TP13: Dynamics of antiviral immunity against hepatitis B virus in the liver

We investigate the impact of antigen levels, circulating antigen as well as presented antigens on the single cell level, on the dynamics of T cell immunity and the outcome of HBV infection. Using sophisticated HBV-infection models in genetically well-defined mice in combination with bioluminescence imaging and multispectral flow cytometry/multiparametric immunohistology, we aim to unravel the decisive factors leading to acute or chronic infection. We will also focus on metabolic, phenotypical and functional differences between virus-specific CD8 T cells in mice with either acute or chronic infection.

PD Dr. Dirk Wohlleber

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)

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

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

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

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

E. Schölzel K, Schildberg FA, Börner C, Geiger S, Kurts C, Heikenwälder M, Knolle PA, Wohlleber D. 2014. Cross-allocation of MHC-class-I molecules among liver sinusoidal cells facilitates hepatic immune surveillance. J Hepatol. 61(3):600-8.

F. Wolf MJ, Adili A, Piotrowitz K, Abdullah Z, Boege Y, Stemmer K, Ringelhan M, Simonavicius N, Egger M, Wohlleber D, Lorentzen A, Einer C, Schulz S, Clavel T, Protzer U, Thiele C, Zischka H, Moch H, Tschöp M, Tumanov AV, Haller D, Unger K, Karin M, Kopf M, Knolle P, Weber A, Heikenwalder M. 2014. Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepa-titis and liver cancer via cross-talk with hepatocytes. Cancer Cell. 26(4):549-64.

G. Huang LR, Wohlleber D, Reisinger F, Jenne CN, Cheng RL, Abdullah Z, Schildberg FA, Odenthal M, Dienes HP, van Rooijen N, Schmitt E, Garbi N, Croft M, Kurts C, Kubes P, Protzer U, Heikenwalder M, Knolle PA. 2013. Intrahepatic myeloid-cell aggregates enable local proliferation of CD8+ T cells and successful immunotherapy against chronic viral liver infection. Nat Immunol. 14(6):574-83.

H. Wohlleber D, Kashkar H, Gärtner K, Frings MK, Odenthal M, Hegenbarth S, Börner C, Arnold B, Hämmerling G, Nieswandt B, van Rooijen N, Limmer A, Cederbrant K, Heikenwalder M, Pasparakis M, Protzer U, Dienes HP, Kurts C, Krönke M, Knolle PA. 2012. TNF-induced target cell killing by CTL acti-vated through cross-presentation. Cell Rep. 2(3):478-87.

I. Hösel M, Broxtermann M, Janicki H, Esser K, Arzberger S, Hartmann P, Gillen S, Kleef J, Stabenow D, Odenthal M, Knolle P, Hallek M, Protzer U, Büning H. 2012. Toll-like receptor 2-mediated innate immune response in human nonparenchymal liver cells toward adeno-associated viral vectors. Hepatology. 55(1):287-97.

J. Stabenow D, Frings M, Trück C, Gärtner K, Förster I, Kurts C, Tüting T, Odenthal M, Dienes HP, Ceder-brant K, Protzer U, Knolle PA. 2010. Bioluminescence imaging allows measuring CD8 T cell function in the liver. Hepatology 51: 1430-1437.

Project in detail +

Infections by the hepatitis B virus (HBV) are controlled by the adaptive immune system. However, approx. 5% of infected subjects develop chronic hepatitis B for so far poorly understood reasons. The dynamics of the induction of innate as well as adaptive immunity is believed to be crucial for control of HBV infection. During the last funding period we have identified key parameters for the outcome of antiviral immunity in the liver: hepatocyte-specific antigen expression may lead to insufficient priming of virus-specific CD8 T cells and low antigen expression of virus-infected hepatocytes leads to an escape from CD8 T cell effector func-tion. Building on these results, during the next funding period we will continue working on the dynamics of antiviral immunity with a focus on HBV-specific CD8 T cells by pursuing two aims. In the first aim we will decipher reasons for the development of chronic HBV infection and associated impared antiviral immunity. Failure to control HBV infection may result from insufficient priming of HBV-specific CD8 T cells or from exhaustion/dysfunction of those cells during the effector phase. As HBV has a strong hepatotropism and behaves like a stealth virus, i.e. being not recognized by innate immune receptors, it is not clear where HBV-specific CD8 T cells are primed. Therefore, we will unravel in which organs priming of HBV-specific CD8 T cells takes place and which antigen-presenting cells are involved (with Knolle and Heikenwälder). In addition, HBV gene expression and replication may be decisive for the amount of presented and secreted antigens. High antigen amount and high number of antigen-presenting hepatocytes are associated with dysfunction-al/exhausted CD8 T cells. In contrast, low amount of presented antigen may cause neglection of virus-infected hepatocytes by HBV-specific CD8 T cells. Therefore, we aim to quantify the amount of presented antigen on virus-infected hepatocytes by mass spectrometry (with Binder/Pichlmair) and antibodies recog-nizing peptide-MHC-I complexes. By generating viruses with tunable HBcAg expression, we will be able to identify the antigen threshold responsible for either elimination of virus-infected hepatocytes or develop-ment of dysfunctional HBV-specific CD8 T cells. To link CD8 T cell responses to disease outcome, we will deliver engineered HBV genomes encoding for luciferase expressed under control of the HBV core promot-er via an adenoviral system into murine hepatocytes enabeling daily virus quantification. This will allow to analyse the dynamics of antiviral immunity. To this end, we will also replace multispectral-optoacoustic to-mography, because of a lack of sensitivity, by in vivo microscopy (with Bartenschlager). In addition, in col-laboration with Bengsch, we aim to connect the metabolic state of CD8 T cells to the disease outcome.
In the second aim we try to overcome chronic HBV infection in mice caused by high antigen load. We will modulate viral antigen expression and stimulate immunity by interferon-beta Flow/Mass-cytometry data (with Z02) and scRNAseq data (with Z03N) of HBV-specific CD8 T cells from acute, chronic and interferon-beta-treated mice will give insights into genes expressed during HBV elimination. These results will be com-pared to data obtained from human HBV-specific CD8 T cells (with Z03N, Thimme, Böttler and Hofmann). In conclusion, this proposal will shed light on the dynamics of HBV-specific immune responses and unravel mechanisms or pathways, which will decide whether an acute HBV infection is cleared or persists.

Milestones of the first funding period:
- Characterization of the dynamics of antiviral T cell immunity during acute viral infection of the liver
- Impact of persistent viral infection on the dynamics of virus-specific T cell immunity
- Refinement of optoacoustic imaging to provide a tool for following T cell responses in vivo