Z02: High-resolution multi-parametric cell and tissue analysisTo unravel the mechanisms of hepatitis virus persistence, TRR179 will employ an interdisciplinary approach. Z2 will provide a central platform for validated, cutting edge immune monitoring and high-speed cell sorting as well as bioinformatics analyses. In addition, Z2 will also develop further these techniques and adapt them to the specific needs of TRR179. In these respects, Z2 will fill technological gaps, provide essential help to harmonize protocols and methods and improve the methods while avoiding parallel and repeated developments. Another major goal of Z2 is to ensure training and education in immune monitoring, cell sorting and bioinformatics for members of this consortium, thus critically contributing also to the IRTG of TRR179.
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. Wührl M, Ringelhan M, Ehmer U, Schneider J, Kager J, Lahmer T, Schneider A, Weichert W, Mogler C. (2023) (Seltene) infektiöse Hepatitiden als wichtige Differenzialdiagnose der unklaren Hepatopathie. Pathologie (Heidelb). 44(1):53-62. German. doi: 10.1007/s00292-022-01167-0.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.Schreiber S, Honz M, Mamozai W, Kurktschiev P, Schiemann M, Witter K, Moore E, Zielinski C, Sette A, Protzer U, Wisskirchen K. (2021) Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors. Mol Ther Methods Clin Dev. 29;23:476-489. doi: 10.1016/j.omtm.2021.10.012.Flommersfeld S, Böttcher JP, Ersching J, Flossdorf M, Meiser P, Pachmayr LO, Leube J, Hensel I, Jarosch S, Zhang Q, Zeeshan Chaudhry M, Andrae I, Schiemann M, Busch DH, Cicin-Sain L, Sun JC, Gasteiger G, Victora GD, Höfer T, Buchholz VR, Grassmann S. (2021) Fate mapping of single NK cells identifies a type 1 innate lymphoid-like lineage that bridges innate and adaptive recognition of viral infection.Immunity. 54:2288-2304 e2287. doi: 10.1016/j.immuni.2021.08.002.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.Winkler F, Bengsch B. (2020) Use of Mass Cytometry to Profile Human T Cell Exhaustion. Front Immunol. 22;10:3039. doi: 10.3389/fimmu.2019.03039.Andrä I, Ulrich H, Dürr S, Soll D, Henkel L, Angerpointner C, Ritter J, Przibilla S, Stadler H, Effenberger M, Busch DH, Schiemann M. 2020. An Evaluation of T-Cell Functionality After Flow Cytometry Sorting Revealed p38 MAPK Activation. Cytometry A. 97(2):171-183. doi: 10.1002/cyto.a.23964. (Z02) Smits M, Zoldan K, Ishaque N, Gu Z, Jechow K, Wieland D, Conrad C, Eils R, Fauvelle C, Baumert TF, Emmerich F, Bengsch B, Neumann-Haefelin C, Hofmann M, Thimme R, Boettler T. 2020. Follicular T helper cells shape the HCV-specific CD4 T cell repertoire after viral elimination. J Clin Invest. 130(2):998-1009. (TP01, TP01A, TP02, TP04, Z02)Schuch A, Salimi Alizei E, Heim K, Wieland D, Kiraithe MM, Kemming J, Llewellyn-Lacey S, Sogukpinar Ö, Ni Y, Urban S, Zimmermann P, Nassal M, Emmerich F, Price DA, Bengsch B, Luxenburger H, Neu-mann-Haefelin C, Hofmann M, Thimme R. 2019. Phenotypic and functional differences of HBV core-specific versus HBV polymerase-specific CD8+ T cells in chronically HBV-infected patients with low viral load. Gut. 68(5):905-915Karimzadeh H, Kiraithe MM, Oberhardt V, Salimi Alizei E, Bockmann J, Schulze Zur Wiesch J, Budeus 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, Hof-mann 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 Population Level. Gast-roenterology. 156(6):1820-1833.Grassmann S, Pachmayr LO, Leube J, Mihatsch L, Andrae I, Flommersfeld S, Oduro J, Cicin-Sain L, Schiemann M, Flossdorf M, Buchholz VR. 2019. Distinct Surface Expression of Activating Receptor Ly49H Drives Differential Expansion of NK Cell Clones upon Murine Cytomegalovirus Infection. Immuni-ty. 50(6):1391-1400 Steiger K, Ballke S, Yen HY, Seelbach O, Alkhamas A, Boxberg M, Schwamborn K, Knolle PA, Weichert W, Mogler C. 2019. Histopathological research laboratories in translational research: Conception and in-tegration into the infrastructure of pathological institutes. Der Pathologe. 40(2):172-178.Bengsch B, Ohtani T, Khan O, Setty M, Manne S, O'Brien S, Gherardini PF, Herati RS, Huang AC, Chang KM, Newell EW, Bovenschen N, Pe'er D, Albelda SM, Wherry EJ. 2018a. Epigenomic-Guided Mass Cy-tometry Profiling Reveals Disease-Specific Features of Exhausted CD8 T Cells. Immunity. 48(5):1029-1045.Bengsch B, Ohtani T, Herati RS, Bovenschen N, Chang KM, Wherry EJ. 2018b. Deep immune profiling by mass cytometry links human T and NK cell differentiation and cytotoxic molecule expression pat-terns. J Immunol Methods. 453:3-10.Mogler C, König C, Wieland M, Runge A, Besemfelder E, Komljenovic D, Longerich T, Schirmacher P, Augustin HG. 2017. Hepatic stellate cells limit hepatocellular carcinoma progression through the orphan receptor endosialin. EMBO Mol Med. 9(6):741-749. 