METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF FIBROSIS WITH AGENTS CAPABLE OF INHIBITING THE ACTIVATION OF MUCOSAL-ASSOCIATED INVARIANT T (MAIT) CELLS

Abstract

Persistent inflammation is a driving force of fibrosis progression. Mucosal-Associated Invariant T (MAIT) cells are non-conventional T cells that display altered functions during chronic inflammatory diseases. Here, the inventors report a loss of circulating MAIT cells in cirrhotic patients and their hepatic accumulation in an activated phenotype within the fibrotic septa. Using two models of chronic liver injury, the inventors demonstrate that mice enriched in MAIT cells (Vα19TCRTg) show exacerbated liver fibrosis and higher number of hepatic fibrogenic cells than wild type counterparts, whereas MAIT cell-deficient mice (MR1.sup.−/−mice) are resistant. The results highlight the profibrogenic functions of MAIT cells and suggest that 1 targeting MAIT cells may constitute an attractive antifibrogenic strategy during chronic liver injury. Accordingly, the present invention relates to a method of treating fibrosis in a patient in need thereof comprising administering to the subject a therapeutically effective amount of an agent capable of inhibiting the activation of MAIT cells.

Claims

1. A method of treating fibrosis in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an agent capable of inhibiting the activation of MAIT cells.

2. The method of claim 1 wherein the fibrosis affects at least one organ selected from the group consisting of skin, eye, intestine, heart, liver, lung, and kidney.

3. The method of claim 1 wherein the patient suffers from dermal scar formation, keloids, liver fibrosis, lung fibrosis, kidney fibrosis, glomerulosclerosis, pulmonary fibrosis, renal fibrosis, intestinal fibrosis, interstitial fibrosis, cystic fibrosis of the pancreas and lungs, injection fibrosis, endomyocardial fibrosis, mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, progressive massive fibrosis, or nephrogenic systemic fibrosis.

4. The method of claim 1 wherein the patient suffers from liver fibrosis.

5. The method of claim 4 wherein the liver fibrosis results from chronic alcohol consumption, overfeeding, insulin resistance, type 2 diabetes, non-alcoholic fatty liver disease, NASH, steatosis, idiopathic portal hypertension, autoimmune hepatitis, primary sclerosing cholangitis, or primary biliary cirrhosis.

6. The method of claim 4 wherein the liver fibrosis is associated with liver steatosis.

7. The method of claim 1 wherein the agent capable of inhibiting the activation of MATT cells is an antibody.

8. The method of claim 1 wherein the agent is an antibody that depletes MAIT cells.

9. The method of claim 1 wherein the agent is an anti-Vα7.2-Jα33 depleting antibody.

10. The method of claim 8 wherein the antibody that depletes MATT cells mediates antibody-dependent-cellular-cytotoxicity (ADCC).

11. The method of claim 8 wherein the antibody antibody that depletes MAIT cells is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof.

12. The method of claim 1 wherein the agent is an antibody that blocks the interaction between MR1 and Vα7.2-Jα33 receptors.

13. The method of claim 1 wherein the agent is an anti-MR1 neutralizing antibody.

14. The method of claim 1 wherein the agent is an anti-Vα7.2-Jα33 neutralizing antibody.

15. The method of claim 13 wherein the anti-MR1 neutralizing antibody does not mediate antibody-dependent cell-mediated cytotoxicity and thus does not comprise an Fe portion that induces antibody dependent cellular cytotoxicity (ADCC).

16. The method of claim 1 wherein the agent capable of inhibiting the activation of MAIT cells is a small organic molecule.

17. The method of claim 16 wherein the small organic molecule is selected from the group consisting of 6-formyl pterin, acetyl-6-formylpterin (Ac-6-FP), 3-formylsalicylic acid (3-F-SA), 5-formylsalicylic acid (5-F-SA) and 2-hydroxy-1-naphthaldehyde (2-OH-1-NA).

