Foxo1-activity controls effector function of CXCR6+CD8+ T cells and prevents liver immune pathology during viral hepatitis and non-alcoholic steatohepatitis

Introduction:

Recognizing foreign antigens as peptides in a complex with MHC class I is indispensable for CD8+ T-lymphocytes to eliminate infected cells. Tight cellular co-regulation of metabolism and immunity is required to control effector function, but the mechanisms regulating organ-specific immunity in tissues rich in nutrients such as liver remained unclear. CD8+ T cells expressing the chemokine receptor CXCR6 are important for staying as tissue-resident memory T cells (Trm) in the liver and providing front-line defense for infections. Trm express high levels of effector molecules like GzmB or IFN-γ but how Trm regulates their effector function in order to avoid immune pathology is largely unknown. Here we identify Foxo1-activity in CXCR6+CD8+ T cells as critical regulator of CXCR6 expression, metabolism and effector function during liver disease states.

Material and Methods:

Extracellular flux analysis, cytokine expression, cytotoxicity assays were performed to study co-regulation of metabolism and immunity of CXCR6+CD8+ T cells and its dependence on Foxo1 in vitro and ex vivo studies. Murine models of viral hepatitis and non-alcoholic steatohepatitis (NASH) were used to explore Foxo1-dependent T cell immunopathology.

Results:

RNA-seq and KEGG pathway analysis of CXCR6+ memory CD8+ T cells of the liver compared to memory CD8+ T cells from other tissues revealed high GzmB level accompanied with downregulation of Foxo1-dependent pathways. Flow cytometric analysis of liver-specific lymphocytes revealed a Foxo1lowCXCR6+GzmBhighCD69+CD8+ T cell population. Using germ-free mice and in vitro studies we found that high GzmB level in Foxo1lowCXCR6+ CD8+ T cells were dependent on the microbiota. We further identified that TGFβ and IL15 are critical cytokines to downregulate Foxo1 via PI3K/pAkt pathway and inducing CXCR6+CD8+ T cells derived from CXCR6-CD122+ CD8+ T cells. To address the functional consequence of CXCR6+Foxo1lowGzmbhigh CD8 T cells we performed in vitro killing assays of infected and non-infected hepatocytes demonstrating antigen-independent immune pathology against hepatocytes in the presence of elevated levels IL15 and acetate. This development of autoimmune CD8 T cell effector function could was also be observed in NASH where we detected high numbers of Foxo1lowCXCR6+GzmBhighCD69+CD8+ T cells that caused liver damage. Since TCR sequencing of hepatic T cells did not reveal presence of particular T cell-clones in NASH, we assume that increased effector function in CXCR6+ CD8+ T cells in the absence of Foxo1-control caused antigen-independent hepatic immunopathology.

Conclusion:

Our results provide evidence for a critical role of Foxo1 in controlling metabolism in CD8 T cells that is required to prevent liver immunopathology and may explain metabolic CD8 T cell activation in NASH causing sterile inflammation.