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Due to the consumption of high caloric food combined with an increased sedentary lifestyle, the incidence of overweight and obesity is growing rapidly in western cultures, like the USA, Europe and notably also in developing countries (e.g. India, China) - as a consequence of adaptation to the western lifestyle. Thus, obesity-related pathologies like metabolic syndrome have become a major issue in modern medicine, and thus far therapeutic options are limited. The liver - the major metabolic organ of the body - is particularly affected by constant high caloric food intake. Consequently, the liver undergoes dramatic changes, including the development of fatty liver disease, termed non-alcoholic fatty liver disease (NAFLD). In 25% of all NAFLD cases, progression to a more severe pathology termed non-alcoholic steatohepatitis (NASH) can be observed. Immune cell activation in NASH leads to liver fibrosis and subsequently to hepatocellular carcinoma (HCC). Today, more than 90 million people in the USA and 30 million people in Europe are affected by NAFLD. Although chronic viral infections with Hepatitis B or C are the leading etiology causing HCC, it has become clear that NASH is an increasingly important factor for HCC development, a notion supported by the fact that HCC is currently the fastest rising cancer in the USA, with a similar trend in Europe. At the same time, knowledge about the key mechanisms causing NASH and NASH-triggered HCC are scarce and therefore efficient therapies to treat this diseases are lacking. The group of Professor Heikenwälder generated a mouse model of NASH and NASH-driven HCC in the context of a chronic metabolic syndrome6. Mice fed a long-term choline-deficient high fat diet (CD-HFD) develop obesity, steatosis, fibrosis, NASH and NASH-triggered HCC, recapitulating most of the key features found in human patients. In this model, activated CD8+ and NKT-cells drive NASH and HCC through cytokine-mediated crosstalk with hepatocytes. Remarkably, a similar T-cell activation, cytokine and immune cell pattern was found in NASH patients - establishing the clinical relevance of the CD-HFD mouse model. Aim 1: Deciphering TCR signaling in metabolically activated T-cells in NASH and liver cancer development In this scientific context, I investigated the role of T-cells in greater detail and T-cell receptor (TCR) dependent signaling in NASH development and NASH-induced HCC. By utilizing different dietary (high fat diet (HFD), CD-HFD and western-style diet with trans-fat (WD-HTF), genetic mouse models (C57Bl6, TCRβδ-/-, OT-1, Prf1-/-, Jα18-/-, CD1d-/-) and interventional antibody (α-CD8, α-PD-1, α-NK1.1) -based strategies in dietary mouse models, I shed new light on the key mechanisms of NASH pathology and its progression towards HCC. The data of this PhD thesis indicates that from early time points onwards, activation of subsets of CD8+ T-cells correlate with NASH pathology of different severity. Further, with progression of NASH towards advanced stages, CD8+ T-cells of animals fed NASH-inducing diets (CD-HFD or WD-HTF) expressed increasing amounts of early activation marker CD69 and activation/exhaustion marker PD-1. Also, PD-1+ CD8+ T-cells expressed different TCR variable β chains (TCR vβ), indicating that distinct exhausted repertoires develop over the course of NASH. Next, I showed that NASH pathology is dependent on a functional TCRαβ repertoire with intact TCRαβ effector function, but less on natural killer T-cell (NKT) cell-dependent mechanisms. Consistent with TCRαβ dependency in NASH development, I demonstrated a differential role of CD8+ T-cells depending on the progression state of NASH pathology, with a protective function of CD8+ T-cells in early states of NASH. However, in advanced NASH CD8+ T-cells drive hepatic immune-related adverse effects (irAEs), resulting in liver damage and tumor formation potentially involving a TNF-a-mediated mechanism. Aim 2: Platelet GPIbα is a mediator and potential interventional target for NASH and subsequent liver cancer After deciphering the role of T-cells in NASH and NASH-induced hepatocarcinogenesis, I investigated alternative ways to target hepatic inflammation in NASH without targeting potentially inflammation-driving immune cell populations directly. Thus, I investigated the key mechanisms of cell-immune cell interactions driving early NASH pathogenesis and identified platelets and platelet activation as major contributors to NASH pathology and subsequent HCC development. Further, I deciphered that platelets could be targeted by antiplatelet therapy (therapeutic Ticagrelor), thereby ameliorating NASH pathology and potentially subsequent hepatocarcinogenesis. I could show that platelet interaction with Kupffer cells and CD44-hyaluronan are key mechanisms of NASH progression and that GPIbα is an interventional target for NASH therapy. I published these results as a co-first author in Nature Medicine in April 2019 (https://doi.org/10.1038/s41591-019-0379-5).