Description

FIGURES

[0036] FIG. 1: Inhibition of MAIT-cell induced profibrogenic effect in vitro by neutralising MR1 and in vivo in MR1 deficient mice. (A) DNA synthesis in hepatic myofibroblasts pre-treated with MR1 neutralizing antibody or isotype, and co-cultured with either activated or non-activated MAIT cells. Results show a representative experiment and is the mean±SEM of quadruplicate determinations. Similar results were obtained in 2 independent experiments with MAIT cell from 2 different donors. (B) Representative images and quantification of Sirius red and α-SMA areas, and hepatic TGF-b1 secretion in MAIT cell-deficient (MR1.sup.−/−) mice (n=6) and their WT littermates (n=5) chronically administered CCl.sub.4.

EXAMPLE

[0037] Material & Methods

[0038] Human Blood Samples.

[0039] Blood samples were obtained with written informed consent from 39 patients with stable biopsy-proven cirrhosis, from alcoholic or non-alcoholic fatty liver disease-related cirrhosis hospitalized at Hopital Beaujon (Clichy, France) (Supplementary Table 1). The study was approved by the local ethics committees (Comité d'Evaluation de l'Ethique des projets de Recherche Biomédicale (CEERB) Paris Nord: n°16-039). Blood from healthy volunteers (n=29) was obtained through a formalized agreement with Etablissement Français du Sang (agreement n° (n°2015012778).

[0040] Human Liver Samples Non tumoral livers (n=7 and n=5 control and n=7 and n=5 cirrhotic patients for FACS analysis and immunohistochemistry, respectively) were obtained from surgical samples (resection or liver transplantation) at distance, when present, from tumor nodule. Liver tissue was processed as rapidly as possible after resection, frozen in liquid nitrogen, and stored at −80° C. (Biobank Pathology Dpt, Beaujon hospital, DC-2009-938). For all cases, fibrosis staging was assessed according to Metavir system.sup.16. All patients signed an informed consent form and the study was approved by the local Ethics Committee.

[0041] Peripheral Blood Mononuclear Cells (PBMC) Isolation.

[0042] PBMC were prepared from the blood using ficoll hypacque (GE Healthcare) density gradient centrifugation, as previously described.sup.17, and freshly used for the analysis of surface phenotype of MAIT cells. For the detection of cytokine production, PBMC were stimulated for 6 hours at 37° C. with PMA and ionomycin (Sigma-Aldrich) at 25 ng/ml and 1 μg/ml, respectively, in the presence of brefeldin A at 10 μg/ml (Biolegend) in RPMI medium supplemented with 10% fetal bovine serum (Life Technologies).

[0043] Flow Cytometric Analysis

[0044] Flurochrome conjugated Anti-CD3, anti-CD4 (OKT4), anti-CD161(HP-3G10), anti-Vα7.2.sup.+(3C10) , anti-CD25 (BC96), anti-CD69 (FN50) anti-CCR6 (G034E3), and anti-Bcl-2 (clone 100) Anti-IFN-γ (45.B3), anti-Granzyme B (GB11), anti-TNF-α (MAb11), anti-IL-17 (BL168), anti-Ki-67 (B56) antibodies were obtained from Biolegend France. Anti-CD8α (SK1), anti-TCRγδ (B1) antibodies were obtained from BD Biosciences. Dead cells were excluded from the analysis using the fixable viability dye eflour 506 (eBiosciensces). MAIT cells were identified by multicolour flow cytometry as CD3.sup.+CD4.sup.−CD8.sup.+CD161.sup.hiVα7.2.sup.+ cells. Intracellular cytokines were analyzed using PMA/ionomycin/BrefeldinA treated PBMC using the intracellular cytokine staining kit (BD Biosciences) according to manufacture's instruction. Analysis of Ki-67 and bc12 in PBMC were performed using the intracellular transcription factor staining kit (eBioscience). Data acquisition were performed using a BD Biosciences LSR Fortessa cytometer and data were analysed using FlowJo analysis software (Tree Star).

