TP05: The impact of hepatic myeloid cells on antiviral CD8 T cell immunity in the liver

We investigate the role of myeloid cells that are recruited after TLR9-L application for development of anti-HBV immunity. We explore the longevity of virus-specific CD8 T cells after TLR9-L application and whether their cytotoxic effector potential remains elevated in a memory-like state. We will characterize the mechanisms underlying the metabolic activation of virus-specific CD8 T cells through hepatic myeloid cells. Finally, we will characterize the impact of liver damage on shut-down of virus-specific CD8 T cells and how this negative feedback loop impacts on clearance vs persistence of HBV infection in hepatocytes.

Prof. Dr. Percy A. Knolle

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)

Hackstein CP*, Spitzer J, Symeonidis K, Horvatic H, Bedke T, Steglich B, Klein S, Assmus LM, Odainic A, Szlapa J, Kessler N, Beyer M, Schmithausen R, Latz E, Flavell RA, Garbi N, Kurts C, Kummerer BM, Trebicka J, Roers A, Huber S, Schmidt SV, Knolle PA*, Abdullah Z*. (2023) Interferon-induced IL-10 drives systemic T-cell dysfunction during chronic liver injury. J Hepatol. S0168-8278(23)00158-7. doi: 10.1016/j.jhep.2023.02.026.

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.

Su J, Brunner L, Ates Oz E, Sacherl J, Frank G, Kerth HA, Thiele F, Wiegand M, Mogler C, Aguilar JC, Knolle PA, Collin N, Kosinska AD, Protzer U. (2023) Activation of CD4 T cells during prime immunization determines the success of a therapeutic hepatitis B vaccine in HBV-carrier mouse models. J Hepatol. 78(4):717-730. doi: 10.1016/j.jhep.2022.12.013.

Esser K, Cheng X, Wettengel JM, Lucifora J, Hansen-Palmus L, Austen K, Roca Suarez AA, Heintz S, Testoni B, Nebioglu F, Pham MT, Yang S, Zernecke A, Wohlleber D, Ringelhan M, Broxtermann M, Hartmann D, Hüser N, Mergner J, Pichlmair A, Thasler WE, Heikenwalder M, Gasteiger G, Blutke A, Walch A, Knolle PA, Bartenschlager R, Protzer U. (2023) Hepatitis B virus targets lipid transport pathways to infect hepatocytes,Cellular and Molecular Gastroenterology and Hepatology, 2023,ISSN 2352-345X,https://doi.org/10.1016/j.jcmgh.2023.03.011.

Dudek M, Lohr K, Donakonda S, Baumann T, Lüdemann M, Hegenbarth S, Dübbel L, Eberhagen C, Michailidou S, Yassin A, Prinz M, Popper B, Rose-John S, Zischka H, Knolle PA. (2022) IL-6-induced FOXO1 activity determines the dynamics of metabolism in CD8 T cells cross-primed by liver sinusoidal endothelial cells. Cell Rep. 15;38(7):110389. doi: 10.1016/j.celrep.2022.110389.

Wratil PR, Stern M, Priller A, Willmann A, Almanzar G, Vogel E, Feuerherd M, Cheng CC, Yazici S, Christa C, Jeske S, Lupoli G, Vogt T, Albanese M, Mejías-Pérez E, Bauernfried S, Graf N, Mijocevic H, Vu M, Tinnefeld K, Wettengel J, Hoffmann D, Muenchhoff M, Daechert C, Mairhofer H, Krebs S, Fingerle V, Graf A, Steininger P, Blum H, Hornung V, Liebl B, Überla K, Prelog M, Knolle P, Keppler OT, Protzer U. (2022) Three exposures to the spike protein of SARS-CoV-2 by either infection or vaccination elicit superior neutralizing immunity to all variants of concern. Nat Med. 28(3):496-503. doi: 10.1038/s41591-022-01715-4.

Koerber N, Priller A , Yazici S, Bauer T, Cheng CC, Mijočević H, Wintersteller H, Jeske S, Vogel E, Feuerherd M, Tinnefeld K, Winter C, Ruland J, Gerhard M, Haller B, Christa C, Zelger O, Roggendorf H, Halle M, Erber J, Lingor P, Keppler O, Zehn D, Protzer U, Knolle PA. (2022) Dynamics of spike-and nucleocapsid specific immunity during long-term follow-up and vaccination of SARS-CoV-2 convalescents. Nat Commun 13, 153. https://doi.org/10.1038/s41467-021-27649-y.

