Cell for identifying MAIT cell ligand, and method for screening MAIT cell ligand using said cell

Abstract

Disclosed in the present description, as an embodiment, are T cells that co-express MHC-related-1 (MR1) molecules and TCRs of mucosal-associated invariant T (MAIT) cells, and that express a reporter protein in response to the activation of the TCRs of MAIT cells. Also disclosed is a method for screening the ligands of MAIT cells in which method said T cells are used.

Description

Cells for identifying MAIT cell ligands and screening method for MAIT cell ligands using said cells

[0001] The present invention provides, as embodiments thereof, cells for identifying ligands of mucosal-associated invariant T (MAIT) cells, and a method for screening ligands of MAIT cells using the cells, which are useful, for example, in the field of medicine.

[0002] MAIT cells, classified as innate immune T cells, are the largest T cell population, accounting for a few percent of human T cells. They recognize a bacterial metabolite presented on MHC-related-1 (MR1) molecules as an antigen. The antigen, 5-(2-oxopropylidene-amino)-6-D-ribitylaminouracil (5-OP-RU), is an intermediate in the riboflavin metabolism found in many bacteria, and this system can elicit an immune response against a wide spectrum of bacteria (Non-Patent Document 1).

[0003] Corbett, Nature 2014 Awad, Immunol. Cell Biol. 2018 Constantinides, Science 2019 Legoux, Science 2019 Lepore, eLife 2017 Crowther, Nat. Immunol. 2020

[0004] However, the above antigens bind only to a portion of the vast MR1 molecular pocket (Non-Patent Document 2), suggesting the existence of other unidentified antigens. Furthermore, the above antigens are thought to be responsible for the thymic differentiation of MAIT cells (Non-Patent Documents 3 and 4), but from a different perspective, the existence of endogenous antigens of MAIT cells has also been suggested (Non-Patent Documents 5 and 6). Therefore, there has been a strong demand for the development of a screening method that can efficiently search for and identify ligands for MAIT cells.

[0005] MR1 molecules are present in almost all cells in the body, but under steady-state conditions, only a small proportion are expressed on the cell surface, with the majority being localized within the intracellular endoplasmic reticulum. However, under conditions such as infection, MR1 molecules that present their antigens (e.g., 5-OP-RU) are translocated to and expressed on the cell surface, where they present the antigen to MAIT cells and induce MAIT cell activation. The present inventors have discovered that if it is possible to simultaneously evaluate, in a single cell, both 1) the expression of MR1 molecules on the cell surface due to binding of a test substance to the MR1 molecule, and 2) the subsequent induction of MAIT cell TCR activation, it will be possible to efficiently search for and identify new MAIT cell antigens (activating ligands), and as a result of extensive research, they have completed the present invention. Specific embodiments of the present invention are as follows. However, the present invention is not limited thereto.

[0006] [1] A T cell that co-expresses an MR1 molecule and a TCR of a MAIT cell and expresses a reporter protein in response to TCR stimulation. [2] The T cell according to [1] above, wherein the reporter protein is selected from green fluorescent protein (GFP), luciferase, and β-glucuronidase. [3] The T cell according to [1] above, wherein the reporter protein is GFP. [4] The T cell according to any of [1] to [3] above, wherein the MR1 molecule and the TCR of the MAIT cell are of human origin.

[0007] [5] A screening method for MAIT cell agonists or antagonists, comprising at least the following steps: (1) contacting the T cell according to any one of [1] to [4] above with a test substance; (2) observing activation of the TCR of the MAIT cell and expression of the MR1 molecule on the cell surface, and (i) determining a test substance that activates the TCR of the MAIT cell as an agonist of the MAIT cell, and (ii) determining a test substance that enhances expression of the MR1 molecule on the cell surface without activating the TCR of the MAIT cell as an antagonist of the MAIT cell. [6] The screening method according to [5] above, in which activation of the TCR of the MAIT cell is determined based on the intensity of GFP emission. [7] The screening method according to [5] or [6] above, in which activation of the TCR of the MAIT cell and expression of the MR1 molecule on the cell surface are observed by flow cytometry. [8] The screening method according to any one of [5] to [7] above, wherein the test substance is a compound group sample.

