Stimulatory MAIT cell antigens reach the circulation and are efficiently metabolised and presented by human liver cells

Introduction

Mucosal-associated invariant T (MAIT) cells are the most abundant population of innate-like T cells in humans; they comprise up to 5% of T cells in peripheral blood and are found in high numbers in the liver and mucosal tissues.1–3 MAIT cells are restricted to the highly conserved major histocompatibility complex (MHC)-class I related molecule MR1.2 They express a semi-invariant T cell receptor (TCR) containing the Vα7.2 variable chain (Vα7.2-Jα33/12/20) paired with an oligoclonal TCRβ repertoire.2 4 MAIT cells recognise MR1-bound metabolites of the riboflavin (vitamin B2) biosynthesis pathway produced by many pathogenic and commensal bacteria.2 5 6 The most potent stimulatory MAIT cell antigen (Ag) is the pyrimidine 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU). It is formed by non-enzymatic condensation of the bacteria-derived precursor 5-amino-6-ribitylaminouracil (5-A-RU) with methylglyoxal derived from the glycolysis pathway.7 On TCR activation, MAIT cells secrete pro-inflammatory cytokines like interferon γ (IFN-γ) and tumour necrosis factor alpha (TNF-α) and acquire potent killing capacity; they consequently exert important immunoregulatory and antimicrobial functions.8 9 MAIT cells are further characterised by high cell-surface expression of CD161, interleukin-18 receptor (IL-18R) and IL-12R. Thus they can be activated by respective cytokines in TCR-independent manner.1 10–12 These features of MAIT cells provide protective innate immunity before an adaptive immune response has formed.

MAIT cells constitute up to 40% of human liver residing T cells,1 3 13 14 indicating their importance in liver physiology and pathogenesis of liver diseases. Liver MAIT cells secrete large amounts of IFN-γ, TNF-α and IL-17. Secretion of the latter follows repetitive IL-12 stimulation or occurs in response to IL-7.13–17 IL-17 acts as a fibrogenic cytokine that activates hepatic stellate cells (HSCs).18 The aforementioned cytokine expression profile suggests a detrimental role of MAIT cells in liver inflammation and fibrogenesis. This is supported by recent findings that lack of MAIT cells in MR1−/− mice protects against liver fibrosis, and that MAIT cells from patients with liver cirrhosis show alterations consistent with profibrotic activity.16 17

Apart from the locally produced endogenous cytokines, the liver receives cytokines and bacterial products originating from the gut and systemic circulation. Bacterial products may also enter the liver via the biliary tree. The responsiveness to bacterial products places MAIT cells at a central position in the immunological gut-liver axis, a notion supported by the finding that biliary epithelial cells (BECs) present Escherichia coli-derived Ag to MAIT cells.14 Recent studies, performed in mice, also argue for a potential of the MAIT cell Ag 5-OP-RU or its precursor to cross the intestinal barrier.19 20 Furthermore, plasma from patients with alcoholic liver disease (ALD), having increased gut leakiness, was found to activate MAIT cells more strongly than plasma from healthy subjects.21

It has been proposed that various hepatic parenchymal and non-parenchymal cell types act as liver-resident antigen presenting cells (APCs) for MHC class I/II and CD1d-dependent Ag-presentation.14 22 However, it has not yet been investigated whether cells from the sinusoidal environment, including hepatocytes, HSCs and liver sinusoidal endothelial cells (LSECs), that is, cells in direct contact with the blood entering through the portal vein, can present metabolite Ags to MAIT cells via MR1.

In the present study, we determine the localisation of MAIT cells within the human liver and characterise the potential of human liver primary cells to promote formation of active Ag from its bacterial precursor and to function as APCs stimulating MAIT cells. We examine the potential of serum derived from patients suffering from portal hypertension or inflammatory bowel disease (IBD) to stimulate MAIT cells in MR1-dependent manner. We test the ability of non-stimulatory ligands to block MAIT cell activation by liver APCs as a therapeutic intervention interfering with the profibrogenic function of MAIT cells.

Materials and methods

Resources used are listed in online supplemental tables 1-3. Further details of resources and methods not described in the main text (including preparation of bacterial products and synthetic MAIT cell Ag; quantitative real-time PCR; ELISA; flow cytometry analysis; mass spectrometry analysis; and D-lactate assay) are in online supplemental data file.