[0045] Human MAIT Cell Isolation

[0046] MAIT cells were isolated from PBMC prepared from buffy coats of healthy donors, as previously described.sup.9. Briefly, monocytes and CD4 T cells were sequentially depleted from PBMC by adhesion on polystyrene culture flasks for three hours at 37° C., and anti CD4 microbeads (Miltenyi Biotech France), respectively. Vα7.2.sup.+ cells were isolated from the non-monocyte and non-CD4 PBMC fraction, using an anti Vα7.2.sup.+ antibody conjugated to FITC, followed by a positive selection with anti-FITC microbeads (Miltenyi Biotech, France) and used as enriched MAIT cells for co-culture experiments. The purity of isolated Vα7.2.sup.+ cells was >80% and >95% of the isolated Vα7.2.sup.+ cells were co-expressing CD161. >95% of the isolated CD161.sup.hi Vα7.2.sup.+ cells were positive to APC conjugated 5-OP-RU loaded MR1 tetramers (.sup.18, NIH tetramer Core Facility, Emory University Vaccine Center, Atlanta, USA) (Extended data FIG. 4a). MAIT cells added in co-culture experiments were either left non-activated or were activated using anti-CD3 (HIT3a) at 2.5 μg/ml, soluble anti CD28 (CD28.2) (1 g/μml) and IL-7 10 ng/ml (Bio legend).sup.19.

[0047] Co-Culture of Human MAIT Cells with Human Hepatic Myofibroblasts

[0048] Human hepatic myofibroblasts (HMF) were obtained by outgrowth of explants prepared from surgical specimen of normal human liver, as we previously described.sup.20. This procedure was performed in accordance with ethical regulations imposed by the French legislation. The fibrogenic phenotype of these cells has been extensively characterized.sup.20. Cells were grown to 80% confluency in RPMI containing 10% fetal calf serum and serum-deprived for two days before adding freshly isolated non-activated or activated MAIT cells, at the ratio of 1:10 (HMF: MAIT cells).

[0049] DNA Synthesis Assay

[0050] BrdU (Roche, France) was added at the bottom of the well (transwell experiments), or to the co-culture medium for 18 hours. In co-culture experiments, MAIT cells were then carefully aspirated, and adherent HMF were washed once with 1× PBS and their DNA synthesis estimated by a colorimetric BrdU ELISA test (Roche, France) as per the manufacturer's instruction. For the experiments with MR1 blocking antibodies, human hepatic myofibroblasts were incubated with purified anti MR1 antibody at 20 μg/ml (26.5, Biolegend, France) or with isotype control antibody for 2 hours at 37° C. Pre-activated or non-activated MAIT cell were washed and co-cultured with anti-MR1 (26.5, Biolegend)-exposed HMF. Cells were then processed for DNA synthesis as described above. In separate experiments, FACS analysis of Ki-67 was performed in HMF upon co-culture with MAIT cells, using phycoerythrin (PE) conjugated anti-Ki67 (Biolegend, France) by intracellular staining as per the manufacturer's instructions.

[0051] Analysis of expression of MR1 on hepatic myofibroblasts by immunocytochemistry HMF were fixed in 4% paraformaldyhyde, followed by incubation with a blocking buffer 1% BSA-PBS (0,1% Triton x100) and with anti-MR1 antibody (clone 26.5, mouse IgG2a, kappa, 1:25, Biolegend,) and Goat-anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 555 (1:1000, Invitrogen,). Nuclear staining was performed using Prolong Gold antifade mountant with DAPI (Invitrogen,). No staining was observed with the isotype (clone MOPC-173, mouse IgG2a, kappa, 1:25, Biolegend,). Cells were visualized using confocal microscopy (Confocal Zeiss LSM 780).