[0008] [9] A biomarker for testing for inflammatory diseases caused by cholestasis, comprising CA3S (cholic acid 3-sulfate) or CA7S (cholic acid 7-sulfate).

[10] The biomarker according to [9] above, wherein the inflammatory disease caused by cholestasis is primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC).

[11] A method for testing for inflammatory diseases caused by cholestasis, comprising measuring the concentration of CA3S or CA7S in bile collected from a subject.

[0009] The present invention provides, as one embodiment, cells for identifying MAIT cell ligands with high sensitivity and an efficient screening method for MAIT cell ligands using the cells, and, as another embodiment, a biomarker for testing inflammatory diseases caused by cholestasis.

[0010] Figure 1 shows a schematic diagram of reporter cells co-expressing the MR1 molecule and the TCR of MAIT cells ("co-expressing reporter cells") (see Example 1 below). Figure 2 shows the results of validation of the co-expressing reporter cells as a screening system using existing ligands of MAIT cells (see Example 1 below). Figure 3 shows a schematic diagram of a screening method for MAIT cell ligands using the co-expressing reporter cells (see Example 2 below). Figure 4 shows the results of reporter cell evaluation of fractions obtained by separating SPF mouse intestinal extract and 5-OP-RU by reverse-phase column chromatography (see Example 2 below). Figure 5 shows the results of high-resolution mass spectrometry (HRMS) evaluation of activated molecules in fractions #84-45 (see Example 2 below). Figure 6 shows the results of NMR spectroscopy analysis of activated molecules in fractions #84-45 (see Example 2 below). Figure 7 shows the structure of cholic acid 7-sulfate (CA7S). Figure 8 shows the results of HRMS / MS, confirming that the activated molecules in fraction #84-45 have sulfate groups (see Example 2 below). Figure 9 shows the results of evaluation of CA7S using co-expressing reporter cells (see Example 2 below). Figure 10 compares the agonistic activity of CA7S and ribityllumazine (RL-7-Me) against MAIT cells, demonstrating that CA7S is a weak agonist against MAIT cells (see Example 2 below). Figure 11 shows that CA7S increased the expression of MR1 on the cell surface (see Example 2 below). Figure 12 shows that the activity of CA7S was also confirmed in the TCR of mouse (m) and human (h)-derived MAIT cells in the presence of mouse or human MR1 (see Example 2 below). Figure 13, related to the results of the study in Example 3 described below, shows that cholic acid (CA) did not increase the expression of either the TCR or MR1 on the cell surface of MAIT cells (Figures 13A and 13B), that CA7S and CA3S activated the TCR of MAIT cells, but CA12S did not (Figure 13, upper panel), and that CA7S, CA3S, and CA12S all increased the expression of the MR1 molecule on the cell surface (Figure 13, lower panel). Figure 14 shows the structures of CA7S and its analogs examined in the examples. Figure 15 shows the results of sc-TCR-RNA-seq analysis after long-term stimulation with CA7S, CA3S, and 5-OP-RU (Example 4 described below).Figure 16 shows the results of sc-TCR-RNA-seq analysis after long-term stimulation with CA7S, CA3S, and 5-OP-RU (see Example 4 below). Figure 17 shows the results of evaluating the bile CA3S concentration (pmol / ml) in subjects in a healthy state and a cholestatic state, demonstrating that the CA3S concentration is higher in the cholestatic state than in the healthy state. Figure 18 shows the results of an investigation into the effects of CA7S and its analogs on TCR activation of MAIT cells and activation of cell surface expression of MR1 molecules, as examined in Example 6 below. The results of flow cytometry analysis of MAIT cell expression in MR1-5-OP-RU tetramer-enriched thymocytes in Sult2a-deficient mice treated with and without CA7S are shown.

[0011] The present invention will now be described with reference to specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0012] [Co-expression reporter cell] One embodiment of the present invention is a "T cell that co-expresses an MR1 molecule and the TCR of a MAIT cell, and that expresses a reporter protein in response to activation of the TCR of the MAIT cell" (hereinafter also referred to as a "co-expression reporter cell"). Because the "co-expression reporter cell" co-expresses the MR1 molecule and the TCR of a MAIT cell, it is possible to simultaneously evaluate, using a single cell, both 1) the expression of the MR1 molecule on the cell surface due to binding of a test substance to the MR1 molecule, and 2) the subsequent induction of activation of the TCR of the MAIT cell. Because MAIT cells are activated via activation of their TCR, it has become possible to efficiently search for and identify activating ligands for MAIT cells based on this evaluation.