Human samples

Written informed consent was obtained from each patient. The study protocol conforms to ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the local ethics committees. Information about patient characteristics of liver biopsy/resection samples used for analyses, patients with a history of transjugular intrahepatic portosystemic shunt stent (TIPSS) placement and patients with IBD, are in online supplemental tables 4-6.

Patient and public involvement statement

Patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research.

Preparation of primary cells from human samples

Primary liver cells (hepatocytes, HSCs, LSECs and BECs) were prepared from liver samples from patients undergoing liver resection at University Hospitals in Basel and Bern, Switzerland. Isolation and purification of cells followed established published procedures. Adipocytes and human umbilical vein endothelial cells (HUVECs) were extracted using published protocols. Identity of purified cells was verified by flow cytometry and bright field or immunofluorescence (IF) microscopy.

To enrich for blood-derived MAIT cells from healthy donors, either CD8+ cells were isolated using the Miltenyi positive selection kit, or negative selection was performed using Abs against CD45RA, CD62L, CD19, CD14, CD36 and TCRγδ.

For detailed protocols and references, see the online supplemental data file.

IF and immunohistochemistry staining of human liver sections and analysis of cell distribution

Cryopreserved human liver biopsy or resection samples from the biobank at the Department of Biomedicine (University of Basel, Switzerland) and the GI biobank (Translational Gastroenterology Unit and the Oxford University Hospitals Trust, Oxford, UK) were used. MAIT cells were either visualised by combined IF staining for CD3, Vα7.2 and IL-18R-α, or by chip cytometry including also additional markers (CD161, CD4, CD8). For visualisation of natural killer (NK) cells, defined as CD57+CD3− cells, paraffin embedded liver sections were used. For detailed staining protocols and antibodies used, see online supplemental data file and online supplemental table 1.

MAIT cell, non-MAIT T cell, NK cell detection and calculation of their distance to regions of interest (ROIs) within liver sample images was performed using QuPath. Using the ‘distance to annotation 2D’ spatial analysis tool, the distance of cells belonging to different cell classes to the ROI was calculated. Only liver biopsies with an intact structure and presenting enough ROIs were used for distance calculations.

Cell lines and MAIT cell clones

All cell lines and MAIT cell clones are listed in online supplemental table 2. This list also includes two liver-derived MAIT cell lines (MAIT-BEL-10, MAIT-BSL-19) generated in this work from fluorescence-activated cell sorting (FACS)-sorted MAIT cells obtained from normal liver tissue samples of two patients undergoing surgery for colorectal cancer metastasis.

Ag presentation assays

All assays with primary human cells used as APCs were performed in Roswell Park Memorial Institute (RPMI) medium containing 10% fetal calf serum (FCS; always the same lot number) to exclude non-specific effects of variable culture media. E. coli lysate or synthetic Ag was added to the APCs at indicated concentration for 2 hours prior to addition of MAIT cells at an APC to MAIT cell ratio of 1:4. Primary MAIT cells were used right after isolation, MAIT cell clone SMC3 and liver-derived MAIT cell lines at day 14 after restimulation. MAIT cell activation and cytokine secretion was evaluated after 16 hours of co-culture. Further details of APC assays with primary cells and established cell lines are described in online supplemental data file.

Measurement of antigenic potential of human serum

Serum from peripheral blood was collected from healthy donors, from patients with IBD and from patients with a history of or in need of TIPSS. Portal blood was drawn during the TIPSS procedure. For Ag presentation assays, 104 K562-MR1 cells were seeded in RPMI medium without FCS and 50 µl of human serum was added. In experiments testing inhibition by acetyl-6-FP, K562-MR1 cells were incubated with 5 µM acetyl-6-FP for 1 hour before serum addition. After 2 hours incubation with serum, 35 μg/ml anti-MR1 Ab was added when indicated, followed by 1 hour incubation before the addition of 5×104 SMC3 MAIT cells and further incubation for 16 hours. IFN-γ secretion was measured by ELISA. To calculate 5-OP-RU equivalents, control MAIT cell activation assays containing different concentrations of 5-OP-RU (0.006–25 pM) were included in each plate.

When measuring intracellular IFN-γ staining in MAIT SMC3 cells, experimental conditions were similar to those used for ELISA, except that final incubation after the SMC3 MAIT cell addition was for 7 hours in presence of 5 µg/mL Brefeldin A. In control experiments, 5-OP-RU (0.1–10 pM) was added instead of the human serum. IFN-γ positive MAIT cells were assessed by FACS.