Brugger M, Laschinger M, Lampl S, Schneider A, Manske K, Esfandyari D, Hüser N, Hartmann D, Steiger K, Engelhardt S, Wohlleber D, Knolle PA. (2022) High precision-cut liver slice model to study cell-autonomous antifviral defense of hepatocytes within their microenvironment. JEHP Rep. 6;4(56):100465. doi: 10.1016/j.hepr.2022.100465.

Wagner KI, Mateyka LM, Jarosch S, Grass V, Weber S, Schober K, Hammel M, Burrell T, Kalali B, Poppert H, Beyer H, Schambeck S, Holdenrieder S, Strötges-Achatz A, Haselmann V, Neumaier M, Erber J, Priller A, Yazici S, Roggendorf H, Odendahl M, Tonn T, Dick A, Witter K, Mijočević H, Protzer U, Knolle PA, Pichlmair A, Crowell CS, Gerhard M, D'Ippolito E, Busch DH. (2022) Recruitment of highly cytotoxic CD8+ T cell receptors in mild SARS-CoV-2 infection. Cell Rep. 11;38(2):110214. doi: 10.1016/j.celrep.2021.110214.

Knolle PA, Huang LR, Kosinska A, Wohlleber D, Protzer U. (2021) Improving therapeutic vaccination against Hepatitis B-insights from preclinical models of immune therapy against persistent Hepatitis B virus infection. Vaccines (Basel). 16;9(11):1333. doi: 10.3390/vaccines9111333.

Manske K, Schneider A, Ko C, Knolle PA, Steiger K, Protzer U, Wohlleber D. (2021) In vivo bioluminescence imaging of HBV replicating hepatocytes allows for the monitoring of anti-viral immunity viruses. 13(11): 2273. doi: 10.3390/v13112273.

Dudek M, Pfister D, Donakonda S, ....Thimme R, Böttler T, Tacke F, Dufour J-F, Haller D, Murray PJ, Heeren R, Zehn D, Böttcher JP, Heikenwälder M, Knolle PA. (2021) Auto-aggressive CXCR6+ CD8 T cells cause liver immune pathology in NASH. Nature. 592, 444–449. doi: 10.1038/s41586-021-03568-2.

Pfister D, Núñez NG, Pinyol R, ... Knolle P, ... Heikenwalder M. (2021) NASH limits anti-tumour surveillance in immunotherapy-treated HCC. Nature. 592(7854):450-456. doi.org/10.1038/s41586-021-03362-0.

Conlon TM, John-Schuster G, Heide D, Pfister D, Lehmann M, Hu Y, Ertüz Z, Lopez MA, Ansari M, Strunz M, Mayr C, Ciminieri C, Costa R, Kohlhepp MS, Guillot A, Günes G, Jeridi A, Funk MC, Beroshvili G, Prokosch S, Hetzer J, Verleden SE, Alsafadi H, Lindner M, Burgstaller G, Becker L, Irmler M, Dudek M, Janzen J, Goffin E, Gosens R, Knolle P, Pirotte B, Stoeger T, Beckers J, Wagner D, Singh I, Theis FJ, de Angelis MH, O'Connor T, Tacke F, Boutros M, Dejardin E, Eickelberg O, Schiller HB, Königshoff M, Heikenwalder M, Yildirim AÖ. Inhibition of LTβR signalling activates WNT-induced regeneration in lung. Nature. 2020 Nov 4. doi: 10.1038/s41586-020-2882-8. (TP05, TP06)

Lampl S, Janas MK, Donakonda S, Brugger M, Lohr K, Schneider A, Manske K, Sperl LE, Kläger S, Küster B, Wettmarshausen J, Müller C, Laschinger M, Hartmann D, Hüser N, Perocchi F, Schmitt-Kopplin P, Hagn F, Zender L, Hornung V, Borner C, Pichlmair A, Kashkar H, Klingenspor M, Prinz M, Schreiner S, Conrad M, Jost PJ, Zischka H, Steiger K, Krönke M, Zehn D, Protzer U, Heikenwälder M, Knolle PA, Wohlleber D. Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes. J Hepatol. 2020 Jun 26:S0168-8278(20)30398-6. doi: 10.1016/j.jhep.2020.06.026. (TP05, TP06, TP10, TP11, TP13, TP16, TP18)

