, Bengsch B, Boettler T, Kochs G, Schwemmle M, Thimme R, Neumann-Haefelin C, Hofmann M. (2022) COVID-19 mRNA booster vaccine induces transient CD8+ T effector cell responses while conserving the memory pool for subsequent reactivation. Nat Commun. 13(1):4631. doi: 10.1038/s41467-022-32324-x.

Barsch M, Salié H, Schlaak AE, Zhang Z, Hess M, Mayer LS, Tauber C, Otto-Mora P, Ohtani T, Nilsson T, Wischer L, Winkler F, Manne S, Rech A, Schmitt-Graeff A, Bronsert P, Hofmann M, Neumann-Haefelin C, Boettler T, Fichtner-Feigl S, van Boemmel F, Berg T, Rimassa L, Di Tommaso L, Saeed A, D'Alessio A, Pinato DJ, Bettinger D, Binder H, Wherry JE, Schultheiss M, Thimme R, Bengsch B. (2022) T-cell exhaustion and residency dynamics inform clinical outcomes in hepatocellular carcinoma. J Hepatol. 77(2):397-409. doi: 10.1016/j.jhep.2022.02.032.

Kemming J, Gundlach S, Panning M, Huzly D, Huang J, Lütgehetmann M, Pischke S, Schulze zur Wiesch J, Emmerich F, Llewellyn-Lacey S, Price DA, Tanriver Y, Warnatz K, Boettler T, Thimme R, Hofmann M, Fischer N, Neumann-Haefelin C. (2022) Mechanisms of CD8+ T-cell failure in chronic hepatitis E virus infection. J Hepatol. 77(4):978-990. https://doi.org/10.1016/j.jhep.2022.05.019.

Lang-Meli J, Luxenburger H, Wild K, Karl V, Oberhardt V, Salimi Alizei E, Graeser A, Reinscheid M, Roehlen N, Reeg DB, Giese S, Ciminski K, Götz V, August D, Rieg S, Waller CF, Wengenmayer T, Staudacher D, Huzly D, Bengsch B, Kochs G, Schwemmle M, Emmerich F, Boettler T, Thimme R, Hofmann M, Neumann-Haefelin C. (2022) SARS-CoV-2-specific T-cell epitope repertoire in convalescent and mRNA-vaccinated individuals. Nat Microbiol. 7(5):675-679. doi: 10.1038/s41564-022-01106-y.

Boettler T, Csernalabics B, Salié H, Luxenburger H, Wischer L, Alizei ES , Zoldan K, Krimmel L, Bronsert P, Schwabenland M, Prinz M, Mogler C, Neumann-Haefelin C, Thimme R, Hofmann M, Bengsch B. (2022) SARS-CoV-2 vaccination can elicit a CD8 T-cell dominant hepatitis. J Hepatol. 77(3):653-659 https://doi.org/10.1016/j.jhep.2022.03.040.

Oberhardt V, Hofmann M, Thimme R, Neumann-Haefelin C. (2022) Adaptive immune responses, immune escape and immune-mediated pathogenesis during HDV infection. Viruses. 20;14(2):198. doi: 10.3390/v14020198.

Wild K, Smits M, Killmer S, Strohmeier S, Neumann-Haefelin C, Bengsch B, Krammer F, Schwemmle M, Hofmann M, Thimme R, Zoldan K, Boettler T. (2021) Pre-existing immunity and vaccine history determine hemagglutinin-specific CD4 T cell and IgG response following seasonal influenza vaccination. Nat Commun. 18;12(1):6720. doi: 10.1038/s41467-021-27064-3.

Salimi Alizei E, Hofmann M, Thimme R, Neumann-Haefelin C. (2021) Mutational escape from cellular immunity in viral hepatitis: variations on a theme. Curr Opin Virol. 50:110-118. doi: 10.1016/j.coviro.2021.08.002.

Zoldan K, Ehrlich S, Killmer S, Wild K, Smits M, Russ M, Globig AM, Hofmann M, Thimme R, Boettler T. (2021) Th1-biased Hepatitis C virus-specific follicular T helper-like cells effectively support B cells after antiviral therapy. Front Immunol. 30;12:742061. doi: 10.3389/fimmu.2021.742061.

Schulien I, Kemming J, Oberhardt V, Wild K, Seidel LM, Killmer S, Sagar, Daul F, Salvat Lago M, Decker A, Luxenburger H, Binder B, Bettinger D, Sogukpinar O, Rieg S, Panning M, Huzly D, Schwemmle M, Kochs G, Waller CF, Nieters A, Duerschmied D, Emmerich F, Mei HE, Schulz AR, Llewellyn-Lacey S, Price DA, Boettler T, Bengsch B, Thimme R, Hofmann M, Neumann-Haefelin C. (2021) Characterization of pre-existing and induced SARS-CoV-2-specific CD8+ T cells. Nat Med. 27(1):78-85. doi: 10.1038/s41591-020-01143-2.