Statistical analysis

Unless indicated otherwise, all graphs presented in the figures represent data from three or more independent experiments. Exact numbers of repetitions are indicated in figure legends. Unless indicated otherwise, values plotted in the column graphs are means+SD. Statistical analyses were performed in GraphPad Prism V.7 or R V.4.1.2. In order to use parametric tests, we log-transformed the response variables or applied square root transformation. We used t-test when comparing the means of two groups, otherwise we applied one-way analysis of variance (ANOVA) with Dunnet post-hoc test for cases where we compared multiple groups to single reference group, or we applied two-way ANOVA with Dunnet post-hoc test in order to take into account a paring variable. In figures depicting distances to ROIs, significance levels were obtained from one-way ANOVA with selected post-hoc comparisons (t-test), p values were adjusted with Benjamini-Hochberg correction for ‘multiple testing’.

ResultsMAIT cells are dispersed within the parenchyma in healthy human liver

We investigated, by IF staining, localisation of MAIT cells using human liver samples without apparent histopathological abnormalities, originating from 15 donors (see online supplemental table 4). MAIT cells were identified as cells positive for CD3, TCR Vα7.2 and IL-18Rα, a robust combination of markers as IL-18Rα parallels high CD161 expression on MAIT cells (figure 1, online supplemental figure 1A–C).9 MAIT cells were distributed throughout the parenchymal space in the liver (figure 1A and online supplemental figures 1A,B). MAIT cells represented 5%–33% of liver T cells, considerably exceeding the frequency of Vα7.2-positive conventional T cells (figure 1B). MAIT cells were found in or in the immediate proximity of the sinusoids, and occasionally also within portal fields. To assess their zonal distribution, we performed digital analysis, measuring distances of MAIT cells and non-MAIT CD3+ cells to their closest portal fields and central veins. MAIT cells showed dispersed localisation, with >75% localising at least 200 µm away from either central veins or portal fields, confining most of them to the intermediary zone 2 of the hepatic lobule, with enrichment towards portal fields (figure 1C,D). In detail, 5% were in portal fields and 7% within 100 µm of portal fields. In contrast, non-MAIT T cells were more frequently (~30%) found within portal fields (figure 1D). We also examined distribution of NK cells, another immune cell population present in liver and found them to be dispersed, like MAIT cells, but without proneness towards portal fields (online supplemental figure 1D,E).

Figure 1Figure 1Figure 1

MAIT cells localise dispersedly to the parenchymal space in the healthy human liver. (A) Representative IF analysis of tissue section from a liver biopsy without histopathological abnormalities (patient C3). Colocalisation of CD3, TCR Vα7.2 and IL18-Rα (see higher magnification lower panels) identifies MAIT cells (yellow arrowheads). White arrow and blue arrowheads point at a portal field and central veins, respectively. Lower panels also show MAIT cells in proximity of TCR Vα7.2-negative and IL18-Rα-negative T cells (high magnification panels in colour and greyscale are shown). (B) Percentages of MAIT cells and non-MAIT Vα7.2+cells vs total CD3 +T cells in the healthy human liver (n=15), assessed as shown in (A). ****P<0.0001, Wilcoxon matched-pairs signed rank test. (C) Representative IF analysis (left panel), computational analysis (middle panel) and H&E staining (right panel) of a tissue section from a liver biopsy without histopathological abnormalities (patient C6). Yellow arrowheads in the left panel depict localisation of MAIT cells. The middle panel shows the same section with the regions of interest (ROIs) portal fields (depicted with brown circles) and central veins (depicted with green circles), as well as a colour heat map of cell distance (ie, any detected nucleus) to MAIT cells. (D) Upper panel: Violin plot of MAIT cell and non-MAIT CD3 +T cell distance to ROIs portal field and central vein. The box plots indicate the median and quartiles. Data were obtained from analysis of nine different liver tissues without histopathological abnormalities (five biopsies and four resection specimens). Significance levels obtained from one-way analysis of variance with selected post-hoc comparisons (t-test), p values were adjusted with Benjamini-Hochberg correction for multiple testing. The response variable ‘distance’ was transformed (square root) to keep also 0 distances in the analysis. Lower panel: Bar histogram pointing to percentage of MAIT cells localising at indicated distance to either portal fields or central veins. IF, immunofluorescence; IL 18R, interleukin-18 receptor; MAIT, mucosal-associated invariant T; TCR, T cell receptor.

