Application of transporter SLC5A12 protein in preparation of product for diagnosing, preventing or treating acute graft versus host disease

Abstract

The invention discloses an application of transporter SLC5A12 protein in preparation of a product for diagnosing, preventing or treating acute graft versus host disease. Through targeted treatment of the SLC5A12 protein, the transport of glutamic acid and glycine into CD4 + T cells is reduced, the synthesis of glutathione in the CD4 + T cells is reduced, the accumulation of a large amount of ROS is promoted, and the ability of the CD4 + T cells to deal with low oxygen pressure and metabolic reprogramming is reduced, so that the apoptosis of donor CD4 + T cells activated in aGVHD is promoted, the activation of the donor CD4 + T cells and the generation of proinflammatory cytokines are inhibited, and the aGVHD condition is relieved. The marker SLC5A12 protein provides a new thought for preventing or treating aGVHD.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceutical preparation, and in particular to the use of the transporter SLC5A12 protein in the preparation of products for diagnosing, preventing or treating acute graft-versus-host disease. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has become an effective comprehensive treatment measure for leukemia, myelodysplastic syndrome, multiple myeloma, aplastic anemia, congenital immunodeficiency and other diseases. In allogeneic hematopoietic stem cell transplantation, donor-derived allogeneic reactive T cells play a very important graft-versus-leukemia (GVL) effect, effectively reducing the recurrence rate and opportunistic infection of the disease after transplantation. However, the allogeneic reactive activity of donor T cells in the anti-host direction also brings the risk of acute graft-versus-host disease (aGVHD) to the recipient. aGVHD is the most common fatal complication after allogeneic hematopoietic stem cell transplantation. It is an immunopathological damage to the recipient's target organs mediated by the immune active cells (mainly T lymphocytes) in the donor graft. The current classic regimen for the prevention and treatment of aGVHD in clinical practice is cyclosporine (CSA) + short-course methotrexate (MTX). However, this treatment method affects the reconstruction of the recipient's entire immune system after transplantation, and patients are prone to post-transplantation infections and leukemia relapse, which seriously affects the patient's prognosis. How to effectively prevent and treat aGVHD has become a difficult problem that urgently needs to be overcome.

[0003] SLC5A12 (Solute Carrier Family 5 Member 12, also known as SMCT2) is a sodium-dependent monocarboxylate transporter that is primarily responsible for the transmembrane transport of monocarboxylates such as lactate, propionate, and butyrate. It drives solutes into cells through the concentration gradient of sodium ions and is widely expressed in tissues such as the kidney and intestine, playing an important role in regulating acid-base balance, energy metabolism, and monocarboxylate absorption. Summary of the invention

[0004] In order to solve the problems of the prior art, the present invention aims to provide an application of the transporter SLC5A12 protein in the preparation of a product for diagnosing, preventing or treating acute graft-versus-host disease. The present invention reduces the transport of glutamate and glycine to CD4 + Trafficking within T cells, reducing CD4 + The synthesis of glutathione in T cells promotes the accumulation of ROS and reduces CD4 + The ability of T cells to cope with hypoxic stress and metabolic reprogramming promotes activated donor CD4 +The apoptosis of T cells can be inhibited, and the activation and production of proinflammatory cytokines can be inhibited, thus alleviating the condition of aGVHD. The marker SLC5A12 protein provides a new idea for the prevention or treatment of aGVHD.

[0005] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0006] Use of SLC5A12 protein, a marker of acute graft-versus-host disease, in the preparation of a product for diagnosing acute graft-versus-host disease.

[0007] As an improvement, the product for diagnosing acute graft-versus-host disease is a detection reagent using SLC5A12 protein as a detection target.

[0008] As an improvement, the detection reagent includes a reagent for monitoring the expression level of the SLC5A12 protein according to claim 1 in the subject's acute graft-versus-host disease tissue, and comparing it with the expression level of the SLC5A12 protein in the tissue without acute graft-versus-host disease, if the expression level of the SLC5A12 protein in the acute graft-versus-host disease tissue is significantly increased, it is judged that the subject has acute graft-versus-host disease.

[0009] Use of a SLC5A12 protein inhibitor in preparing a drug for preventing or treating acute graft-versus-host disease.

[0010] As an improvement, the SLC5A12 protein inhibitor can inhibit the expression of SLC5A12 protein or inhibit the function of SLC5A12 protein, promote the activation of CD4 + T cell apoptosis and inhibition of CD4 + The pro-inflammatory effect of T cells can significantly prevent or treat acute graft-versus-host disease.

