Abstract A132: Nonalcoholic fatty liver disease causes selective CD4+ lymphocytes loss and promotes hepatocarcinogenesis
Authors: Ma Chi , Kesarwala Aparna , McVicar Daniel , Weber Achim , Heikenwaelder Mathias , Greten Tim 
Published: May 2016

Abstract
Introduction: Hepatocellular carcinoma (HCC) is the second most common cause of cancer related death. Non-alcoholic fatty liver disease (NAFLD), which comprises a spectrum of liver pathology ranging from simple steatosis to the non-alcoholic steatohepatitis (NASH), affects a large proportion of the US population and is considered a metabolic predisposition to liver cancer. Until recently, adaptive immune responses in NAFLD and HCC have just begun to be understood. This study was designed to better understand how metabolic changes observed in NAFLD affect immune surveillance and promote hepatocarcinogenesis.
Methods: Methionine choline deficient (MCD) diet, choline-deficient and amino acid-defined (CDAA) diet and high fat (HF) diet were used to induce NASH/NAFLD in Lap-tTA-myc mice, which have liver-specific MYC oncogene expression after withdrawing doxycycline and develop HCC, or C57BL/6 mice challenged with dientylnotrisamine (DEN), a carcinogen known to induced HCC. Difference immune cell subsets were identified by flow cytometry. Cell death was measured by 7AAD/Annexin V staining. GK1.5 antibody was used to deplete CD4 T cells. Gas chromatography–mass spectrometry was performed to identify fatty acids. Gene change profile was identified by microarray. Oxygen consumption assay was performed to monitor cell metabolic changes. N-acetylcysteine was used to block ROS in vitro and in vivo.
Results: A robust selective loss of intrahepatic CD4+ but not CD8+ lymphocytes was observed in NASH/NAFLD mice fed with all three diets. The three NASH/NAFLD diets promoted HCC development in both Lap-tTA-myc and DEN-mice companying with the selective intrahepatic CD4+ lymphocyte reduction. Depletion of CD4+ lymphocytes further accelerated NAFLD-promoted HCC suggesting the contribution of hepatic CD4+ lymphocyte lose in HCC development. As expected higher level of cell death was detected in hepatic CD4+ lymphocytes after NASH/NAFLD. Next, we found that lipid-laden hepatocytes from NASH mice induced selective CD4+ but not CD8+ lymphocyte death through a contact-independent mechanism. FFAs were found from hepatocyte-conditioned medium to be responsible, and linoleic acid recapitulates the selective toxicity to CD4+ lymphocytes. The accumulation of linoleic acid in liver was confirmed by hepatic fatty acid profiling. CD4+ lymphocytes have greater mitochondrial mass than CD8+ lymphocytes and generate higher levels of mitochondrially-derived ROS. Accordingly, oxygen consumption assay plus directly measuring ROS production found that disruption of mitochondrial function by linoleic acid caused more severe oxidative damage in CD4+ lymphocytes. In vivo blockade of ROS using N-acetylcysteine reversed NASH-induced hepatic CD4+ lymphocytes decrease, and delayed NASH-promoted HCC.
Conclusion: Our results suggest the critical role of CD4+ lymphocytes in the disease progression of NASH to HCC, and provide a new link between lipid dysregulation and impaired anti-tumor surveillance.
Citation Format: Chi Ma, Aparna Kesarwala, Daniel McVicar, Achim Weber, Mathias Heikenwaelder, Tim Greten. Nonalcoholic fatty liver disease causes selective CD4+ lymphocytes loss and promotes hepatocarcinogenesis. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A132.