Baumann T, Dunkel A, Schmid C, Schmitt S, Hiltensperger M, Lohr K, Laketa V, Donakonda S, Ahting U, Lorenz-Depiereux B, Heil JE, Schredelseker J, Simeoni L, Fecher C, Körber N, Bauer T, Hüser N, Hartmann D, Laschinger M, Eyerich K, Eyerich S, Anton M, Streeter M, Wang T, Schraven B, Spiegel D, Assaad F, Misgeld T, Zischka H, Murray PJ, Heine A, Heikenwälder M, Korn T, Dawid C, Hofmann T, Knolle PA, Höchst B. 2020. Regulatory myeloid cells paralyze T cells through cell-cell transfer of the metabolite methylglyoxal. Nat Immunol. 21(5):555-566. (TP05, TP06)

Namineni S, O'Connor T, Faure-Dupuy S, Johansen P, Riedl T, Liu K, Xu H, Singh I, Shinde P, Li F, Pandyra A, Sharma P, Ringelhan M, Muschaweckh A, Borst K, Blank P, Lampl S, Durantel D, Farhat R, Weber A, Lenggenhager D, Kündig TM, Staeheli P, Protzer U, Wohlleber D, Holzmann B, Binder M, Breuhahn K, Assmus LM, Nattermann J, Abdullah Z, Rolland M, Dejardin E, Lang PA, Lang KS, Karin M, Lucifora J, Kalinke U, Knolle PA, Heikenwalder M. 2020. A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control. J Hepatol. 72(5):960-975. (TP05, TP06, TP11, TP13, TP14)

Michler T, Kosinska AD, Festag J, Bunse T, Su J, Ringelhan M, Imhof H, Grimm D, Steiger K, Mogler C, Heikenwalder M, Michel ML, Guzman CA, Milstein S, Sepp-Lorenzino L, Knolle P, Protzer U. 2020. Knockdown of Virus Antigen Expression Increases Therapeutic Vaccine Efficacy in High-titer HBV Carrier Mice. Gastroenterology. 158(6):1762-1775. (TP05, TP06, TP18)

Schneider A, S Kurz, K Manske, M Janas, M Heikenwalder, T Misgeld, M Aichler, SF Weissmann, H Zischka, PA Knolle, D Wohlleber. 2019. Single organelle analysis to characterize mitochondrial function and crosstalk during viral infection. Sci Rep 9(1): 8492.

Alfei F, K Kanev, M Hofmann, M Wu, HE Ghoneim, P Roelli, DT Utzschneider, M von Hoesslin, JG Cullen, Y Fan, V Eisenberg, D Wohlleber, K Steiger, D Merkler, M Delorenzi, PA Knolle, CJ Cohen, R Thimme*, B Youngblood*, D Zehn*. 2019. TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection. Nature 571(7764): 265-269.

Malehmir M, D Pfister, … N Djouder, P Kubes, PA Knolle, K Unger, L Zender, B Nieswandt, M Gawaz, A Weber, M Heikenwalder. 2019. Platelet GPIbalpha is a mediator and potential interventional target for NASH and subsequent liver cancer. Nat Med 25(4): 641-655.

Kosinska AD, A Moeed, N Kallin, J Festag, J Su, K Steiger, ML Michel, U Protzer, PA Knolle. 2019. Synergy of therapeutic heterologous prime-boost hepatitis B vaccination with CpG-application to improve immune control of persistent HBV infection. Sci Rep 9(1): 10808.

Manske K, N Kallin, V Konig, A Schneider, S Kurz, M Bosch, M Welz, RL Cheng, B Bengsch, K Steiger, U Protzer, R Thimme, PA Knolle, D Wohlleber. 2018. Outcome of Antiviral Immunity in the Liver Is Shaped by the Level of Antigen Expressed in Infected Hepatocytes. Hepatology 68(6): 2089-2105.

Welz M, S Eickhoff, Z Abdullah, J Trebicka, KH Gartlan, JA Spicer, AJ Demetris, H Akhlaghi, M Anton, K Manske, C Kurts, JA Trapani, PA Knolle*, D Wohlleber*, W Kastenmuller*. 2018. Perforin inhibition protects from lethal endothelial damage during fulminant viral hepatitis. Nat Comm 9(1): 4805.