[0011] Beneficial effects:

[0012] Compared with the prior art, the application of the transporter SLC5A12 protein in the preparation of products for diagnosing, preventing or treating acute graft-versus-host disease has found that the peripheral blood CD4 + T cells highly expressed SLC5A12 protein, and donor CD4 + T cells also highly express SLC5A12. Anti-SLC5A12 antibodies reduce the expression of glutamate and glycine to CD4 + Transport within T cells significantly reduces CD4 + The glutathione content in T cells, which in turn causes CD4 + ROS accumulates in large quantities in T cells. At the same time, CD4 +The ability of T cells to cope with hypoxic stress and metabolic reprogramming. Ultimately, the high level of ROS and poor ability to cope with hypoxia caused by anti-SLC5A12 antibodies significantly promoted the CD4 + The apoptosis of T cells was inhibited, and the activation and release of pro-inflammatory effector molecules were inhibited, which significantly prolonged the survival time of aGVHD mice and alleviated the symptoms of aGVHD, which has good application prospects. In addition, knocking out the gene of SLC5A12 protein can also significantly inhibit the donor CD4 + Activation of T cells, reduction of their pro-inflammatory cytokine expression, and reduction of CD4 + Glutathione content in T cells and promotes activated CD4 + T cell apoptosis. That is, targeting SLC5A12 significantly prolonged the survival of aGVHD mice, and the marker SLC5A12 protein provided a new idea for the prevention or treatment of aGVHD. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 CD4 activated in vivo and in vitro + T cells highly expressed SLC5A12. A is the donor CD4 + The expression level of SLC5A12 in T cells was significantly increased; B is the donor CD4 + The expression level of SLC5A12 in T cells was significantly increased; C is the donor CD4 + The expression level of SLC5A12 in T cells was significantly increased; D is the CD4 + The expression of SLC5A12 in T cells showed an upward trend; E is CD4 activated by CD3 / CD28 antibody + The expression level of SLC5A12 in T cells was significantly increased; F is the mouse CD4 activated by allogeneic antigen in the in vitro MLR system + The expression level of SLC5A12 in T cells was significantly increased;

[0014] Figure 2 Anti-SLC5A12 antibodies significantly inhibited CD4 + The expression of proinflammatory cytokines in T cells and the inhibition of the differentiation of corresponding cell subsets. A is the inhibition of CD4 by anti-SLC5A12 antibody + T cell IFN-γ secretion; B is anti-SLC5A12 antibody inhibits CD4 + T cell TNF-α secretion; C is anti-SLC5A12 antibody inhibits CD4 +T cell IL-17A secretion; D is that anti-SLC5A12 antibody can inhibit the differentiation of Th1 cell subsets; E is that anti-SLC5A12 antibody can inhibit the differentiation of Th17 cell subsets; F is that anti-SLC5A12 antibody can inhibit the differentiation of Th-GM-CSF cell subsets;

[0015] Figure 3 Anti-SLC5A12 antibody significantly inhibited mouse CD4 + T cell activation, A: Anti-SLC5A12 antibody reduces CD69 in mice + CD4 + T cells (early activated CD4 + B and C show that anti-SLC5A12 antibody reduces the CD25 + CD4 + T cells and PD-1 + CD4 + T cells (late activated CD4 + T cells) ratio; D is anti-SLC5A12 antibody reduces the human CD69 + CD4 + T cells (early activated CD4 + T cell) ratio.

[0016] Figure 4 Anti-SLC5A12 antibody significantly reduced CD4 + The ability of T cells to cope with hypoxic stress and metabolic reprogramming. A is the CD4 + GO clustering analysis of T cell transcriptome sequencing; B is anti-SLC5A12 antibody reduces CD4 + The expression levels of HIF1α and its target genes in T cells; C is the expression level of CD4 + OCR detection of T cells; D is anti-SLC5A12 antibody reduces CD4 + T cell basal oxygen consumption; E is CD4 after anti-SLC5A12 antibody + T cell glycolysis rate detection; F is anti-SLC5A12 antibody reduces CD4 + T cell maximum respiratory capacity; G is anti-SLC5A12 antibody reduces CD4 + T cell basal glycolysis level; H is anti-SLC5A12 antibody reduces CD4 + T cell compensatory glycolytic capacity; I is anti-SLC5A12 antibody reduces CD4 + ATP levels in T cells.

[0017] Figure 5 Anti-SLC5A12 antibody against CD4 +Effects of anti-SLC5A12 antibody on T cell metabolites. A is CD4 + Changes in metabolites in T cells; B is the changes in CD4 + Changes in metabolites in T cell culture supernatant; C shows that anti-SLC5A12 antibody affects CD4 + Amino acid metabolism of T cells; D is anti-SLC5A12 antibody reduces CD4 + GSH levels in T cells.

[0018] Figure 6 Infusion of anti-SLC5A12 antibody significantly improved aGVHD in mice. A shows the effect of infusion of anti-SLC5A12 antibody on the survival of mice. B shows the effect of infusion of anti-SLC5A12 antibody on the clinical score of aGVHD in mice. C shows the effect of anti-SLC5A12 antibody on the target organs of aGVHD in mice after Allo-HSCT (scale bar 50μm).

[0019] Figure 7 Anti-SLC5A12 antibody reduces donor CD4 + T cell infiltration in aGVHD target tissues. A: Anti-SLC5A12 antibody reduces donor CD4 + The proportion of T cells; B is anti-SLC5A12 antibody reduces the donor CD4 + The proportion and absolute count of T cells; C is the reduction of donor activated CD4 + The proportion and absolute count of T cells.

