ABSTRACT
Background & Aims Tissue-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.
Methods Liver 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.
Results CD69 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.
Conclusions Intermediate and high CD69 expression demarcates two discrete intrahepatic CD4+ T cell subsets with distinct developmental and functional profiles.
Highlights
CD69HI (CXCR6+CD49a+S1PR1-PD-1+) are the CD4+ TRM of the human liver
Hepatic CD69INTCD4+ T-cells are distinct, activated, and recirculation-competent
Stimulation evokes respective IFN-γ and IL-4 responses in CD69HI and CD69INT cells
CD69INT cell frequencies correlate with worsening fibrosis in chronic HBV patients
Liver slice cultures allow differentiation of CD69INT and CD69HI cells from blood
Lay summary Tissue-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.
Competing Interest Statement
BGW collaborated with and received funding from Bioniz. LJP has consulted for Gilead Sciences. KA is funded by a studentship with Dr Falk. MKM has received research funding from Gilead, Hoffmann La Roche and Immunocore. MKM has sat on advisory boards/provided consultancy for Gilead, Hoffmann La Roche, Immunocore, VIR, Galapagos NV, GSK, Abbvie, Freeline. ZS collaborated with Bioniz and AstraZeneca and has consulted for Boehringer Ingelheim. All other authors declare no conflict of interest.
Footnotes
↵* Co-first authors
↵# Co-last authors
Professor Mala Maini, Division of Infection and Immunity, Rayne building, 5 University street, London, UK, WC1E 6JF, m.maini{at}ucl.ac.uk
Professor Yuehua Huang, Department of Infectious Diseases and Guangdong Provincial Key Laboratory of Liver Disease Research, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, huangyh53{at}mail.sysu.edu.cn
List of abbreviations
- ALD
- alcoholic liver disease
- ALT
- alanine aminotransferase
- BEC
- biliary epithelial cells
- CHB
- chronic hepatitis B virus
- CTLA-4
- cytotoxic T-lymphocyte-associated protein-4
- FNA
- fine needle aspirates
- HAI-NI
- histology activity index necroinflammation score
- HBeAg
- hepatitis B e antigen
- HBV
- hepatitis B virus
- HCC
- hepatocellular carcinoma
- HSEC
- hepatic sinusoidal endothelial cells
- IFN-γ
- interferon-gamma
- IHL
- intrahepatic leukocytes
- IL
- interleukin
- KLRG-1
- killer cell lectin-like receptor-G1
- MFI
- median fluorescence intensity
- NASH
- non-alcoholic steatohepatitis
- PBC
- primary biliary cholangitis
- PD-1
- programmed cell death protein-1;
- PSC
- primary sclerosing cholangitis
- S1PR1
- sphingosine-1-phosphate receptor-1
- TCM
- central memory T cell
- TCR
- T cell receptor
- TEM
- effector memory T cell
- TGFβ
- transforming growth factor beta
- TN
- naïve T cell
- TNFα
- tumour necrosis factor-alpha
- TREG
- regulatory T cell
- TRM
- tissue-resident memory T cell.