[0013] (Cells, etc.) The T cells used in this embodiment are not particularly limited as long as they can co-express the MR1 molecule and the TCR of MAIT cells, and may be either human-derived T cells or animal-derived (e.g., mouse-derived) T cells. Furthermore, the MR1 molecule and the TCR of MAIT cells used in this embodiment are also not particularly limited as long as they can co-express, and may be either human-derived or animal-derived (e.g., mouse-derived) T cells, although human-derived T cells are preferred from the perspective of clinical application.

[0014] (Reporter Protein) A preferred embodiment of the "co-expressing reporter cell" is one that expresses a reporter protein in response to TCR activation of MAIT cells, in order to detect TCR activation of MAIT cells efficiently and with high sensitivity. The reporter protein to be expressed is not particularly limited as long as its expression can be easily observed, and any protein commonly used in the art can be used. For example, green fluorescent protein (GFP), luciferase, and β-glucuronidase, which are commonly used in the art and allow easy observation of the expression of the protein, can be used. Among these, a protein that allows the level of reporter protein expression to be observed based on the intensity of luminescence is preferred, with GFP being particularly preferred. The reporter protein must be expressed in response to TCR activation of MAIT cells; for example, a system in which the reporter protein is expressed downstream of a TCR activation signal can be used.

[0015] (Creation of Co-expression Reporter Cells) Those skilled in the art can create the above-mentioned "co-expression reporter cells" by, for example, using genetic engineering techniques commonly used in the art (e.g., gene recombination techniques, gene transfer techniques, etc.) to prepare T cells into which the gene for the MR1 molecule, the gene for the TCR of an MAIT cell, and a gene that expresses a reporter protein in response to TCR stimulation have been co-transfected. For example, those skilled in the art can appropriately create such "co-expression reporter cells" by referring to the method described in Example 1 below.

[0016] [Method for screening for MAIT cell ligands] Another embodiment of the present invention is a method for screening for MAIT cell agonists or antagonists. Specifically, the method for screening for MAIT cell agonists or antagonists comprises at least the following steps: (1) contacting the above-mentioned "co-expression reporter cells" with a test substance, and (2) observing activation of the TCR of MAIT cells and expression of MR1 molecules on the cell surface, and (i) determining that a test substance that activates the TCR of MAIT cells is an agonist of MAIT cells, and (ii) determining that a test substance that enhances the expression of MR1 molecules without activating the TCR of MAIT cells is an antagonist of MAIT cells.

[0017] (Determination of Ligand) In the screening method of this embodiment, if a test substance activates the TCR of MAIT cells, the test substance is determined to be an agonist of MAIT cells (step (2)(i)). Note that if the test substance promotes the expression of MR1 molecules on the cell surface, it is considered to be an agonist with stronger binding to MR1 molecules. In this determination, for example, if the level of TCR activation (e.g., NFAT-GFP (%)) when stimulated with the test substance is three or more times higher than the level of activation when not stimulated, the test substance can be determined to be an agonist. In the screening method of this embodiment, if a test substance enhances the expression of MR1 molecules on the cell surface but does not activate the TCR of MAIT cells, the test substance is determined to be an antagonist of MAIT cells (step (2)(ii)). In this determination, for example, a test substance can be determined to be an antagonist if the expression level of the MR1 molecule (e.g., MFI value) following stimulation with the test substance is three or more times higher than the level without stimulation. As described above, the screening method using the present "co-expressing reporter cells" has an excellent feature in that it can search for and identify not only MAIT cell agonists but also antagonists. In the above determination, the expression level of the TCR in MAIT cells can be determined, for example, by evaluating the luminescence intensity of the reporter protein using flow cytometry. Furthermore, the expression level of the MR1 molecule on the cell surface can be determined, for example, by performing fluorescent antibody staining and then evaluating it using flow cytometry. In this screening, it is preferable that the expression level of the TCR in MAIT cells and the expression level of the MR1 molecule on the cell surface can be determined using the same evaluation method. Therefore, it is preferable to determine the expression system of the reporter protein taking this into consideration.