[0052] Analysis of Surface Expression of MR1 on Hepatic Myofibroblasts by Flow Cytometry

[0053] Hepatic myofibroblast cultures or HMF/MAIT cell co-cultures were exposed to 1 μM Acetyp 6-formylpterin (Ac 6-FP) (Schircks Laboratories, Switzerland) for 2 hrs at 37° C. Cells were then washed, trypsinised, labeled with PE-conjugated anti MR1 antibody (26.5)/isotype control and subjected to flow cytometric analysis.

[0054] Analysis of cytokine and chemokine production by hepatic myofibroblasts Hepatic myofibroblasts were co-cultured with either non-activated or activated MAIT cells for 18 hours. Brefeldin A (10 μg/ml) was added for the last 3 hours, and cells were analysed for intracellular cytokines and chemokines, using the intracellular cytokine staining kit (BD Biosciences). When indicated, IL-17 neutralizing antibody (64CAP17, eBioscience) or isotype control antibody was added to the co-culture.

[0055] Animals MR1.sup.−/−

[0056] Animals MR1.sup.−/− C57BL/6.sup.27, Vα19 Tg C57BL/6 mice.sup.40 and their WT counterparts were generated as described in.sup.27,40. Since MR1 is the restriction molecule required for MAIT thymic development, MR1.sup.−/− C57BL/6 do not have MAIT cells. On the contrary, mice expressing the Vα19-Jα33 TCR transgene have a 10-fold increase in MAIT cell frequency in the various tissues such as spleen, liver, colon, lymph nodes.

[0057] Mice Models of Liver Fibrosis

[0058] Animals were housed in pathogen-free animal facility and fed ad libitum. Liver fibrosis was induced in male mice by either repeated injections of carbon tetrachloride (CCl.sub.4, 0.5 ml/kg body weight, 1:10 dilution in mineral oil [MO; Sigma, France), twice a week for 4 weeks (Vα19 TCR transgenic, n=9; WT littermates n=10; MR1.sup.−/−, n=6; WT littermates n=5), or bile duct ligation and section (Vα19 TCR transgenic, n=9; WT littermates n=3), as we previously described.sup.21,22. Animals were sacrificed 24 h after the last CCl.sub.4 injection or 12 days after surgery in BDL mice. There was no difference in the frequency of B lymphocytes, neutrophils, macrophages and dendritic cells between the two groups in the different models. Moreover, the extent of injury was similar between groups in all the models, as reflected by similar increases in serum transaminases measurements (data not shown). Experiments were performed in accordance with protocols approved by the Paris-Nord ethical committee C2EA 121 (authorization number 02529.02).

[0059] Histological Analysis

[0060] Hematoxylin and eosin and Sirius Red staining were performed on 4-μm thick formalin-fixed paraffin-embedded tissue sections at the Pathology Department of Hopital Bichat, Paris, France. Sirius Red-stained areas from 10 fields (magnification ×20) from each mouse were quantified with Image J.

[0061] Immunohistochemistry

[0062] Mice liver. Immunohistochemical detection of ACTA2 was carried out as previously described.sup.21 on paraffin-embedded mice liver tissue sections (4 μm) using the MOM immunodetection kit (Vector, PK2002) and a mouse monoclonal anti-ACTA2 antibody (1:1000, Sigma, 2547) according to manufacturer's instructions. ACTA2 positive area from ten fields (magnification ×20) from 3-7 mice/group were quantified with ImageJ. No staining was observed when the primary antibody was omitted.

[0063] Human liver. Va7.2 immunodetection was performed in frozen sections of normal or subnormal liver samples (n=5) from patients that underwent resection surgeries for non-hepatocellular primary tumor (n=3) or colorectal cancer liver metastasis (n=2), and showed no (F0) or mild (F1) fibrosis and no alteration in liver biological tests. Liver samples from patients with cirrhosis, that were obtained from non-tumoral part of HCC resection (n=3) and liver explant during liver transplantation (n=2). Briefly, tissue sections were fixed in 4% paraformaldehyde for 15 min, and incubated overnight at 4 ° C. with purified anti-human Va7.2 antibody (1:50, #351702, Biolegend), following a blocking step (PBS containing 10% goat serum, 1% BSA and 0.2% Triton X-100). After washes, sections were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:200, A-11001, Life Technologies) for 1 h at room temperature. Nuclear counterstaining was obtained with DAPI (1:10000, #62248, Thermo). Va7.2-positive cells were semi-quantitatively assessed (0: absent; +:positive; ++ strongly positive) and their location determined (sinusoidal space and mesenchymal space).