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In depth characterization of immune responses in vivo requires specific methodologies that are optimized for low cell numbers, sparse tissue samples and noisy data. To tackle these challenges, the central aim of project Z02 is the establishment of highly sensitive immunoassays and histopathological analyses that will be used to support members of TRR179. During the first funding period, Z02 focused on the most im-portant analytical challenges, which were the scarcity of relevant immune cells that can be isolated from infected patients or mouse tissues, the high background inherent to the analyses of these low cell numbers and limitations imposed by high biosafety regulations. In the course of this work continuous improvements in high-speed cell sorting of rare cell populations, such as antigen-specific T cells or NK cells, were made (Grassmann et al., 2019). In addition, methods were adapted to allow e.g. the identification of T cell recep-tors against HBV epitopes (Wisskirchen et al., 2017), which will lay the ground for CAR T-cell approaches aiming at HBV cure. Moreover, as part of the immune monitoring activities, a database of patients with HBV, HCV, and HDV infection has been established and used for multiple collaborative projects characteriz-ing important parameters of immune cells in patients with chronic hepatitis virus infection1-3 (Grassmann et al., 2019; Karimzadeh et al., 2019). Another important development was the establishment of methods allow-ing transcriptional profiling of rare T cell populations isolated from patients with chronic viral infection and the characterization of their T cell exhaustion status4,5. Building on these achievements, and taking into ac-count the increasing demand for high-resolution, ideally single cell profiling and the new technological de-velopments in this field, we decided to focus Z02 onto histopathology and sorting of rare cell populations and to integrate cutting-edge mass cytometry. Bioinformatics analysis (former part of Z02) will be combined with single cell transcriptomic and (epi)genomic analyses as new project Z03N that will address a rapidly growing field offering unprecedented possibilities to profile immune cells and to dissect their heterogeneity, which is at the heart of TRR179. Regarding Z02, during the next funding period we will continue the devel-opment of cutting-edge sorting approaches such as FRET biosensors to characterize signaling pathways in immune or infected cells, improve further BSL3-conform cell sorting and integrate tissue-based technical services. These include histology and immunohistochemistry, laser microdissection, a histopathological slide consultation service for all TRR179 members (for mouse and human samples) and access to human liver tissue, individually adopted to the projects of the consortium (in collaboration with the biobank located at the institute of pathology at Technical University Munich). A very important new addition to Z02 is the provision of cutting-edge mass cytometry. This method combines the advantages of single-cell high-speed flow cytometry with the ability to resolve more than 40 different parameters, thus allowing very detailed phe-notypic and functional analyses. This is particularly crucial in the context of immune cell populations that require a high-dimensional approach for their precise characterization, such as exhausted T cells as studied in TRR179. For instance, mass cytometry was required to identify subpopulations of health- and disease-associated exhausted T cells based on their combinatorial expression profile in patients (Bengsch et al., 2018a). Likewise, HBV-specific T cells associated with control of infection after DAA therapy were identified by mass cytometry6. Moreover, mass cytometry was also crucial to identify the T cell differentiation and exhaustion profiles of HBV-specific T cells responding to epitopes presented by an Asian HLA allele7. Of note, in addition to these applications, the technology allows highly efficient use of rare samples, which is a frequent limitation when working with in vivo material. While in all these respects, Z02 provides important technical and scientific support to TRR179 projects, it will also participate in training and education by offer-ing method-oriented workshops and hands-on experience, thus complementing the training program of the IRTG “Immunovirology” of TRR179.
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 interferon-stimulated genes on virus-specific CD8+ T cells and NK cells in viral hepatitis
Impact of the enteric stage of hepatitis A virus and hepatitis E virus on the outcome of liver infection