[0018] (Test Substance) The test substance evaluated in the screening method of this embodiment may be an isolated and purified single-compound sample, or a compound group sample consisting of a group of many single compounds. For example, it may be a sample containing a large number of compounds to be evaluated, such as an extract from tissue such as an organ or a library of in vivo metabolites. When a "single-compound sample" is used, it is possible to directly determine whether the test substance is a MAIT cell agonist or antagonist. When a "compound group sample" is used, it is first possible to determine the presence or absence of candidate compounds that may be MAIT cell agonists or antagonists within the compound group. These compounds can then be purified and isolated using methods commonly used in the art to narrow down the candidate compounds and determine their structures. When a "compound group sample" is used, it is preferable to first fractionate the compound group into a predetermined number of fractions using an appropriate purification method (e.g., HPLC), and then evaluate each of these fractions using the screening method. By combining this with prior purification of the evaluation sample, it is possible to efficiently and sensitively search for and identify MAIT cell agonists or antagonists from a "compound group sample" containing a large number of compounds. In particular, when a group of in vivo metabolites is used, it is possible to identify endogenous agonists and antagonists, which is expected to lead to effective application in drug discovery.

[0019] More specifically, the method described in Example 2 below can be used to search for MAIT cell agonists using the above-mentioned "compound group sample." Each fraction is subjected to evaluation using "co-expressing reporter cells" to select fractions containing a test substance that activates the TCR of MAIT cells. Candidate compounds are identified from these fractions, and the identified compounds are further evaluated using "co-expressing reporter cells" to confirm that they are agonists according to the above criteria, thereby obtaining the desired agonist. It is also possible to synthesize or purchase derivatives of the compounds identified above as agonists or antagonists, and further evaluate these derivatives using the present screening method to obtain additional agonists or antagonists.

[0020] (Contact between Test Substance and Co-expressed Reporter Cells) The conditions (temperature, time, specific procedures) for stimulating the co-expressed reporter cells by contacting the test substance with the co-expressed reporter cells can be appropriately determined by one skilled in the art, depending on the test substance to be evaluated, etc. A simple example is to culture "co-expressed reporter cells" in an appropriate medium in each well of a 96-well plate, and then add the test substance thereto. The test substance may be prepared in a solution state using an appropriate solvent (water, alcohol, etc.) as needed. The contact can be carried out, for example, within a temperature range of 25 to 45°C. After contact for, for example, 1 to 24 hours, the activation of the TCR of MAIT cells and the expression of MR1 molecules on the cell surface can be observed to determine agonist or antagonist activity.

[0021] [Bile Acid Metabolites] As described in Examples 2 and 3 below, the present inventors used the above-described screening method to search for MAIT cell ligands from mouse intestinal extracts containing a vast number of metabolites. As a result, they were the first to identify CA7S and CA3S, which constitute bile acid metabolites, major components of enteric secretions from the gallbladder, as MAIT cell agonists. These findings clearly demonstrate the usefulness of the above-described screening method. As shown in Example 3 below, other analogs differing in the position of sulfate conjugation did not have agonistic activity on MAIT cells, confirming that the activity was specific to CA7S and CA3S. Furthermore, as shown in Example 4 below, it was also revealed that CA7S and CA3S, unlike the conventional exogenous agonist 5-OP-RU, possess properties that contribute to the development and survival of MAIT cells. Until now, no endogenous MAIT cell agonists were known, and it is highly anticipated that the new agonist discovered this time will be utilized in future drug discovery research and in clinical settings.

[0022] [CA3S and Cholestasis] Cholic acid (CA) is a precursor to sulfated cholic acid (CAS), which is conjugated by the sulfotransferase sulfotransferase 2a (Sult2a). Sult2a has eight isoforms, 2a1-2a8, in mice. Sult2a is particularly highly expressed, and Sult2a8 adds a sulfate group to position 7 of CA, resulting in abundant CA7S. In contrast, humans express only SULT2A1, which adds a sulfate group to position 3, resulting in the majority of CA3S. Therefore, of the newly discovered MAIT cell agonists CA7S and CA3S, CA3S was considered more suitable for clinical application in humans, and further investigation was conducted into the clinical application of CA3S.