[0064] Statistical Analysis

[0065] Results are expressed as mean±standard error of the mean (SEM). Nonparametric tests were performed using Mann-Whitney U test or Student t test, as appropriate. All P values are 2-sided, and P values less than 0.05 were considered to be statistically significant. Analyses were performed using GraphPad Prism version 6.

[0066] Results

[0067] The Frequency and Functions of Circulating MAIT Cells are Altered in Cirrhotic Patients

[0068] We evaluated the frequency of circulating T cell subsets in the peripheral blood mononuclear cells (PBMC) from cirrhotic patients with alcoholic and non-alcoholic fatty liver disease (n=39), and compared to that of healthy donors (n=29). There was a decrease in CD8.sup.+ positive cells and a slight, but significant increase in the CD4.sup.+ population in cirrhotic patients. Detailed analysis of innate-like T cell populations showed a small decrease in the frequency of iNKT cells in cirrhotic patients, and no change in γδT cells. However, as compared to healthy donors, the median MIT cell frequency, identified as CD3.sup.+CD4.sup.−CD161.sup.hiVα7.2.sup.+ cells within the CD3.sup.+ population, was strongly decreased in cirrhotic patients (3.010%±0.38 in healthy donors, within the range reported in other studies vs 0.39%±0.11%, in cirrhotic patients). The majority of MAIT cells from healthy donors and cirrhotic patients were either CD8α.sup.+ (nearly 80%) or double negative (up to 20%). Blood MAIT cells from cirrhotic patients displayed an activated phenotype, characterized by higher frequencies of CD25.sup.+ and CD69.sup.+ MAIT cells vs healthy donors, that were negatively correlated with MAIT cell frequency. They also produced more IL-17 and granzyme B than healthy MAIT cells, whereas the frequency of IFN-γ or TNF-α-positive cells was high, but not different between the two groups. The decrease in peripheral MAIT cell frequency did not result from activation-induced cell death or exhaustion, since there was no difference in the mean fluorescence intensity of the survival marker Bc12 and the T cell exhaustion markers TIM-3 and PD-1 in MAIT cells from the 2 groups. Rather, cirrhotic MAIT cells showed increased proliferation, as indicated by a higher frequency of Ki-67.sup.+ MAIT cells compared to healthy donors. Finally, MAIT cells from healthy donors exposed to the plasma of cirrhotic patients showed an activated phenotype, characterized by an up-regulation of CD25 and CD69 expressions, whereas healthy MAIT cells exposed to healthy plasma had no effect. These data suggested that activation of blood MAIT cells may be due to soluble factor(s) present in the plasma of cirrhotic patients.

[0069] MAIT cells accumulate along the fibrotic septa in livers from cirrhotic patients We also studied the fate of MAIT cells in the cirrhotic liver, using isolated intrahepatic leukocytes from liver explants of patients undergoing transplantation. As expected.sup.6,7, the percentage of CD161.sup.+ Vα7.2.sup.+ MAIT cells was higher in the liver than in the blood in both control and cirrhotic livers (3.010%±0.38 in blood vs 12% in liver in healthy donors and 0.39%±0.11 vs 2.7% in liver in cirrhotic patients), but hepatic MAIT cell frequency was similar between cirrhotic patients and controls. However, immunohistochemistry of cirrhotic liver sections showed significant accumulation of CD3.sup.+Vα7.2.sup.+ positive cells along the fibrotic septa, with discrete or even no staining in the sinusoid, whereas control samples showed CD3.sup.+Vα7.2 immunoreactivity within the sinusoidal space. Cirrhotic liver MAIT cells showed an activated phenotype, characterized by higher frequencies of IL-17-producing MAIT cells, and a tendency to increase for Granzyme B, that did not reach significance. The number of IFN-γ and TNF-α positive MAIT cells was high but not different between the two groups. In addition, MAIT cells expressed high levels of CD25 and CD69 in cirrhotic and healthy livers, but the frequencies of CD25.sup.+ and CD69.sup.+ MAIT cells were similar between the two groups. Finally, the frequencies of iNKT and γδT cells did not differ between the two groups.