[0020] Figure 8 Anti-SLC5A12 antibody reduces donor CD4 in aGVHD target organs and spleen + The expression of proinflammatory cytokines of T cell inflammatory factors, among which, anti-SLC5A12 antibody infusion decreased: A is the donor CD4 + T cell IFN-γ expression level; B is the donor CD4 + The expression level of TNF-α in T cells; C is the expression level of CD4 + T cell GM-CSF expression level; D is the CD4 + T cell GM-CSF expression level.

[0021] Fig. 9 Anti-SLC5A12 antibody promotes intestinal infiltration of donor CD4+ T cells in aGVHD mice + T cell apoptosis, A is anti-SLC5A12 antibody promotes donor CD4 + T cell apoptosis; B is anti-SLC5A12 antibody promotes donor CD69 +CD4 + T cell apoptosis; C is anti-SLC5A12 antibody promotes donor effector CD4 + T cell apoptosis; D is immunofluorescence showing that anti-SLC5A12 antibody promotes CD4 + T cell apoptosis.

[0022] Fig.10 CD4 + The loss of SLC5A12 in T cells significantly prolonged the survival of aGVHD mice. A is a schematic diagram of the construction of the aGVHD mouse model; B is the donor CD4 + Effect of SLC5A12 deficiency in T cells on the survival of aGVHD mice.

[0023] Fig.11 The effect of SLC5A12 deficiency on donor CD4 + T cells play an immune regulatory role. A is the donor CD4 + T cell ratio; B is the activated donor CD4 + T cell ratio; C is the donor CD4 + T cell ratio; D is the activated donor CD4 + T cell ratio; E is the activated donor CD4 + T cell ratio; F. Donor CD4 + Apoptosis of T cells; G is activated donor CD4 + Apoptosis of T cells; H is the donor CD4 + Apoptosis of T cells; I is activated donor CD4 + Apoptosis of T cells.

[0024] Fig.12 Targeted knockdown of SLC5A12 expression in human CD4 + The immune regulation of T cells, A is the Cas9 gene editing system in human CD4 + Transfection efficiency in T cells; B is the transfection efficiency in human CD4 T cells after targeted knockdown of SLC5A12 + The activation ratio of T cells; C is the ratio of human CD4 + Apoptosis of T cells; D is the apoptosis of activated CD4 + Apoptosis of T cells. DETAILED DESCRIPTION

[0025] Example 1

[0026] 1. Animals

[0027] 6-8 weeks old female C57BL / 6 (H-2K b) mice and female BALB / c (H-2K d ) mice were purchased from Beijing Weitonglihua Company. C57BL / 6(H-2K b ) background of SLC5A12 - / - The mice were raised by Jiangsu Jicui Pharmaceutical Co., Ltd. All mice were raised and bred in an SPF-level isolation environment at the Experimental Animal Center of Soochow University. The drinking water was acidified water (pH 4.0) after high temperature and high pressure sterilization. The feed and bedding were all sterilized by the Soochow University Irradiation Center. 60 The animals were sterilized by irradiation with 300,000 Gy of Co. The animal experiments were approved by the Experimental and Animal Ethics Committee of Soochow University.

[0028] 2. Clinical samples

[0029] Peripheral blood mononuclear cell (PBMC) samples were collected from 45 patients who developed aGVHD about 30 days after allogeneic hematopoietic stem cell transplantation and 40 patients who did not develop GVHD in the Department of Hematology of the First Affiliated Hospital of Soochow University from September 2021 to May 2022 to determine the expression level of SLC5A12. The collection of clinical samples was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University and was carried out with the informed consent of the patients themselves or their guardians.

[0030] 3. Establishment of mouse syngeneic hematopoietic stem cell transplantation (syn-HSCT), allo-HSCT and aGVHD models

[0031] The syn-HSCT model was based on C57BL / 6 (H-2K b ) Female mice were used as recipient mice;

[0032] Allo-HSCT and aGVHD models were performed in BALB / c (H-2K d ) female mice were used as recipient mice. Gentomycin (80,000 U / 200 ml) was added to the drinking water of the recipient mice one week before the transplantation for pretreatment before the transplantation. 6-8 week old C57BL / 6 (H-2K b ) female mice were used as donor mice. Each recipient mouse received Co Myeloablative irradiation of 650 cGy.

[0033] Each recipient mouse of the syn-HSCT and aGVHD models was infused with 300 μl of 6×10 6 T cell-depleted donor mouse bone marrow cells + 1×10 6In the allo-HSCT model, each recipient mouse was infused with 300 μl of 6×10 6 The T-cell-removed donor bone marrow cell transplants were used to calculate the amount of each cell suspension according to the number of recipient mice in each model, and each model transplant was prepared with 1×PBS.

[0034] 4. aGVHD Scoring of Recipient Mice

[0035] Starting from the day of transplantation, the body weight of the recipient mice was recorded every 2 days and the aGVHD clinical score was performed; the specific scoring criteria for aGVHD in recipient mice are as follows:

[0036]

[0037] 5. Histopathological Examination of Recipient Mice

[0038] aGVHD pathology mainly studies the degree of damage to the three target organs: liver, intestine, and skin. ， On the 16th day after transplantation, liver, part of the intestine and skin tissue specimens of the recipient mice were removed using ophthalmic scissors and forceps, fixed with 4% paraformaldehyde solution, and sent to Wuhan Saiweier Biological Company for paraffin sectioning and HE staining, and then observed under an optical microscope.