[0023] Regarding inflammatory diseases associated with cholestasis, it has been reported that (1) there is a correlation between primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) and the activation of MAIT cells (Am. J. Pathol. 987, 192, 629-641 (2022); J. Autoimmun. 90, 64-75 (2018)), (2) MAIT cells are activated in the livers of PBC and PSC patients (J. Autoimmun. 2018; Eur. J. Immunol. 2018), and (3) bile from PBC patients activates MAIT cells (Am. J. Pathol. 2022). However, although it is speculated that excessive activation of MAIT cells may be the cause of the disease, it has not been clarified what components (molecular entities) in bile activate MAIT cells. Based on the novel finding that CA3S is an endogenous agonist of MAIT cells, the present inventors conceived the novel idea that CA3S could be used as a diagnostic marker for the onset of cholestasis. They compared the bile CA3S concentrations (pmol / ml) in humans in healthy and cholestasis-affected states. As shown in Example 5 below, it was confirmed that the CA3S concentration increased approximately fourfold in cholestasis-affected states compared to healthy states. As described above, it has been demonstrated that CA3S can be used as a biomarker for the diagnosis of cholestasis. Therefore, it can also be used for the diagnosis of inflammatory diseases caused by cholestasis. When used as a biomarker, CA3S can be used alone or in the form of a composition containing a carrier commonly used in the pharmaceutical field. Those skilled in the art can select the appropriate form depending on the purpose. Such a test (diagnosis) can be performed, for example, by measuring the concentration of CA3S in bile collected from a patient using equipment commonly used in the art (e.g., a mass spectrometer). If the measured value is significantly elevated compared to a healthy state, it can be diagnosed as a possible inflammatory disease. CA7S is also expected to be useful as a biomarker.

[0024] The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto. Those skilled in the art may modify the embodiments of the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also within the scope of the present invention.

[0025] Example 1: Generation of T cells co-expressing the MR1 molecule and the TCR of MAIT cells in combination with a reporter assay and validation as a screening system (1) Generation of co-expressing T cells. The MAIT TCR and MR1 molecule of mouse (Tiloy et al., J. Exp. Med. 1999) or human (4L4T, Protein Data Bank) were genetically synthesized and inserted into retroviral vectors (pMX-IRES-ratCD2, pMX-IRES-humanCD8), respectively. These plasmids were co-transfected into Phoenix packaging cells using PEI MAX (Polysciences). A TCR-deficient mouse T cell hybridoma (Matsumoto, BBRC. 2020) carrying an NFAT-GFP reporter gene was infected with the supernatant containing the retrovirus. Using anti-mouse TCRβ (H57-597), anti-human CD3 (HIT3a), and anti-human / mouse / rat MR1 (26.5) antibodies, T cells co-expressing MAIT TCR and MR1 molecules (hereinafter also referred to as "co-expressing reporter cells") were selected. Figure 1 shows a conceptual diagram of the "co-expressing reporter cells." (2) Validation as a screening system Using 5-OP-RU, which is known as an antigen of MAIT cells, and acetyl-6-formylpterin (Ac-6-FP), which is known as a non-stimulatory MR1 molecule ligand, it was verified that the "co-expressing reporter cells" produced in (1) can be used as a screening system for MAIT cell ligands. 5-OP-RU was prepared using 5-A-RU (Toronto Research Chemical) and methylglyoxal (Sigma-Aldrich) as previously described (Nature. 509, 361-365 (2014)). Ac-6-FP (Cat. No. 11.418) was purchased from Schircks Laboratories. Verification experiments evaluated GFP expression and MR1 molecule surface expression in co-expressing reporter cells 6 hours after stimulation with vehicle control, 5-OP-RU, or Ac-6-FP. GFP expression was assessed by flow cytometry. MR1 molecule surface expression was expressed as the MFI value of stimulated cells.As shown in Figure 2, 5-OP-RU enhanced GFP expression in a dose-dependent manner. On the other hand, Ac-6-FP strongly increased the expression of MR1 molecules on the cell surface without enhancing GFP expression. This demonstrates that the above-mentioned "co-expressing reporter cells" can be used to evaluate the agonistic or antagonistic effects of test substances on MAIT cells.

