The human liver microenvironment shapes the homing and function of CD4+ T-cell populations
Background & AimsTissue-resident memory T cells (TRM) are important immune sentinels that provide efficient in situ immunity. Liver-resident CD8+ TRM have been previously described, and contribute to viral control in persistent hepatotropic infections. However, little is known regarding liver CD4+ TRM cells. Here we profiled resident and non-resident intrahepatic CD4+ T cell subsets, assessing their phenotype, function, differential generation requirements and roles in hepatotropic infection. MethodsLiver tissue was obtained from 173 subjects with (n=109) or without (n=64) hepatic pathology. Multiparametric flow cytometry and immunofluorescence imaging examined T cell phenotype, functionality and location. Liver T cell function was determined after stimulation with anti-CD3/CD28 and PMA/Ionomycin. Co-cultures of blood-derived lymphocytes with hepatocyte cell lines, primary biliary epithelial cells, and precision-cut autologous liver slices were used to investigate the acquisition of liver-resident phenotypes. ResultsCD69 expression delineated two distinct subsets in the human liver. CD69HI cells were identified as CD4+ TRM due to exclusion from the circulation, a residency-associated phenotype (CXCR6+CD49a+S1PR1-PD-1+), restriction to specific liver niches, and ability to produce robust type-1 multifunctional cytokine responses. Conversely, CD69INT were an activated T cell population also found in the peripheral circulation, with a distinct homing profile (CX3CR1+CXCR3+CXCR1+), and a bias towards IL-4 production. Frequencies of CD69INT cells correlated with the degree of fibrosis in chronic hepatitis B virus infection. Interaction with hepatic epithelia was sufficient to generate CD69INT cells, while additional signals from the liver microenvironment were required to generate liver-resident CD69HI cells. ConclusionsIntermediate and high CD69 expression demarcates two discrete intrahepatic CD4+ T cell subsets with distinct developmental and functional profiles. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY HighlightsO_LICD69HI (CXCR6+CD49a+S1PR1-PD-1+) are the CD4+ TRM of the human liver C_LIO_LIHepatic CD69INTCD4+ T-cells are distinct, activated, and recirculation-competent C_LIO_LIStimulation evokes respective IFN-{gamma} and IL-4 responses in CD69HI and CD69INT cells C_LIO_LICD69INT cell frequencies correlate with worsening fibrosis in chronic HBV patients C_LIO_LILiver slice cultures allow differentiation of CD69INT and CD69HI cells from blood C_LI Lay summaryTissue-resident memory T cells (TRM) orchestrate regional immune responses, but much of the biology of liver-resident CD4+ TRM remains unknown. We found high expression of cell-surface protein CD69 defined hepatic CD4+ TRM, while simultaneously uncovering a distinct novel recirculatory CD69INT CD4+ T cell subset. Both subsets displayed unique immune receptor profiles, were functionally skewed towards type-1 and type-2 responses respectively, and had distinct generation requirements, highlighting the potential for differential roles in the immunopathology of chronic liver diseases.