[0039] The severity of aGVHD in each experimental group was evaluated by histopathological examination: pathological observation of liver aGVHD showed lymphocyte infiltration in the portal area and bile duct necrosis; pathological observation of intestinal aGVHD showed inflammatory cell infiltration, goblet cell degeneration and apoptosis, purulent secretions on the crypt surface, and mucosal damage and ulceration; pathological observation of skin aGVHD showed inflammatory cell infiltration, vacuolar degeneration of epidermal cells, focal dissolution of the basal layer of the epidermis, and ulcer formation.

[0040] 6. Establishment of mixed lymphocyte reaction (MLR) system

[0041] Take 6-8 week old C57BL / 6 (H-2K b ) female mice and 6-8 week old BALB / c (H-2K d ) mice to prepare spleen cell suspension. Treatment of stimulatory cells: the concentration was 1×10 7 / ml BALB / c (H-2K d ) mouse spleen or C57BL / 6 (H-2K b ) Mouse spleen cell suspension was added with 10 μg / ml mitomycin C and treated in a cell culture incubator at 37°C for 30 min; a large volume of 1× PBS was centrifuged at 1300 rpm at 4°C for 5 min, and washed twice; after counting, the concentration was adjusted to 1×10 6 / ml for standby use. Treatment of effector cells: The remaining C57BL / 6 (H-2K b) Mouse spleen cell suspension was counted and the concentration was adjusted to 1×10 6 / ml for standby use. Group plating: Take a 96-well U-shaped plate and place BALB / c (H-2K d ) mouse-derived stimulator cells and effector cells were mixed in equal volumes as heterozygous genomes, 200 μl / well; C57BL / 6 (H-2K b ) Mouse-derived stimulator cells and effector cells were mixed in equal volumes as the isogenic control group, 200 μl / well. Centrifuged at 250 g for 4 min at 4°C and placed in a 37°C, 5% CO2 incubator for 72 h.

[0042] 7.CD4 + In vitro stimulation of T cells

[0043] Take a 96-well U-shaped plate, coat it with 2 μg / ml CD3 and 0.4 μg / ml CD28 antibodies (Biolegend, USA) one day in advance, place it in a 4°C refrigerator overnight, and take 6-8 week old C57BL / 6 (H-2K b ) Prepare spleen cell suspension from female mice and use stem cell magnetic bead sorting kit to sort CD4 + The coated 96-well U-shaped plate was washed 3 times with 1× PBS to remove excess antibody and CD4 + T cells 200 μl / well (control group: cells plus 50 IU / ml interleukin 2 (IL-2, R&D, USA) were added to wells not coated with CD3 antibodies or CD28 antibodies); centrifuged at 4°C 250g for 4 min and placed in a 37°C, 5% CO2 incubator for 48 h.

[0044] 8. Flow cytometry

[0045] 8.1 Cell membrane staining

[0046] The cell samples to be tested were centrifuged at 4°C 1300rpm for 5min, the supernatant was discarded, resuspended with 1×PBS, centrifuged at 4°C 1300rpm for 5min, and washed twice; 100μl / well CD16 / CD32 antibody was added to block Fc receptors, mixed, incubated at 4°C in the dark for 20min, 100μl / well cell membrane antibody was added, mixed, incubated at 4°C in the dark for 30min, centrifuged with staining buffer (1×PBS containing 1% fetal bovine serum) at 4°C 1300rpm for 5min, washed twice, 200μl / well staining buffer was added to resuspend the cells, and tested on the machine.

[0047] 8.2 Intracellular staining

[0048] Add RPMI medium containing 10% FBS containing 10 μg / ml BFA (Brefeldin A, protein transport inhibitor) to the cells to be tested, and stimulate them in a cell culture incubator at 37°C and 5% CO2 for 4 hours. Centrifuge with staining buffer at 4°C, 1300 rpm for 5 minutes, wash twice, add 100 μl / well CD16 / CD32 antibody to block Fc receptors, mix well, incubate at 4°C in the dark for 20 minutes, add 100 μl / well cell membrane antibody, mix well, incubate at 4°C in the dark for 30 minutes, centrifuge with staining buffer at 4°C, 1300 rpm for 5 minutes, and wash twice. Add 200 μl / well Fixation / Permeabilization solution, mix well, incubate at 4℃ in dark for 30 min, dilute 10× wash buffer to 1× with double distilled water, centrifuge at 4℃ 1300 rpm for 5 min, wash twice, add 100 μl / well intracellular antibody, mix well, incubate at 4℃ in dark for 30 min; centrifuge at 4℃ 1300 rpm for 5 min, wash twice, add 200 μl / well staining buffer to resuspend the cells, and detect on the machine.