[0026] Example 2: Screening for MAIT cell ligands from mouse intestinal extracts (1) To screen for host-derived ligands for MAIT cells, a "co-expression reporter cell" line was used to screen fractionated extracts from the intestines of specific pathogen-free (SPF) mice (CLEA Japan). Figure 3 shows a conceptual diagram of the MAIT cell ligand screening process used in this example. Of the 100 fractions obtained by separating the mouse intestinal extracts using reverse-phase column chromatography, three large peaks were detected near fractions #11, #42, and #84 (Figure 4, top panel). To determine which peaks corresponded to known microbial antigens, 5-OP-RU was subjected to the same column separation process. Activity was again observed in fractions #11 and #42 (Figure 4, bottom panel), corresponding to the expected retention times of 5-OP-RU and its related lumazine derivatives, respectively. In the above screening, to stimulate the "co-expressing reporter cells," tissue extract samples and synthetic compounds were dissolved in water and added to each well of a 96-well plate in the absence of methylglyoxal. Some water-insoluble samples were dissolved in ethanol or chloroform / methanol (2:1, vol / vol) and coated onto the plate. The "co-expressing reporter cells" (1-3 × 10 ４The cells (100 μL / well) were cultured at 37°C for 6-20 hours, and the expression of GFP and MR1 molecules was analyzed by flow cytometry. This procedure is repeated in subsequent examples. (2) To determine the chemical structure of the activated molecule contained in fraction #84, this fraction was further purified by hydrophilic interaction chromatography (HILIC), and a single fraction (designated #84-45) was collected. This activity was resistant to nucleases and proteases, suggesting that it was a metabolite other than polypeptides or oligonucleotides. High-resolution mass spectrometry (HRMS) detected a major m / z peak at 487.2368 in negative ion mode, indicating that it is a low-molecular-weight compound that is easily negatively ionized (Figure 5). (3) Next, 1D and 2D-NMR spectroscopy were performed. The results are shown in Figure 6. The H and C NMR spectra showed signals characteristic of cholic acid analogs (three methyl protons at δ 0.73, 0.93, and 0.97 ppm, two methylene protons at δ 2.32 and 2.45 ppm, and three methine proton peaks at δ 3.52, 4.07, and 4.51 ppm in the H NMR spectrum, and a carbonyl carbon peak at δ 180.8 ppm in the C NMR spectrum). Extensive analysis, including 2D-NMR, identified two peaks at δ 4.07 and 4.51 ppm in the H NMR spectrum as methine protons H-12 and H-7, respectively. The H-7 signal appeared more deshielded than expected, indicating that the hydroxyl group at C7 of the cholic acid backbone was functionalized. Furthermore, the molecular formula estimated from the main peak at m / z 487.2368 is C ２４ H ４０ O ８S[M-H], suggesting that the major component of this fraction was cholic acid 7-sulfate (CA7S) (Figure 7). Indeed, HRMS / MS analysis confirmed the presence of sulfate groups (Figure 8). (4) To confirm the predicted structure, CA7S was synthesized according to a previous report (Chem. Pharm. Bull. 27, 1402-1411 (1979)) and its assignment was confirmed. The 1H and 13C NMR spectra of fraction #84-45 matched those of the synthesized CA7S, and liquid chromatography-HRMS (LC / HRMS) analysis confirmed that the retention times of fraction #84-45 and the CA7S standard were identical. As expected, the synthetic CA7S activated reporter cells expressing the MAIT TCR (Figure 9). Because the absolute concentration of unstable 5-OP-RU, converted from 5-amino-6-D-ribitylaminouracil (5-A-RU), cannot be accurately estimated, a synthetic version of the more stable ribityllumazine (RL-7-Me) was used for comparison (ChemBioChem. 22, 672-678 (2021)). The activity of synthetic CA7S was approximately 1 / 50 to 1 / 20 of that of RL-7-Me, indicating that CA7S is a weak agonist for MAIT cells (Figure 10). Synthetic CA7S also increased cell surface MR1 molecule expression (Figure 11). CA7S activated mouse (m) and human (h) MAIT TCRs regardless of the MR1 molecule species (Figure 12). These results demonstrate that CA7S is a novel ligand for MAIT cells.

