Biliary epithelium and liver B cells exposed to bacteria activate intrahepatic MAIT cells through MR1

Abstract

Background & aims: Mucosal-Associated Invariant T (MAIT) cells are innate-like T cells characterised by the invariant TCR-chain, Vα7.2-Jα33, and are restricted by MR1, which presents bacterial vitamin B metabolites. They are important for antibacterial immunity at mucosal sites; however, detailed characteristics of liver-infiltrating MAIT (LI-MAIT) and their role in biliary immune surveillance remain unexplored.

Methods: The phenotype and intrahepatic localisation of human LI-MAIT cells was examined in diseased and normal livers. MAIT cell activation in response to E. coli-exposed macrophages, biliary epithelial cells (BEC) and liver B cells was assessed with/without anti-MR1.

Results: Intrahepatic MAIT cells predominantly localised to bile ducts in the portal tracts. Consistent with this distribution, they expressed biliary tropic chemokine receptors CCR6, CXCR6, and integrin αEβ7. LI-MAIT cells were also present in the hepatic sinusoids and possessed tissue-homing chemokine receptor CXCR3 and integrins LFA-1 and VLA-4, suggesting their recruitment via hepatic sinusoids. LI-MAIT cells were enriched in the parenchyma of acute liver failure livers compared to chronic diseased livers. LI-MAIT cells had an activated, effector memory phenotype, expressed α4β7 and receptors for IL-12, IL-18, and IL-23. Importantly, in response to E. coli-exposed macrophages, liver B cells and BEC, MAIT cells upregulated IFN-γ and CD40 Ligand and degranulated in an MR1-dependent, cytokine-independent manner. In addition, diseased liver MAIT cells expressed T-bet and RORγt and the cytokines IFN-γ, TNF-α, and IL-17.

Conclusions: Our findings provide the first evidence of an immune surveillance effector response for MAIT cells towards BEC in human liver; thus they could be manipulated for treatment of biliary disease in the future.

Keywords: Biliary epithelium; Biliary firewall; E. coli; Human liver; Immune response; Mucosal-associated invariant T cells.

Fig. 1

Peri-biliary localisation of Vα7.2⁺ cells in chronic liver diseases. (A) Representative staining for Vα7.2 on frozen liver sections viewed at 10× (i and iii) or 40× (ii, iv, v, and vi) magnification. Distribution of Vα7.2+ cells in the parenchyma (i and iv) and portal tract (i, ii, and v) in PSC and in the parenchyma (iii and vi) in seronegative acute liver failure. (B) Densities of Vα7.2+ cells in parenchyma and portal tracts of normal and chronically diseased livers (∗∗p <0.01; ∗∗∗p <0.0001 by Mann-Whitney U test). (C) Vα7.2+ cell density data according to diseases. Data are median ± interquartile range.

Fig. 2

CD3⁺CD161⁺Va7.2⁺ cells reside close to bile ducts in portal tracts. Representative confocal immunofluorescence staining for CD3, CD161, and Va7.2 on frozen sections from explanted human livers diagnosed with Alcoholic liver disease (A) and Primary Biliary Cirrhosis (B). DAPI nuclear stain reveals liver architecture indicating sites of bile ducts. Images are representative of staining of four different diseased livers, scale bar shows 100 μm.

Fig. 3

Flow cytometric analysis of MAIT cell frequencies in chronic liver disease and their correlations with other immune subsets. Representative FACS plots (A) and summary frequency data for total CD3⁺ MAIT (B) and CD4⁺, CD8⁺ and CD4⁻ CD8⁻ (DN) MAIT subsets in normal and diseased blood (C) and liver (D). (E) Frequencies of intrahepatic MAIT and other immune cells. (F) Correlation of CD3⁺ MAIT cell frequencies with total CD4⁺, CD8⁺, DN, and CD161⁺ T cell frequencies in normal and diseased livers. Data are median ± interquartile range. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 by Mann-Whitney U test (B–E) or Spearman’s rank correlation (F).

Fig. 4

Expressions of tissue-homing chemokine, integrin and cytokine receptors on intrahepatic MAIT cells in normal and chronic liver diseases. Chemokine receptor and integrin expression profiles (A, B, and C) and cytokine receptor profiles (D) were determined by flow cytometry gating on the total CD3⁺ MAIT cell population. Representative overlays for marker (line) and isotype control (grey shading) and total summary data are shown. Summary data are median ± interquartile range. ∗p <0.05; ∗∗p <0.01; ∗∗∗p <0.001 by Mann-Whitney U test.

Fig. 5

Cytokine and cytolytic factor expression by liver-infiltrating MAIT cells. Intrahepatic MAIT cell production of Th1 (IFN-γ, TNF-α), Th2 (IL-4, IL-5, IL-13) and Th17 (IL-17, IL-22) cytokines was examined by flow cytometry. Representative dot-plots for each cytokine vs. IFN-γ, expression (A) and summary data by MAIT cells (B) and MAIT cell subsets (C and D) are shown. Summary data for ex vivo granzyme B expression in total MAIT and MAIT cell subsets (E). Data are median ± interquartile range. ∗p <0.05; ∗∗p <0.01; by Dunn’s post hoc test following Friedman’s test.

Fig. 6

MR1-dependent activation of blood and liver MAIT cells by professional and non-professional antigen presenting cells (APC) exposed to bacteria. APC exposed to E. coli were co-cultured with MAIT cells and their activation examined by flow cytometry for CD107a, IFN-γ, TNF-α, CD40L. Dependency on MR1 or IL-12 and IL-18 was assessed by antibody blocking. Activation of blood MAIT by blood macrophages (A) and activation of liver MAIT by THP1 cells (B) and autologous intrahepatic B cells (C) were analyzed. Data are mean ± SEM (A) and median ± interquartile range (B and C). In overlays: untreated (shaded); E. coli (solid line); E. coli + αMR1 (dotted line). ∗p <0.05; ∗∗∗p <0.001 by paired t test. UT = untreated.

Fig. 7

Bacterially-exposed primary human biliary epithelial cells activate both blood and liver MAIT cells in an MR1 dependent manner. Biliary epithelial cells exposed to E. coli were co-cultured with sorted blood CD8⁺ (A and B) or liver CD3⁺ (C) T cells and activation of CD161⁺⁺ Va7.2⁺ MAIT cells examined by flow cytometry for CD107a, IFNγ-, TNFα, CD40L. Dependency on MR1 or cytokines IL-12 and IL-18 was assessed using function-blocking antibodies. Data are mean ± SEM (A and B) and median ± interquartile range (C). In overlays: untreated (shaded); E. coli (solid line); E. coli + αMR1 (dotted line). ∗p <0.05; ∗∗∗p <0.001 by t test. UT = untreated.