[0049] 8.3 Intranuclear staining

[0050] The cell samples to be tested were centrifuged at 4℃1300rpm for 5min, the supernatant was discarded, resuspended in 1×PBS, centrifuged at 4℃1300rpm for 5min, and washed twice. 100μl / well CD16 / CD32 antibody was added to block Fc receptors, mixed, incubated at 4℃ in the dark for 20min, 100μl / well membrane antibody was added, mixed, incubated at 4℃ in the dark for 30min, centrifuged at 4℃1300rpm for 5min with staining buffer, washed twice, Fixation / Permeabilization Concentrate and Fixation / Permeabilization Diluent were prepared at a ratio of 1:3, 200μl / well cells were resuspended, and incubated at 4℃ in the dark for 30min. Dilute 10× Permeabilization Buffer to 1× with double distilled water, centrifuge at 1300 rpm at 4°C for 5 min, wash twice, add 100 μl / well nuclear antibody, mix well, incubate at 4°C in the dark for 30 min, centrifuge at 1300 rpm at 4°C for 5 min, wash twice with staining buffer, add 200 μl / well staining buffer to resuspend the cells, and detect on the machine.

[0051] 9. Immunofluorescence Detection

[0052] On the 16th day after transplantation, ophthalmic scissors and forceps were used to remove part of the intestinal tissue specimens of the recipient mice and fixed with 4% paraformaldehyde solution. Alcohol concentration gradient was used to replace the excess water in the organs. After treatment with a transparent agent, the specimens were embedded in paraffin and sliced. The slices were baked in an oven for 3 hours and dewaxed with gradient alcohol. The sections were infiltrated with the repair solution, heated to boiling and then adjusted to medium heat, repaired for 10 minutes, and washed 3 times with 1×PBS. Blocked with 5% goat serum for 30 minutes. CD4 antibody and Tunnel dye were diluted and mixed, and incubated in a refrigerator at 4°C overnight. After washing 3 times with 1×TBST, the corresponding secondary antibody was incubated at room temperature in the dark for 1 hour. Washed 3 times with 1×TBST, DAPI was added and incubated in the dark for 10 minutes. Washed 3 times with 1×TBST, the liquid on the slices was wiped dry with absorbent paper, and the slices were sealed with a sealing solution containing an anti-fluorescence quencher. The slices were observed and images were collected under a fluorescence microscope.

[0053] 10. Glutathione detection (using micro-reduced GSH assay kit, Jiancheng Biotechnology, China)

[0054] Collect the cell samples to be tested, centrifuge at 4℃1300rpm for 5min, discard the supernatant, resuspend with 1×PBS, centrifuge at 4℃1300rpm for 5min, discard the supernatant, resuspend with 1×PBS, and ultrasonically disrupt the cell samples. Take a portion of the sample and measure the protein concentration using the BCA protein concentration assay kit. Add equal volume of reagent 1 to the remaining sample, mix well, centrifuge at 4℃3500rpm for 10min, take the supernatant, and test.

[0055] Dissolve and dilute the GSH standard to 20 μM according to the instructions; add samples to the ELISA plate in the kit according to the table below:

[0056]

[0057] The well plate was gently shaken to mix, and then allowed to stand for 5 min. The absorbance value A at 405 nm was detected using a microplate reader. The GSH concentration was calculated according to the following formula: GSH concentration (μmol / gprot) = (A assay - A blank) ÷ (A standard - A blank) × 2 ÷ Cpr; Cpr is the sample protein concentration (g / l).

[0058] 11. Glycolysis rate test

[0059] Take out the coated cell plate from the 4°C refrigerator, equilibrate to room temperature, and prepare the detection solution according to the table below (taking 30 ml as an example)

[0060]

[0061] Place the prepared test solution in a 37°C, CO2-free incubator for incubation.

[0062] Collect mouse spleen CD4+ Add T cells to the test solution, centrifuge at 4℃200g for 5min, resuspend each group of cells with the test solution and adjust to the same concentration. Add 100μl / well of each group of cells to the cell plate, and add 100μl / well of the test solution to the background well. Centrifuge at room temperature at 200g (speed 6, speed 0) for 1min, and place the cell plate in a 37℃, CO2-free incubator for 30min. Add 400μl / well of the test solution to the cell plate; place it in a 37℃, CO2-free incubator for 15-25min.

[0063] Take a pack of glycolytic rate test drugs, which contains two drugs: Rot / AA (red cover) and 2-DG (green cover); use the test solution to prepare each drug and mix thoroughly to make the concentration of Rot / AA working solution 5μM and 2-DG working solution concentration 500mM. Take out the hydrated probe plate from the 37℃, CO2-free cell culture incubator, remove the cover and Hydro Booster, add 56μl / well Rot / AA to well A, and add 62μl / well 2-DG to well B. Run on the machine: open and run the Glycolytic Rate Test template on the Wave software; after the plate tray pops up, put the probe plate in and start calibration; after the calibration is completed, continue to run the software, wait for the tray to pop up, take out the hydrated plate, and take out the cell plate from the 37℃, CO2-free incubator and place it on the tray; continue to run the test program; after the test is completed, export the data for analysis.