Liver cell lines differ in their ability to activate MAIT cells

We analysed several parenchymal and non-parenchymal established cell lines for a potential to act as APCs for MAIT cells. Primary blood-derived CD8+ T cells served as MAIT cell source (gating on CD8+CD161++Vα7.2+ MAIT cells, see online supplemental figure 2A) and either riboflavin-synthesising E. coli or synthetic 5-OP-RU as Ags. In contrast to conventional APC line THP-1, we observed neither IFN-γ nor activation marker CD69 expression by MAIT cells co-cultured with hepatoma cells HepG2 and Huh7 exposed to a low concentration of E. coli (figure 2A), whereas higher concentration resulted in increased CD69 expression but no marked IFN-γ or TNF-α production (figure 2B and online supplemental figure 2B). MAIT cell activation by LSEC line TMNK-1 led to IFN-γ and TNF-α expression in response to E. coli while activation by HSC line TWNT-4 was weaker, detectable only at higher E. coli concentrations (figure 2C–D and online supplemental figure 2C). The effects of TMNK-1 and TWNT-4 cell lines were dependent on MR1 and partially on cytokines IL12/18 (figure 2E–F). Finally, we found that BEC H69 cells present Ag in an MR1-restricted manner while HSC line LX-2 had no activating effect (online supplemental figure 2D). All liver-derived cell lines, except LX-2, responded to pure stimulatory Ag 5-OP-RU (figure 2A,C and online supplemental figure 2E). We measured MR1 surface levels and MR1 messenger RNA in different cell lines. HepG2 and Huh7 hepatoma cells were very low in both assays (online supplemental figure 2F–G), consistent with their limited ability to activate MAIT cells.

Figure 2Figure 2Figure 2

Liver cell lines exert limited capacity to activate MAIT cells. (A–D) MAIT cell expression of IFN-γ (left panels) and CD69 (right panels) measured by flow cytometry. Blood-derived CD8 +T cells were co-cultured with different indicated cell lines in the presence of fixed Escherichia coli or 5-OP-RU. (A) THP-1, HepG2 or Huh7 cells exposed to fixed E. coli (1.5e7 CFU/mL) or 10 nM 5-OP-RU (n=4). (B) THP-1, HepG2 or Huh7 cells exposed to increasing concentrations of fixed E. coli (1.3e7–5e8 CFU/mL) (n=3). (C) THP-1, LSEC TMNK-1 or stellate TWNT-4 cells exposed to fixed E. coli (1.5e7 or 5e7 CFU/mL) or 10 nM 5-OP-RU (n=4). (D) TMNK-1 or TWNT-4 cells exposed to increasing concentrations of fixed E. coli (n=3). (E and F) LSEC TMNK-1 (E) and stellate TWNT-4 (F) cells in the presence of fixed E. coli (5e7 CFU/ml) exposed to different combinations of blocking Abs. MAIT cell expression of IFN-γ, TNF-α, and CD69 is shown in left, middle and right panels, respectively, (n≥5). Other details as in panels (A–D). CFU, colony forming unit; IFN, interferon; IL, interleukin; LSECs, liver sinusoidal endothelial cells; MAIT, mucosal-associated invariant T; 5-OP-RU, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil.

All investigated primary liver cells act as APCs stimulating MAIT cells, with hepatocytes being most efficient

We next investigated the APC activity of major types of primary cells present in liver parenchyma. From surgically removed human liver specimens, we isolated primary hepatocytes, hepatic myofibroblasts/HSCs, LSECs and BECs and verified their identities by flow cytometry and/or microscopy (online supplemental figure 3A–C). Importantly, we observed surface MR1 expression in all investigated cell types (figure 3A and online supplemental figure 3D), though quantitative assessment was not possible due to high autofluorescence.