Titelblatt und Inhaltsverzeichnis und Literaturverzeichniss und Lebenslauf und Danksagung und Erklärung Einleitung Material und Methoden Ergebnisse Diskussion Zusammenfassung, Ziel der vorliegenden Arbeit war es, Hörstörungen nach HWS- Weichteildistorsionen (HWS-WTD) und deren Lokalisation in der zentralen Hörbahn mittels objektiver Messverfahren nachzuweisen. Im Zeitraum zwischen Mai 2002 bis Januar 2004 wurden 265 Patienten nach akut erlittenen Verkehrsunfällen mit HWS-WTD in einem prospektiven Studiendesign untersucht. Alle Patienten wurden nach dem Unfall innerhalb von 72 Stunden in der Rettungsstelle des UKB bzw. direkt in der HNO-Klinik vorstellig. Dabei wurden sie neurologisch, radiologisch und HNO-ärztlich untersucht. Die Patienten wurden nach 3, 6 und 12 Monaten wieder zur Nachuntersuchung einbestellt. Im Rahmen der vorliegenden Arbeit fanden sich bei wenigen Patienten (n=8) initial diskrete Hörverluste als Innenohrschwerhörigkeit. Diese Hörverluste konnten in der Rein-tonaudiometrie nach 3 Monaten nicht mehr nachgewiesen werden. In der Gruppe der chronifizierten Patienten (n = 31) klagten insgesamt 11 Patienten über subjektive Hörstörungen (Hyperakusis, Tinnitus). Bei 20 der chronifizierten Patienten ließen sich nach 3 Monate noch komplette Stapediusreflexausfälle (zusätzlich 16 Teilausfälle) darstellen. Damit konnte im Rahmen der Untersuchungen eine Schädigung des akustiko-fazialen Reflexbogens nachgewiesen werden [23]. Im Studienverlauf ließen sich bei den chronifizierten Patienten Teilausfälle im TEOAE nachweisen. Mit Hilfe von Suppressionsversuchen der TEOAE im linearen Stimulationsmodus konnte eine nachhaltige Störung des olivo-cochleären Bündels und somit eine Störung der zentral-akustischen Verarbeitung dokumentiert werden [8; 48]. Bei einigen wenigen Patienten (n = 3) kam es im Zuge der Suppressionsversuche zu einer paradoxen Zunahme der OAE-Amplitude bei kontralateraler Stimulation (mit geringen Schallpegeln). Dieses als zentrale Enthemmung bekannte Phänomen in Verbindung mit einer klinisch angegebenen Hyperakusis bestätigt das in der neurotraumatologischen Forschung ansonsten gut belegte Paradigma der central hypersensitivity . Damit stehen die Untersuchungsergebnisse an der zentralen Hörbahn im Einklang mit den von den chronifizierten Patienten ansonsten angegebenen Leitsymptomen, vor allem der deutlich verringerten Schmerzschwelle. Die Variabilität der Ergebnisse der OAE- Suppressions- versuche ist vorrangig Ausdruck einer differenten neuronalen Schädigung ( axonal injury ). Pathologische BERA-Veränderungen ließen sich zu keinem Untersuchungszeitpunkt bei den untersuchten chronifizierten Patienten nachweisen, so dass diese Methode für den Nachweis subtiler neuronaler Veränderungen wenig geeignet zu sein scheint. Die vorliegende Arbeit zeigte, dass durch den Kopfanprall im Zuge einer HWS-WTD an die Kopfstütze oder den Anprall des Kopfes an festen Teilen des PKW (z.B. A-Holm) im Fahrzeuginneren in wenigen Fällen Hörstörungen ausgelöst werden können, die ihren Ursprung vorrangig in der zentralen Hörbahn haben. Durch eine differenzierte objektiv- audiometrische Diagnostik gelingt so eine Topodiagnostik, so dass damit auch gutachterlich eine Abgrenzung zu Aggravation und Simulation nach Unfällen vorgenommen werden kann., It is the aim of the present work to correlate clinical symptoms of auditory dysfunction (tinnitus, hyperacusis) after ablunt trauma of the head with objective audiological test results (otoacoustic emission, auditory brainstem response testing, impedance audiometry).Two houndred sixty and five patients were included. They were largely female.The clinical and otolaryngological examination (including otoscopy)of all patients revealed no pathological abnormalties. Pure tone audiometry showed a sensineural hearing loss at eight patients, this loss vanished entirely after three months.The main auditory symptomps were tinnitus (n=9) and hyperacusis (n=2). The results of testing the central auditory pathway showed that the transiently evoked otoacoustic emissions revealed statistically significant differences between amplitude differences of all patients in the linear, but not in the non-linear stimulation mode. A complette loss of stapedial reflex responses was found in twenty patients and a particular loss in 16 additional patients. Auditory brainstem response (ABR) were normal in all patients, but sixteen had lowered loudness discomfort levels (LDL).Blunt trauma of the head can lead to auditory dysfunction, probably as a result of diffuse axonal injury of the central auditory pathway.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1–5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6, 7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH–HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH–HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH–HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1–5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH–HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH–HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH–HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment., In hepatocellular carcinoma driven by non-alcoholic steatohepatitis, aberrant T cell activation and impaired immune surveillance seem to make hepatocellular carcinoma less responsive to anti-PD1 or anti-PDL1 immunotherapy.