[0064] 12. ATP detection (using ATP detection kit, Bio-Tech, China, operated on ice)

[0065] Prepare 100 μl of ATP working solution for each well to be tested and the standard curve well; prepare ATP working solution according to the required amount at the ratio of ATP detection reagent: ATP detection dilution reagent = 1:9; ice bath for standby use. Dilute 0.5mM ATP standard solution with ATP lysis buffer to 0, 0.016, 0.08, 0.4, 2, and 10 μM. Collect the cell samples to be tested into EP tubes, centrifuge at 4℃1300rpm for 5min, discard the supernatant, resuspend with 1×PBS, centrifuge at 4℃1300rpm for 5min, discard the supernatant, add 40μl / tube lysis buffer (about 1×10 6 Cells), vortex thoroughly, centrifuge at 4℃1200g for 5min, and carefully aspirate the supernatant for testing. According to the number of wells required for the sample to be tested and the standard curve, add 100μl / well ATP detection working solution to the 96-well flat-bottom plate, leave it at room temperature for 3-5min, add 20μl / well of the sample to be tested or the diluted sample, and the diluted standard of each concentration, mix quickly, and detect with an enzyme marker (select chemiluminescence determination). Calculate the standard curve based on the data obtained from the determination of each concentration of the standard, bring the data of the sample to be tested into the standard curve, and obtain the sample concentration.

[0066] Example 2a Donor CD4 in GVHD + T cells highly express SLC5A12

[0067] The aGVHD mouse model was constructed (the construction method is shown in Example 1-3) (donor mice: C57BL / 6 (H-2K b ), recipient mice: Balb / c (H-2K d ), recipient mice infused with donor mouse bone marrow cells and spleen T cells) and mice transplanted with only allogeneic bone marrow cells (allo-HSCT) (donor mice: C57BL / 6 (H-2K b ), recipient mice: BALB / c(H-2K d ), recipient mice were infused with donor mouse bone marrow cells) for comparison.

[0068] The results of flow cytometry showed that after aGVHD occurred, the donor CD4 + The expression level of SLC5A12 transporter in T cells was significantly increased ( Figure 1 A- Figure 1 C) Peripheral blood samples were collected from patients with and without aGVHD about 30 days after allogeneic hematopoietic stem cell transplantation, mononuclear cells were isolated, and CD4 + The expression level of SLC5A12 was detected by reverse transcription real-time quantitative PCR (RT-qPCR). + The expression level of SLC5A12 in T cells showed an upward trend compared with patients without aGVHD (p = 0.05, Figure 1 D).

[0069] RT-PCR

[0070] 1) RNA extraction

[0071] Add 200 μL of chloroform to the sample EP tube containing Trizol, vortex mix for 15 seconds, let stand at room temperature for 10 minutes; centrifuge at 4℃12000g for 15 minutes, and carefully pipette the top colorless liquid into a new RNase-free EP tube. Add an equal volume of isopropanol, invert and mix, let stand at room temperature for 10 minutes; centrifuge at 4℃12000g for 10 minutes, carefully aspirate the supernatant, and retain the white precipitate. Slowly add 1 mL of 75% ethanol prepared with DEPC water, gently invert the EP tube to suspend the precipitate; centrifuge at 4℃7500g for 5 minutes, carefully aspirate the supernatant, and retain the white precipitate. Open the EP tube cap and dry the white precipitate until it is transparent; slowly and gently add an appropriate amount of preheated DEPC water, gently blow to fully dissolve the precipitate, and place the RNA sample on ice for later use or store in a -80℃ refrigerator.

[0072] 2) Reverse transcription to cDNA

[0073] Reverse transcribe the RNA according to the measured concentration, taking 20μl working system as an example:

[0074]

[0075] Reverse transcription conditions: 37°C for 15 min, 85°C for 5 s, 4°C for ∞

[0076] 3) RT-qPCR

[0077] 20μL reaction system:

[0078]

[0079] Reaction conditions:

[0080] 95℃5min; (95℃15s, 60℃30s, 70℃30s)×40 cycles; 95℃15s; 60℃60s; 95℃15s

[0081] Primer sequences

[0082]

[0083] Example 3

[0084] Anti-CD3 and anti-CD28 antibodies were used to stimulate mouse spleen CD4 + T cells are used to simulate the activation state of T cells in an inflammatory environment (see Example 1-7 for specific steps).

[0085] The results showed that compared with the unactivated group which maintained normal growth with only low concentration of IL-2, CD4 + The expression level of SLC5A12 in T cells was significantly increased (p < 0.0001, Figure 1 E). Next, we constructed an MLR experimental system to simulate the allogeneic reaction during allo-HSCT: C57BL / 6 (H-2K b ) mouse spleen cells (effector cells) and BALB / c (H-2K d ) mice spleen cells (stimulator cells) were mixed and cultured as allogeneic genomes (Allo), and C57BL / 6 (H-2K b ) mice spleen cells (effector cells) and C57BL / 6 (H-2K b ) mouse spleen cells (stimulator cells) were co-cultured as the isogenic control group (Syn). + The expression level of SLC5A12 in T cells was significantly higher than that in the syngeneic control group (p=0.0032, Figure 1 F) Further clarify CD4 + The expression of SLC5A12 increases after T cell activation.

[0086] Example 4

[0087] We investigated whether high expression of SLC5A12 could directly affect CD4 + Effector function of T cells. Sorting of CD4 T cells from spleen of C57BL / 6 mice + T cells were stimulated with anti-CD3 / CD28 antibodies (Biolegend, USA), and anti-SLC5A12 antibodies (Atlas, Sweden) or isotype control antibodies (Abcam, USA) were added to the system.