Figure 3Figure 3Figure 3

Primary human parenchymal and non-parenchymal liver cells express MR1 and present antigen to MAIT cells, with hepatocytes representing most potent APCs. (A) FACS histograms showing primary BECs, LSECs, HSCs and hepatocytes (HEPs) stained with anti-MR1 (red) or isotype-matched control antibody (filled grey). (B) IFN-γ production by MAIT cell clone SMC3 on co-culture with hepatocytes incubated with Escherichia coli lysate (3e7 or 3e8 CFU/mL) (n=4). (C) Representative FACS histograms, showing indicated markers on clone SMC3 in response to hepatocytes incubated with E. coli lysate (3e6–3e8 CFU/mL). Filled grey histograms correspond to negative controls (CTRL) lacking lysate. D–G) IFN-γ production by clone SMC3 in response to incubation with indicated liver cells and Ags. (D) Hepatocytes incubated with 5-OP-RU (0.01–10 nM). E. coli lysate (Lys; 3e8 CFU/mL) served as positive control (n=8). (E) HSCs incubated with E. coli lysate (6e7 or 3e8 CFU/mL) (n=5). (F) HSCs incubated with 5-OP-RU (0.01–1 nM) (n=10). (G) LSECs and BECs incubated with E. coli lysate (3e7–3e9 CFU/mL) (n=6). In panels B, D, E, F and G, the data (means±SD of measurements from three independent wells originating from the same patient) exemplify representative experiments out of 4–10 performed with cells obtained from at least three different donors. K562 cells overexpressing MR1 (K562-MR1) served as positive control APCs in panels (B and F). Anti-MR1 antibody (αMR1) was used in panels (B and D–F). (H) Two representative examples of synthetic Ag 5-OP-RU titration (0.01–3 nM) on liver APCs. Three independent measurements per dose are depicted. (I) Pooled results of all experiments performed as in panel (H), additionally including cells from adipose tissue (ADIPO, n=4; assembly of two experiments using adipose stromal cells and two experiments using differentiated adipocytes) and human umbilical vein endothelial cell (n=3). Shown are concentrations of 5-OP-RU needed to reach EC50 of IFN-γ secretion. Statistical significance of differences to HEPs was determined by two-way analysis of variance with Dunnet post-hoc test. (J and K) Representative examples of 5-OP-RU (0.01–10 nM) titration on liver APCs, in the absence or presence of Abs blocking either IL-12/23 (J) or IL-18 (K). One representative experiment out of two is shown in each panel. Other details are as in panel (H). IFN-γ production was always measured by ELISA. ADIPO, adipocytes; APCs, antigen presenting cells; BECs, biliary epithelial cells; CFU, colony forming unit; FACS, fluorescence-activated cell sorting; HSC, hepatic stellate cell; IL, interleukin; IFN, interferon; LSEC, liver sinusoidal endothelial cell; 5-OP-RU, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil.

We first used MAIT cell clone SMC3, representing a pure homogenous MAIT cell population,23 to assess the capacity of primary liver cells to act as APCs. Hepatocytes, when incubated with E. coli lysate, activated the SMC3 clone in MR1-dependent manner as shown by IFN-γ secretion (figure 3B), upregulation of activation markers (figure 3C) and downregulation of TCR (online supplemental figure 4A). Some of the observed shifts were relatively small because of high baseline expression of activation markers on regular restimulations of clone SMC3 to induce its proliferation. K562-MR1 leukemia cells stably overexpressing MR1 served as positive control APCs (figure 3B and F). The robust MR1-restricted MAIT cell activation by hepatocytes was confirmed using pure synthetic Ag 5-OP-RU (figure 3D and online supplemental figure 4B). It was particularly interesting to test whether HSCs are able to activate MAIT cells since HSCs have been postulated as main drivers of fibrogenesis in the liver,18 and MAIT cells were also recently linked to liver fibrosis.16 17 HSCs treated with either E. coli lysate or 5-OP-RU activated MAIT cells in a MR1-dependent way (figure 3E–F and online supplemental figure 4C), arguing for a direct interaction between HSCs and MAIT cells as a factor contributing to the mechanism of their fibrogenic activity. Primary cell populations of BECs and LSECs were also able to present E. coli-derived and synthetic 5-OP-RU Ag to SMC3 cells in MR1-dependent manner (figure 3G–H and online supplemental figure 5A).