Chen Q, Coto-Llerena M, Suslov A, Teixeira RD, Fofana I, Nuciforo S, Hofmann M, Thimme R, Hensel N, Lohmann V, Ng CKY, Rosenberger G, Wieland S, Heim MH. (2021) Interferon lambda 4 impairs hepatitis C viral antigen presentation and attenuates T cell responses. Nat Commun. 12;12(1):4882. doi: 10.1038/s41467-021-25218-x.

Oberhardt V, Luxenburger H, Kemming J, Schulien I, Ciminski K, Giese S, Csernalabics B, Lang-Meli J, Janowska I, Staniek J, Wild K, Basho K, Marinescu MS, Fuchs J, Topfstedt F, Janda A, Sogukpinar O, Hilger H, Stete K, Emmerich F, Bengsch B, Waller CF, Rieg S, Sagar, Boettler T, Zoldan K, Kochs G, Schwemmle M, Rizzi M, Thimme R, Neumann-Haefelin C, Hofmann M. (2021) Rapid and stable mobilization of CD8+ T cells by SARS-CoV-2 mRNA vaccine. Nature. 597(7875):268-273. doi: 10.1038/s41586-021-03841-4.

Rennert C, Tauber C, Fehrenbach P, Heim K, Bettinger D, Sogukpinar Ö, Schuch A, Zecher BF, Bengsch B, Lang SA, Bronsert P, Björkström NK, Fichtner-Feigl S, Schultheiss M, Thimme R, Hofmann M. (2021) Adaptive subsets limit the anti-tumoral NK-cell activity in hepatocellular carcinoma. Cells. 2;10(6):1369. doi: 10.3390/cells10061369.

Schwabenland M, Salié H, Tanevski J, Killmer S, Lago MS, Schlaak AE, Mayer L, Matschke J, Püschel K, Fitzek A, Ondruschka B, Mei HE, Boettler T, Neumann-Haefelin C, Hofmann M, Breithaupt A, Genc N, Stadelmann C, Saez-Rodriguez J, Bronsert P, Knobeloch KP, Blank T, Thimme R, Glatzel M, Prinz M, Bengsch B. (2021) Deep spatial profiling of human COVID-19 brains reveals neuroinflammation with distinct microanatomical microglia-T cell interactions. Immunity. 13;54(7):1594-1610.e11. doi: 10.1016/j.immuni.2021.06.002.

Basho K, Zoldan K, Schultheiss M, Bettinger D, Globig A-M, Bengsch B, Neumann-Haefelin C, Klocperk A, Warnatz K, Hofmann M, Thimme R, Boettler T. (2021) IL-2 contributes to cirrhosis-associated immune dysfunction by impairing follicular T helper cells in advanced cirrhosis. J Hepatol. 74(3):649-660. doi: 10.1016/j.jhep.2020.10.012.

Hensel N, Gu Z, Sagar, Wieland D, Jechow K, Kemming J, Llewellyn-Lacey S, Gostick E, Sogukpinar O, Emmerich F, Price DA, Bengsch B, Boettler T, Neumann-Haefelin C, Eils R, Conrad C, Bartenschlager R, Grün D, Ishaque N, Thimme R, Hofmann M. (2021) Memory-like HCV-specific CD8+ T cells retain a molecular scar after cure of chronic HCV infection. Nat Immunol. 22(2):229-239. doi: 10.1038/s41590-020-00817-w.

Heim K, Binder B, Sagar, Wieland D, Hensel N, Llewellyn-Lacey S, Gostick E, Price DA, Emmerich F, Vingerhoet H, Kraft ARM, Cornberg M, Boettler T, Neumann-Haefelin C, Zehn D, Bengsch B, Hofmann M, Thimme R. (2020) TOX defines the degree of CD8+ T cell dysfunction in distinct phases of chronic HBV infection. Gut. 70(8): 1550–1560. doi: 10.1136/gutjnl-2020-322404

Schulien I, Kemming J, Oberhardt V, Wild K, Seidel LM, Killmer S, Sagar, Daul F, Salvat Lago M, Decker A, Luxenburger H, Binder B, Bettinger D, Sogukpinar O, Rieg S, Panning M, Huzly D, Schwemmle M, Kochs G, Waller CF, Nieters A, Duerschmied D, Emmerich F, Mei HE, Schulz AR, Llewellyn-Lacey S, Price DA, Boettler T, Bengsch B, Thimme R, Hofmann M, Neumann-Haefelin C. Characterization of pre-existing and induced SARS-CoV-2-specific CD8+ T cells. Nat Med. 2020 Nov 12. doi: 10.1038/s41591-020-01143-2. (TP01, TP02, TP11, TP20, TP21)

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

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 contribution

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):1103-1113

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 authorship

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 contribution

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 contribution

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: e01891-17

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):3454-3467

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 authorship

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

Project in detail +

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.