[0088] The experimental results showed that compared with the control group without anti-SLC5A12 antibody, the levels of IFN-γ, TNF-α and IL-17A in the anti-SLC5A12 antibody group were significantly reduced ( Figure 2 AC). It was confirmed that anti-SLC5A12 antibody significantly inhibited CD4 + Activation of T cells and expression of pro-inflammatory effector molecules.

[0089] C57BL / 6 mouse spleens were obtained by magnetic bead sorting CD4 + T cells were selected and cytokines and other reagents were added to induce differentiation into Th1, Th17, Treg and Th-GM-CSF cell subsets. Anti-SLC5A12 antibodies or isotype control antibodies were added to each T cell subset differentiation system, and the induction of each cell subset was detected by flow cytometry. + Effects on T cell subset differentiation.

[0090] The experimental results showed that anti-SLC5A12 antibodies could significantly inhibit the differentiation of Th1 cell subsets and reduce the secretion levels of IFNF-γ and TNF-α ( Figure 2 D); significantly inhibited the differentiation of Th17 and Th-GM-CSF cell subsets ( Figure 2 EF). The above data indicate that anti-SLC5A12 antibodies can directly inhibit CD4 + The differentiation of various pro-inflammatory cell subsets of T cells is inhibited, thereby inhibiting their expression of pro-inflammatory cytokines.

[0091] CD4 was detected in mouse spleen cells sorted in vitro + The molecular markers of early T cell activation (CD69) and late T cell activation (CD25, PD-1) were detected, and it was found that anti-SLC512 antibody significantly reduced the expression levels of these activation molecules ( Figure 3 AC). In human peripheral blood samples, anti-SLC5A12 antibodies were found to significantly reduce CD4 + The activation ratio of T cells ( Figure 3 D).

[0092] Example 5

[0093] The CD4 + Splenic CD4 T cells and isotype control antibodies + T cells were sent for transcriptome sequencing, and GO cluster analysis showed that the top 5 cluster analysis results all pointed to CD4 + The ability of T cells to cope with hypoxic stress is significantly reduced ( Figure 4 A). qPCR verification of differentially expressed genes related to hypoxia stress suggested by sequencing results revealed that the expression levels of hypoxia-inducible factors HIF1α, HIF2α, and HIF1α target genes SLC2A3, BNIP3, RORα, and TGFβ3 were significantly reduced ( Figure 4 B) These results indicate that anti-SLC5A12 antibodies can induce CD4 + While T cell mitochondria were damaged, the cells’ adaptability to hypoxia was not activated, but the CD4 + The ability of T cells to cope with hypoxic stress.

[0094] CD4 detection using hippocampus + The oxygen consumption rate (OCR) of T cells was found to be increased after anti-SLC5A12 treatment. + T cell mitochondrial oxidative phosphorylation function is inhibited ( Figure 4 C), the basal oxygen consumption and maximum respiratory capacity of cells in the anti-SLC5A12 antibody experimental group were significantly lower than those in the control group ( Figure 4DE). Under hypoxic conditions, cells may increase their reliance on glycolysis to obtain more ATP.

[0095] We then tested the effect of anti-SLC5A12 antibody on CD4 + The effect of anti-SLC5A12 antibody on T cell glycolysis was found + The glycolytic activity of T cells was also significantly inhibited ( Figure 4 F), the basal glycolysis level and compensatory glycolysis capacity of cells were significantly decreased ( Figure 4 GH).

[0096] The above results show that after the anti-SLC5A12 antibody is applied, both oxidative phosphorylation and glycolysis, two metabolic pathways that produce ATP, are significantly inhibited. Further testing of the effect of anti-SLC5A12 antibody on intracellular ATP production found that compared with the isotype control antibody, the addition of anti-SLC5A12 antibody significantly reduced CD4 + ATP levels in T cells (p<0.0001, Figure 4 I). Therefore, anti-SLC5A12 antibody significantly reduced activated CD4 + The ability of T cells to metabolically reprogram to reduce ATP production.

[0097] Example 6

[0098] Anti-SLC5A12 antibody affects the transport of glutamate and glycine to CD4 + Transport within T cells and reduce intracellular glutathione content

[0099] CD4 + Metabolomics sequencing was performed on cell lysate samples and culture supernatant samples of T cells. The results showed that compared with the isotype control antibody, two amino acids (glutamic acid and glycine) were increased in CD4 T cells after the addition of anti-SLC5A12 antibody. + The content in T cells was significantly reduced ( Figure 5 A), while the content in the culture supernatant increased significantly ( Figure 5 B), which indicates that the use of anti-SLC5A12 antibodies affects the entry of both into CD4 + Inside T cells.