Use of a MAIT cell clone enabled us to quantitatively compare the potential of different primary cells to activate MAIT cells by determining Ag concentrations needed to reach EC50 of IFN-γ production. For both synthetic 5-OP-RU and bacterial lysate, we found hepatocytes to be the most efficient APCs, also when compared with adipocytes and HUVECs, included as examples of primary cells of non-liver origin (figure 3H–I and online supplemental figures 5B,C. The differences between various liver cells persisted when either IL-12 or IL-18 was blocked (figure 3J–K and online supplemental figures 5D,E). Inclusion of exogenous IL-12 or IL-18 increased MAIT cell activation, a known effect synergistic with TCR-dependent stimulation.10 24 The effect decreased on addition of anti-cytokine antibodies (online supplemental figure 5F,G). Since primary hepatocytes might secrete IL-7,13 15 leading to enhanced MAIT cell activation, we tested its effect using BECs and HSCs; IL-7 had no effect on MAIT cell clone SMC3 activity (online supplemental figure 5H).

Polyclonal blood-derived and liver-derived MAIT cells are also robustly activated by liver APCs

To investigate whether findings for the SMC3 clone apply to primary polyclonal MAIT cell populations, we isolated MAIT cells from human peripheral blood and liver tissue. Starting with peripheral blood mononuclear cells, which contain 2%–5% MAIT cells, negative magnetic bead selection depleted naïve T cells, B cells, monocytes, dendritic cells, platelets and γδ T cells, resulting in fourfold to sevenfold enrichment for MAIT cells (online supplemental figure 6A). Exposure of these enriched polyclonal MAIT cells to 5-OP-RU-loaded primary liver cells resulted in MR1-dependent downregulation of TCR expression, with hepatocytes again being very potent APCs (figure 4A, upper panels, and online supplemental figure 6B). Stimulation of blood MAIT cells by hepatocytes led to upregulation of activation markers CD25, CD69 and CD137, and the proliferation marker CD71 (figure 4A, lower panels). Consistent with the MAIT cell clone findings, BECs and HSCs were less potent APCs for blood-derived MAIT cells (figure 4A and online supplemental figure 6B).

Figure 4Figure 4Figure 4

Robust activation of polyclonal MAIT cells in response to interaction with primary human liver cell subsets. (A) Peripheral blood mononuclear cells, enriched for Vα7.2+CD161++cells by negative selection (see Materials and Methods, and online supplemental figure 6) were co-cultured with indicated liver APCs exposed to 3.3 nM 5-OP-RU. Upper panel: representative example of cell surface expression of CD161 and TCR Vα7.2 as measured by flow cytometry (n=3). Dot plots are gated on CD3 +CD8+CD26+cells. Lower panels: cell surface expression of CD25, CD69, CD137 and CD71, gated on CD3 +CD8+cells that are CD26 +and/or CD161+, as depicted in online supplemental figure 6C. (B and C) Production of IFN-γ (B) and IL-17 (C) by liver-derived MAIT cell line MAIT-BEL-10, measured by ELISA, in response to interaction with different liver APCs exposed to indicated concentrations of 5-OP-RU. Stimulation of MAIT cells with PMA/ionomycin (PMA/Iono) served as positive control. (D) IFN-γ production by liver-derived MAIT cell line MAIT-BSL-19, stimulated by indicated liver APCs exposed to 5-OP-RU (nM). (E) Cell surface staining for CD107a (upper panel), CD25, CD69, CD137 and CD71 (lower panels) on MAIT-BSL-19 cells, stimulated by either hepatocytes or BECs exposed to 3.3 nM (upper panel) or 0.37–3.3 nM 5-OP-RU (lower panels). Data in (D) and (E) are derived from the same experiment, which is representative of two experiments performed. MR1 dependence of activation was assessed with anti-MR1 blocking antibody (αMR1) in panels (A, D and E). Negative control (CTRL) in panels (A and E) lacks the APC. APC, antigen presenting cell; BEC, biliary epithelial cell; HEP, hepatocyte; HSC, hepatic stellate cell; IL, interleukin; IFN, interferon; MAIT, mucosal-associated invariant T; 5-OP-RU, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil; PMA, phytohemagglutinin; TCR, T cell receptor.