[0070] Altogether, these data demonstrate that MAIT cell frequency is strongly decreased in the blood of cirrhotic patients, whereas there is a significant accumulation of MAIT cells along the fibrotic septa in the liver of these patients, in close contact with fibrogenic cells. Both in blood and liver of cirrhotic patients, MAIT cells show an activated phenotype as compared to that of healthy donors. We next investigated whether MAIT cells directly interact with hepatic fibrogenic cells and the functional consequences on their functions.

[0071] Human MAIT Cells Enhance Mitogenic and Proinflammatory Properties of Human Fibrogenic Cells.

[0072] We first assessed in co-culture experiments whether MAIT cells directly stimulate fibrogenic cell proliferation. When human hepatic fibrogenic cells in their fully activated myofibroblastic phenotype.sup.12 were co-cultured with activated (anti-CD3/anti CD28/IL-7-exposed) MAIT cells, they showed enhanced Ki-67 staining or BrdU incorporation. In contrast, non-activated MAIT cells had a marginal effect on the proliferative capacity of hepatic myofibroblasts. Surprisingly, there was no stimulation of hepatic myofibroblast DNA synthesis by MAIT cells in transwell experiments. These data suggested that direct MAIT cell-hepatic myofibroblast contact rather than cytokine/chemokine production underlies MAIT cell-induced DNA synthesis of hepatic myofibroblasts. We hypothesized that TCR-dependent interaction via MR1 may be involved, since MR1 expression was revealed in hepatic myofibroblasts by immunostaining and FACS analysis, and strongly increased upon contact with MAIT cells. In addition, surface expression of MR1 on hepatic myofibroblasts was enhanced in response to Acetyl-6-formyl-pterin (6FP), an MR1 ligand that stabilize MR1 at the plasma membrane.sup.14. MAIT cell-induced proliferation of hepatic myofibroblasts was significantly blunted by a neutralizing anti MR1 antibody, whereas control isotype or anti-CD40 neutralizing antibody had no effect (FIG. 1A). Altogether, these data show that MAIT cells promotes accumulation of fibrogenic cells by stimulating their proliferation via an MR1-dependent pathway.

[0073] Hepatic myofibroblasts are also key contributors of the hepatic inflammatory response by producing chemokines and cytokines.sup.1,2 and secrete proinflammatory mediators when stimulated by IL-17 or TNF-α.sup.13,15. Since MAIT cells are well characterized IL-17-and TNF-α-producing cells.sup.3, we investigated whether hepatic myofibroblasts may adopt a proinflammatory profile when exposed to activated MAIT cells. In co-culture experiments, activated MAIT cells enhanced the production of IL-6, TNF-a and IL-8 by hepatic myofibroblasts. Similar findings were observed in transwell experiments, as shown by FACS analysis and confirmed by ELISA, suggesting that mediators produced by MAIT cells enhance the proinflammatory properties of hepatic myofibroblasts. In keeping, the production of proinflammatory mediators in hepatic myofibroblasts was reduced when adding a TNF-a neutralizing antibody to the co-cultures, whereas the anti-IL17 antibody had no effect.

[0074] Collectively, these data reveal the profibrogenic and proinflammatory functions of MAIT cells, via distinct contact-mediated effect involving MR1, and cytokine-dependent pathways, respectively.