[0027] Example 3: Evaluation of CA7S Analogs (1) CA7S is biosynthesized by sulfate conjugation of cholic acid (CA). CA is a major component of bile acids released from the gallbladder into the intestine and aids in the absorption of dietary lipids through micellization. However, CA (purchased from Nacalai Tesque) itself did not activate either the TCR or MR1 molecule expression on the cell surface of MAIT cells (Figure 13, upper and lower panels), indicating that sulfate groups are necessary for agonist function. In addition to the hydroxyl group (7-OH) at the 7th position of CA, the 3-OH and 12-OH groups are also potential sulfation sites. Therefore, these isomers were also synthesized (sulfated at the 3-OH group: CA3S; sulfated at the 12-OH group: CA12S) according to a previous report (Chem. Pharm. Bull. 27, 1402-1411 (1979)). Although CA3S is present in several mammalian species, whereas CA12S is not, CA3S activated MAIT reporter cells in the presence of MR1 molecules (Fig. 13, upper panel). Furthermore, all three forms of CA sulfate (CA7S, CA3S, and CA12S) increased the expression of MR1 molecules on the cell surface to a similar extent (Fig. 13, lower panel), suggesting that CA sulfate may have the ability to bind to MR1 molecules.

[0028] (2) Because primary bile acids in mice form taurine conjugates (TCAs) in vivo, we synthesized taurine-conjugated CA3S (TCA3S) and taurine-conjugated CA7S (TCA7S) according to a previous report (Chem. Pharm. Bull. 27, 1402-1411 (1979)), and examined the effect of C24 modification on their activity. TCA, TCA3S, and TCA7S showed no activity as antigens or MR1 molecular ligands (Figure 13, bottom panel). This suggests that the formation of an amino acid conjugate of CA with a hydrophilic group at position 24 abolished its MR-binding ability. Primary bile acids are further dehydroxylated by symbiotic bacteria to produce secondary bile acids. Sulfate conjugates of secondary bile acids, such as deoxyCA3S (DCA3S), lysine CA3S (LCA3S), and taurolate CA3S (TLCA3S), showed no activity in reporter cells (Figure 13, upper panel). (3) Taken together, these findings indicate that sulfate conjugation of the 3- or 7-OH of the cholic acid skeleton is important for MAIT cell activation. *) TCA was purchased from Nacalai Tesque, DCA3S from Avanti, LCA3S and TLCA3S from Cayman, and TCA3S and TCA7S were synthesized according to a previous report (Chem. Pharm. Bull. 27, 1402-1411 (1979)). The structures of CA7S and its analogs evaluated above are shown in Figure 14.

[0029] Example 4: Analysis of gene expression signatures. Human peripheral blood mononuclear cells (PBMCs) were stimulated with CA7S and CA3S for several days and then subjected to sc-TCR-RNA-seq analysis. MAIT cells that survived in the presence of CA7S / 3S expressed representative MAIT TCRs (TRAV1-2-TRAJ33 / 12 / 20: TRBV6-4 / 6-1 / 20-1), similar to those stimulated with 5-OP-RU (Figure 15). This suggests that CA7S / 3S is a ligand for MAIT cells with representative MAIT TCRs. Furthermore, in contrast to 5-OP-RU, CA7S / 3S induced genes involved in homeostasis, such as IL7R, KLF2, and TCF7 (Figure 16). These findings indicate that sulfate-conjugated bile acids are host-derived (endogenous) ligands distinct from the microbial-derived (exogenous) antigen 5-OP-RU, and contribute to the survival and tissue repair of MAIT cells.

