Blinding of virus-specific CXCR6+ CD8 T cells in the liver during chronic viral hepatitis causes loss of effector function

Chronic viral hepatitis after infection with hepatotropic viruses like hepatitis B virus (HBV) affects 300 million persons worldwide, which as the result of chronic immune-mediated hepatic inflammation cause liver cirrhosis and cancer being responsible for 800.000 deaths per year3. Chronic viral hepatitis is maintained by failure of the host´s immune response to control viral infection, but the mechanisms for this inability of virus-specific CD8 T cells to eliminate HBV-infected hepatocytes remain unclear. Here, we demonstrate that during persistent experimental infection with hepatotropic viruses and HBV replication in hepatocytes all virus-specific CD8 T cells present in the liver expressed the tissue-residency markers CXCR6 and CD69. However, RNAseq analysis revealed that CXCR6+CD8 T cells during persistent hepatotropic infection were retained in the liver because of antigen-recognition rather than a transcriptional tissue-residency program, in contrast to canonical liver-resident memory CXCR6+CD8 T cells emerging after resolved infection. Whereas during persistent infection with a model virus like lymphocytic choriomeningitis virus with broad tissue and cell tropism exhausted CD8 T cells show graded loss of effector functions, hepatic virus-specific CXCR6+CD8 T cells during persistent infection with hepatotropic viruses were blinded and completely non-responsive to stimulation in absence of a canonical tox exhaustion signature. Rather, in blinded liver CXCR6+CD8 T cells, transcription factor network analysis revealed Crem, the cAMP-responsive-element-modulator, as the only transcription factor discreetly active in CD8 T cells with complete loss of effector function during persistent hepatotropic infection. Similarly, single cell RNA-sequencing of peripheral blood HBcore-specific CD8 T cells from chronic hepatitis B patients also revealed enhanced CREM activity. Notably, knock-out of the inhibitory CREM/ICER gene in T cells failed to rescue protective T cell immunity during persistent infection with hepatotropic viruses pointing towards post-translational mechanisms relevant for enhanced Crem activity and loss of effector function. Indeed, T cell receptor-associated signalling was blocked in blinded antigen-specific CXCR6+CD8 T cells, that in situ during persistent hepatotropic infection were in close proximity to liver sinusoidal endothelial cells producing high amounts of cAMP-inducing prostanoids. Inhibitory cAMP/PKA/CSK activity increased CREM activity and disconnected CXCR6+CD8 T cells from activation signalling through the T cell receptor. Thus, enhanced CREM expression identifies blinded liver CXCR6+CD8 T cells, but loss of effector functions is caused by post-translational prevention of signalling, which identifies novel molecular targets for immune monitoring and immune therapy of chronic hepatitis B. mRNA profiles of P14 cells after either LCMV Clone 13 or LCMV Armstrong infection 27 dpi.