[0075] Profibrogenic Properties of MAIT Cells In Vivo

[0076] Since Vα7.2 +cells accumulated within the fibrotic septa in the cirrhotic liver and because MAIT cells enhance the mitogenic and proinflammatory properties of hepatic myofibroblasts, we investigated whether MAIT cells display fibrogenic properties in vivo, taking advantage of the availability of MAIT cell deficient mice (MR1−/−) and mice carrying a 10-fold increase in MAIT cell number (Vα19TCRTg). MR1-deficient mice showed no difference in liver injury, as shown by similar levels of serum transaminases. However, MR1-deficient mice were resistant to fibrosis induced by CCl.sub.4, as evidenced by decreased morphometry analysis of sirius red staining and lower number of fibrogenic cells (FIG. 1B). Conversely, MAIT cell-enriched mice exposed to CCl.sub.4 displayed exacerbated fibrosis as compared to WT counterparts, as reflected by enhanced sirius red staining and increased number of α-smooth muscle actin-positive cells (α-SMA). Similar increases in Sirius red staining and accumulation of α-SMA positive cells were observed in Vα19TCRTg transgenic animals undergoing bile duct ligation as compared to WT counterparts. In contrast, we found no difference in the hepatic levels of TNF-α between either MR1-deficient or Vα19TCRTg as compared to their WT counterparts. Collectively, these data highlight the profibrogenic properties of MAIT cells in the liver.

[0077] Discussion

[0078] Recent advances in the understanding of liver fibrosis pathogenesis have revealed that dysregulation of the immune system is a key factor leading to cirrhosis and liver failure, and suggested that manipulation of specific immune cell subsets may serve as the basis for antifibrotic strategies, in a pathological context where new therapies are urgently needed.sup.2,3. Combining human data in cirrhotic patients with cell culture experiments and in vivo models of fibrosis in MAIT cell-deficient or MAIT cell-enriched mice, the present study identifies activated MAIT cells as a major actor of the fibrogenic process.

[0079] Our data demonstrate that MAIT cell frequency is decreased in the blood of patients with chronic inflammatory liver diseases. However, MAIT cells from cirrhotic patients display an activated and proinflammatory profile, characterized by increased CD25 and CD69 expression and higher production of IL17 and granzyme B. These findings corroborate data obtained in patients with viral (HIV, HCV) or bacterial infections, or with inflammatory diseases, including inflammatory bowel disease, arthritic disease, systemic lupus erythromatosis, rheumatoid arthritis or type 2 diabetes (refs). The fate of circulating MAIT cells, and in particular whether they die or migrate to the target tissue has not been definitely addressed, due to the lack of appropriate tools to track these cells in vivo. However, our results indicate that the loss of circulating MAIT cells is unlikely due to cell death or exhaustion, as we found no difference in the number of BC12, PDL3 or TIM-3-expressing MAIT cells between healthy and cirrhotic patients. More surprisingly, and although the number of MAIT cells is much higher in the liver than in the blood, we found no difference in MAIT cell number in the liver of cirrhotic individuals as compared to controls, but, as observed in the blood they displayed a proinflammatory phenotype, characterized by a higher frequency of IL17-positive cells. However, despite no change in hepatic MAIT cell frequency in the cirrhotic liver, immunhistochemistry experiments combining Vα7.2 and α-SMA immunostaining indicated that MAIT cells accumulate within the fibrotic septa, in close contact to hepatic fibrogenic cells. In contrast, MAIT cells were located in the sinusoidal space of control liver, in keeping with previous studies, but interestingly, moderate or no expression of MAIT cells was observed in the sinusoidal space of cirrhotic livers. However, additional studies are needed to determine whether local accumulation of activated MAIT cells in a fibrotic environment results from their redistribution within the liver and/or recruitment from the blood, therefore reflecting their decreased circulating frequency in cirrhotic patients. Yet, these data suggested that MAIT cells may interact with fibrogenic cells during chronic liver injury.