[0030] Example 5: Relationship with Cholestasis Bile was collected from humans in a healthy state and a state with cholestasis, and the concentration of CA3S in the bile (pmol / ml) was measured using a mass spectrometer (LC-MS / MS). The results are shown in Figure 17. The CA3S concentration in the state with cholestasis increased to approximately four times the concentration in the healthy state. This indicates that the concentration of CA3S in bile can be used as a diagnostic marker for the onset of cholestasis. Example 6: Evaluation of CA7S Analogs (2) As in Example 3, the effects of CA7S and analogs other than those evaluated in Example 3 on activating the TCR of MAIT cells and activating the expression of MR1 molecules on the cell surface were examined. Figure 18A shows the results of the investigation into the effect of each analog on activating the TCR of MAIT cells, and Figure 18B shows the results of the investigation into the effect of each analog on activating the expression of MR1 molecules on the cell surface. Analogs 11 and 13 activated the TCR of MAIT cells and activated the expression of MR1 molecules on the cell surface, suggesting the possibility that these analogs also function as activating molecules for MAIT cells in vivo. The structures of the analogs evaluated in this example are shown below.

[0031]

[0032]

[0033]

[0034]

[0035] Example 7: Flow cytometry analysis of mice administered MAIT cells in MR1-5-OP-RU tetramer-enriched thymocytes were analyzed by flow cytometry in 2- to 4-week-old wild-type mice, Sult2a-deficient mice administered with CA7S, and Sult2a-deficient mice not administered with CA7S. The results are shown in Figure 19 (Panels A to F). Panel A shows the expression of MAIT cells in MR1-5-OP-RU tetramer-enriched thymocytes. － CD3 ＋ MR1-5-OP-RU tetramer in cells ＋ TCRβ ＋ Representative flow cytometry dot plots of MAIT cells are shown. Panel B shows the absolute number of thymic MAIT cells. Panel C shows the representative CD44 and CD24 expression patterns. Panel D shows the expression patterns of stage 1 (CD44 － CD24 ＋ ), stage 2 (CD44 － CD24 － ), stage 3 (CD44 ＋ CD24 － ) and the frequency of MAIT cells. ＋ CD24 － Representative CD319 and CD138 expression in MAIT cells is shown. Figure F shows MAIT17 cells (CD138 ＋ ) and MAIT1 cells (CD319 ＋ ) are shown. Data were obtained from experiments using three or more mice per group. *P<0.05, **P<0.01, P***<0.005, one-way analysis of variance and Tukey's multiple comparison test were used. These studies demonstrated that administration of CA7S to Sult2a-deficient mice resulted in recovery of MAIT cells. This indicates that CA7S is an autoantigen for MAIT cells.

[0036] The present invention provides, as embodiments thereof, cells for identifying ligands of MAIT cells, and methods for screening ligands of MAIT cells using the cells, which are useful, for example, in the field of medicine.

[0037] This application is based on patent application No. 2023-186971 filed in Japan (filing date: October 31, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A T cell that co-expresses the MR1 molecule and the TCR of a MAIT cell and expresses a reporter protein in response to TCR stimulation.

2. The T cell of claim 1, wherein the reporter protein is selected from green fluorescent protein (GFP), luciferase and β-glucuronidase.

3. The T cell of claim 1, wherein the reporter protein is GFP.

4. The T cell according to any one of claims 1 to 3, wherein the MR1 molecule and the TCR of the MAIT cell are of human origin.

5. A method for screening for an agonist or antagonist of MAIT cells, comprising at least the following steps: (1) contacting the T cell according to claim 1 with a test substance, (2) observing activation of the TCR of MAIT cells and expression of MR1 molecule on the cell surface, and (i) determining that a test substance that activates the TCR of MAIT cells is an agonist of MAIT cells, and (ii) determining that a test substance that enhances expression of MR1 molecule on the cell surface without activating the TCR of MAIT cells is an antagonist of MAIT cells.

6. The screening method according to claim 5, wherein activation of TCR of MAIT cells is determined based on the intensity of GFP luminescence.

7. The screening method according to claim 5 or 6, wherein TCR activation of MAIT cells and expression of MR1 molecules on the cell surface are observed by flow cytometry.

8. The screening method according to claim 5 or 6, wherein the test substance is a compound group sample.

9. Biomarkers for testing for inflammatory diseases caused by cholestasis, including CA3S (cholic acid 3-sulfate) or CA7S (cholic acid 7-sulfate).

10. The biomarker described in claim 9, wherein the inflammatory disease caused by cholestasis is primary biliary cholangitis (PBC) or primary sclerosing cholangitis (PSC).

11. A method for testing for an inflammatory disease caused by cholestasis, comprising measuring the concentration of CA3S or CA7S in bile collected from a subject.

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