Wieland D, J Kemming, A Schuch, F Emmerich, PA Knolle, C Neumann-Haefelin, W Held, D Zehn, M Hofmann, R Thimme. 2017. TCF1(+) hepatitis C virus-specific CD8(+) T cells are maintained after cessation of chronic antigen stimulation. Nat Comm 8: 15050.

Wittlich M, M Dudek, JP Bottcher, O Schanz, S Hegenbarth, T Bopp, E Schmitt, C Kurts, C Garbers, S Rose John, PA Knolle*, D Wohlleber*. 2017. Liver sinusoidal endothelial cell cross-priming is supported by CD4 T cell-derived IL-2. J Hepatol 66(5): 978-986.

Hackstein CP, LM Assmus, M Welz, S Klein, T Schwandt, J Schultze, I Forster, F Gondorf, M Beyer, D Kroy, C Kurts, J Trebicka, W Kastenmuller, PA Knolle*, Z Abdullah*. 2017. Gut microbial translocation corrupts myeloid cell function to control bacterial infection during liver cirrhosis. Gut 66(3): 507-518.

Project in detail +

The liver is known for its capacity to skew immune responses towards T cell tolerance, yet T cell immunity can be mounted under particular conditions. In the last funding period, we have identified the mechanisms that induce T cell immunity in the liver through recruitment of inflammatory monocytes into intrahepatic mye-loid cell aggregates associated with T cell expansion (iMATEs) by Toll-like receptor 9-ligand (TLR9-L) appli-cation. Together with Wohlleber and Heikenwälder we have identified a dual role of tumor necrosis factor (TNF) receptor signaling in recruitment of pro-immunogenic inflammatory monocytes to the liver as well as a direct anti-viral effect of TNF to elicit apoptosis selectively in infected hepatocytes. This revealed a novel cell-autonomous mechanism in antiviral defense in virus-infected hepatocytes that was governed by de-creased mitochondrial resilience leading to mitochondrial permeability transition after TNF challenge. We have further unraveled CD40 as molecular marker that identifies monocytes forming iMATEs and determined the key function of IL-15 for T cell activation within iMATEs, together with Heikenwälder. Moreover, together with Protzer we provided evidence that TLR9-L application leading to intra-hepatic CD8 T cell activation was synergistic with a heterologous prime-boost therapeutic vaccination and established immune control in pre-clinical models of persistent HBV infection. Finally, we have obtained evidence that iMATE-defining cells have a unique metabolic phenotype defined by extraordinary glycolytic activity. In the next funding period, we will explore the contribution of metabolic activation through iMATE-forming myeloid cells for HBV-specific T cell immunity in the liver. We will address the molecular mechanisms that mediate this unique form of metabolic T cell activation. In collaboration with Bengsch and Böttler, we will explore whether human HBV-specific CD8 T cells also share similar features of this metabolic T cell activation. Since CD8 T cells activat-ed and expanded within iMATEs express markers of liver-resident memory CD8 T cells, in particular CD69, CD49a and CXCR6, we will explore with Protzer and Heikenwälder whether activation of CD8 T cells after TLR9-L application is long-lasting. We will investigate whether repetitive TLR9-L application can lead to suc-cessive increase in hepatic CD8 T cell numbers and functionality to reveal establishment of a memory phe-notype of increased effector function in hepatic T cells. Finally, based on pilot data revealing a novel form of T cell tolerance in the liver with loss of TCR expression in the context of liver damage, we will determine whether liver cell death contributes to loss of protective HBV-specific CD8 T cell immunity against HBV-transduced hepatocytes by TCR downregulation and whether this can be overcome by TLR9-L application. Altogether, the results from these experiments will shed light on the molecular mechanisms of metabolic activation of CD8 T cells through a unique population of monocytes after TLR9-L application, how long im-mune protection by this metabolic activation lasts and whether it overcomes different forms of HBV-specific immune tolerance in antigen-specific CD8 T cells.

Milestones of the first funding period:
- Phenotypic analysis of myeloid cells in the liver under physiological steady state and inflammatory conditions
- Characterization of antigen (cross)presenting cell function of myeloid cells in the liver
- Determination of the relevance of myeloid cells for anti-infectious immunity against hepatotropic viruses
- Contribution of myeloid cells for immune control of HBV infection