To assess activation of polyclonal liver-derived MAIT cells, we generated two MAIT cell lines (MAIT-BEL-10 and MAIT-BSL-19) from two liver donors. Consistent with their tissue origin, the liver-derived lines expressed higher baseline levels of tissue residency marker CD69,13 compared with MAIT cell clone SMC3 (online supplemental figure 7A,B; see legend for additional characterisation of MAIT liver lines). In response to 5-OP-RU-treated hepatocytes, BECs or HSCs, the liver-derived lines produced IFN-γ and IL-17 and—as tested with hepatocytes and BECs—showed MR1-dependent upregulation of activation markers CD25 and CD137, and degranulation marker CD107a (figure 4B–E). Ag presentation capacity differed between different liver APCs (figure 4B and D–E). At high concentrations of 5-OP-RU, some of the hepatocytes’ effects were only partially blocked by MR1 antibody, possibly due to very abundant cell surface presence of MR1, caused by its stabilisation by 5-OP-RU,25 or a continual appearance of new MR1 on the hepatocyte surface (figure 4D–E). Moreover, we observed loss of CD25 high MAIT cells on stimulation by hepatocytes exposed to high (3.3 nM) 5-OP-RU, arguing for activation-mediated cell death (figure 4E, lower panel). High CD137 expression by MAIT cells stimulated by BECs confirmed their Ag-presentation capacity; however, induction of IFN-γ secretion was not as efficient as in response to hepatocytes (figure 4D–E).

To mimic an inflammatory context in which increased amounts of bacterial products and inflammatory cytokines reach the liver, synthetic Ag was mixed with E. coli lysate. Under these conditions, hepatocytes were again more efficient APCs than LSECs and BECs (online supplemental figure 7C), and this was paralleled by cytotoxicity elicited by the MAIT-BSL-19 cells (online supplemental figure 7D and online supplemental videos). Thus, as a proof of concept, we demonstrated that hepatocytes can be killed in vitro following Ag-mediated activation of liver-derived MAIT cells. Killing capacity by MAIT cells was also demonstrated for LSECs, using the SMC3 MAIT clone (online supplemental figure 7E).

Inhibition of liver APC-mediated MAIT cell activation by MR1 blocking ligands

We explored whether blocking of MR1 occupancy with non-activating MR1 ligands prevents MAIT cell activation by liver cells, potentially offering an approach to counteract the postulated role of MAIT cells in liver fibrogenesis.16 17 We found that MAIT cell activation by Ag-exposed primary liver cells can be prevented in a dose-dependent manner by pre-treating liver cells with 6-formylpterin (6-FP) or acetyl-6-FP, known to enter the MR1 5-OP-RU binding pocket but not activate MAIT cells (figure 5A,B).5 26 The acetylsalicylic acid derivative 5-formyl-salicylic acid (5-F-SA) was found previously to stabilise MR1 on the cell surface, without stimulating Jurkat T cells overexpressing the MAIT TCR.27 We found that 5-F-SA decreased MAIT cell activation by liver APCs (figure 5B).

Figure 5Figure 5Figure 5

Non-activating MR1 ligands prevent MAIT cell activation by liver-derived APCs. MAIT cell clone SMC3 was co-cultured with indicated liver cells pretreated with different non-activating MR1 ligands for 1 hour before addition of 5-OP-RU. IFN-γ secretion was assessed by ELISA. (A) Hepatocytes either treated with 0.1–1 nM 5-OP-RU alone (CTRL) or pretreated with 50 µM 6-FP (left panel) or 5 µM acetyl-6-FP (right panel) before adding 5-OP-RU. (B) HSCs and BECs either treated with 5-OP-RU alone (CTRL), or pretreated with indicated concentrations of 6-FP, acetyl-6-FP, or 5-formyl-salicylic acid (5-F-SA) before 5-OP-RU addition. The mean+SD of measurements from three independent wells originating from the same patient’s cells is shown. One representative experiment out of two, originating from two different donors, is shown. APCs, antigen presenting cells; BEC, biliary epithelial cell; 6-FP, 6-formylpterin; 5-F-SA, 5-formyl-salicylic acid; HSC, hepatic stellate cell; IFN, interferon; MAIT, mucosal-associated invariant T; 5-OP-RU, 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil.

Primary liver cells promote active MAIT cell Ag formation when provided with its precursor

It has been established that the Ag precursor 5-A-RU is not able to bind to MR1 and activate MAIT cells.7 However, when 5-A-RU is used at high concentration, it can conjugate with cellular glycolysis products such as methylglyoxal, yielding active Ag.7 We observed that exposure of liver APCs to high concentration of 5-A-RU also Ieads to MAIT cell activation (figure 6A,B), suggesting that the formation of activatory 5-OP-RU is promoted by the liver APCs. Similarly, as in the case of the 5-OP-RU Ag presentation to MAIT cells (figure 3I), hepatocytes were more responsive to 5-A-RU than other liver and non-liver primary cells (figure 6B).

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