[0080] Combining in vivo studies and co-cultures experiments, our data identify MAIT cells as a novel pro-fibrogenic player. Indeed, MAIT cell-deficient or -overexpressing mice show miror decrease and increase in fibrosis, respectively, associated with a corresponding decrease and increase in the number of a-SMA positive fibrogenic cells. Interestingly, a major finding of our study is that enhanced proliferation of hepatic myofibroblasts by MAIT cells is a critical determinant of the profibrogenic function of MAIT cells. A key feature of the fibrogenic process is the high mitogenic capacity of hepatic fibrogenic cells that leads to their accumulation in the fibrotic septa during chronic liver injury. Proliferation of hepatic myofibroblasts is stimulated by a large variety of growth factors expressed during chronic liver injury, including PDGF (23); vasoconstrictors such as thrombin (24); the metalloproteinase MMP-2 (25); or adhesion molecules such as alphaVbeta3 integrins (26). However, co-culture experiments showed that the promitogenic properties of activated MAIT cells do not result from the release of mitogens for hepatic myofibroblasts but rather from direct cell-cell contact, since it was observed in co-cultures but not in transwell experiments. Because it has been reported that human hepatic fibrogenic cells express members of the human leucocyte antigen (HLA) family (HLA-I and HLA-II), lipid-presenting molecules (CD1b and CD1c) and factors involved in T cell activation (CD40 and CD80) and display features of antigen-presenting cells [22], we postulated that the mitogenic properties of MAIT cells may rely on TCR-dependent effects. These data were corroborated by the identification of the non-classical MHC-related molecule MR1 at the cell membrane, both by FACS analysis and immunohistochemistry, and by the decrease in hepatic myofibroblast DNA synthesis upon incubation of co-cultures with a neutralizing MR1 antibody. Strikingly however, co-culture experiments of hepatic myofibroblasts and in vitro activated MAIT cells showed that MAIT cells stimulate hepatic myofibroblast proliferation in the absence of MAIT ligand into the medium, suggesting that at least initially, MAIT cell-myofibroblast contact occurs via a TCR-dependent, antigen-independent pathway, as described for dendritic cell-T cell interaction. Whether during chronic liver injury, both antigen-dependent and antigen dependent pathways are initiated remains to be evaluated. Indeed, increase in gut permeability, intestinal bacteria overgrowth and dysbiosis are characteristic features of patients with chronic liver diseases from various etiologies, allowing gut bacteria to flow to the liver. Similar findings have been reported in experimental models of chronic liver injury. These data suggest that in cirrhotic patients and in experimental models, bacterial-derived MAIT ligands generated from vitamin B2 metabolites and host-derived methylglyoxal could accumulate in the liver. Nevertheless, enhanced accumulation of hepatic myofibroblasts following MR1-dependent contact appears as a critical determinant of the profibrogenic functions of MAIT cells.

[0081] Another characteristic of hepatic fibrogenic cells is their pro-inflammatory properties. Our findings demonstrate that activated MAIT cells promotes a shift of hepatic myofibroblasts toward a proinflammatory phenotype that relies on the release of mediator produced by MAIT cells. Indeed, MAIT cell-induced production of IL8, IL6 and TNF-a by hepatic myofibroblasts is similarly observed in co-cultures and transwell experiments. Although TNF-a and IL17 were likely candiates release by MAIT cells, analysis of hepatic myofibroblast inflammatory profile showed a reduction in the production of IL8, IL6 and TNF-a by an anti TNF a neutralizing antibody whereas, surprisingly, IL17 had no or a marginal effects. These data indicated that MAIT cells stimulate the proinflammatory functions of hepatic myofibroblasts, via a TNF-a-dependent pathway. Nevertheless, it is likely that other mitogenic/pro-inflammatory mediators produced in low amounts by MAIT cells in culture experiments may contribute to MAIT cell-induced proinflammatory phenotype.

[0082] In conclusion, these data extend our knowledge on the general properties of MAIT cells. They also add to our understanding of the mechanisms underlying inflammation-driven fibrogenesis and unravel this non-conventional T cell subset as a promising target for antifibrogenic therapy.

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[0083] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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