[0100] Glycine and glutamate are the building blocks of glutathione (GSH), which plays a key role as an antioxidant in cells and helps cells cope with cell damage caused by a high ROS environment. Therefore, after the anti-SLC5A12 antibody acts, glutamate and glycine are reduced to CD4 +Transport within T cells, resulting in a decrease in intracellular glutathione levels, which in turn cannot buffer CD4 + ROS, a byproduct of T cell activation, causes activated CD4 + In addition, cluster analysis of metabolomics sequencing showed that many amino acid metabolisms, such as glycine, glutamate, and glutathione metabolism, were significantly inhibited after the use of anti-SLC5A12 antibodies ( Figure 5 C) At the same time, CD4 + After the addition of anti-SLC5A12 antibody, the content of glutathione in T cells was significantly reduced compared with the isotype control antibody group. + The content of GSH in T cells (p<0.0001, Figure 5 D) Through analysis, it can be seen that anti-SLC5A12 antibodies affect the transport of glycine and glutamate into cells, affecting CD4 + Metabolic synthesis and content of glutathione in T cells.

[0101] Example 6

[0102] Anti-SLC5A12 antibody infusion significantly improves aGVHD in mice

[0103] The aGVHD mouse model was established (donor mice: C57BL / 6 (H-2K b ), recipient mice: Balb / c (H-2K d ), recipient mice were infused with donor mouse bone marrow cells and spleen T cells), and starting from the 10th day after allo-HSCT, two anti-SLC5A12 antibodies that can recognize different epitopes were selected at the same time. The antibodies were injected intraperitoneally into the mice once every 5 days. The drug was stopped after 4 injections, and the differences between the anti-SLC5A12 antibody groups and between the anti-SLC5A12 antibody and isotype control antibody groups were observed.

[0104] The results showed that the use of both anti-SLC5A12 antibodies could significantly improve the survival of aGVHD mice. The difference between the antibody group and the control group could be observed earlier and more significantly when the anti-SLC5A12 antibodies were injected into aGVHD mice at a later time ( Figure 6AB). On the 16th day after transplantation, the liver, large and small intestines, and skin tissues of aGVHD mice were collected for HE staining. Through observation, it was found that a large number of inflammatory cells infiltrated in the liver of the control group; the glands on the surface of the large intestinal mucosa were broken, the interstitium was edematous, and the inflammatory cells increased; the structure of the small intestinal villi was disordered, and the infiltration of inflammatory cells was also observed; the stratum corneum of the skin was thickened, and an increase in inflammatory cells was observed in the dermis. The above target organ damage in the anti-SLC5A12 antibody group was significantly reduced compared with the homologous control group, especially in the intestine ( Figure 6 C). The results showed that anti-SLC5A12 antibodies can significantly reduce the pathological damage of target organs in aGVHD.

[0105] Example 7

[0106] A mouse model of aGVHD was constructed. After two infusions of anti-SLC5A12 antibodies, the target organs of aGVHD in mice (liver and intestine) were collected for flow cytometry detection and counting. The detection found that the use of anti-SLC5A12 antibodies can reduce the donor CD4 + The proportion of T cells ( Figure 7 AB), especially in the intestine, donor CD4 + The absolute count of T cells was also significantly reduced (p = 0.0232, Figure 7 B) Further analysis of intestinal immune cells revealed that after anti-SLC5A12 antibody infusion, activated donor CD4 + T cells (CD69 + ) and the absolute counts were also significantly reduced ( Figure 7 C).

[0107] On the 16th day after transplantation, the target organs and spleens of aGVHD mice were collected. The results of flow cytometry showed that in the livers of mice in the anti-SLC5A12 antibody group, donor CD4 + The expression levels of IFN-γ and TNF-α in T cells were significantly decreased ( Figure 8 AB). In addition, donor CD4 + The expression of GM-CSF in T cells was also significantly decreased ( Figure 8 CD). Intestinal immune cells of aGVHD mouse model were collected for detection and it was found that the donor CD4 + Increased T cell apoptosis ( Fig. 9 A), activated donor CD4 + The percentage of T cell apoptosis also increased significantly ( Fig. 9 B), we also have donor effect CD4 + T cells (CD4 + CD44 + CD62L -) observed the pro-apoptotic effect of anti-SLC5A12 antibody ( Fig. 9 C) Intestinal tissues of the two groups of recipient mice were taken for immunofluorescence staining of CD4 and TUNEL. It was also found that injection of anti-SLC5A12 antibody promoted the expression of donor CD4 in the intestine. + T cell apoptosis Fig. 9 D). This test demonstrates that anti-SLC5A12 antibodies play an important immunomodulatory role in aGVHD.

[0108] Example 8

[0109] Donor CD4 + Deletion of SLC5A12 in T cells also significantly improved aGVHD in mice and had an adverse effect on donor CD4 + T cells play an immune regulatory role

[0110] C57BL / 6(H-2K b ) background of SLC5A12 - / - The mice were constructed by Jiangsu Jicui Pharmaceutical Co., Ltd. (the SLC5A12 small guide RNA (sgRNA) was designed and constructed, and after the in vitro activity test of sgRNA, the fertilized eggs were microinjected, the F0 generation mice born were identified, the positive F0 generation mice were further mated and bred with wild-type mice, the F1 generation mice born were identified, and the F1 generation mice with successful knockout were preserved and established). The mice and wild-type C57BL / 6 mice were used as donor mice, and myeloablative irradiated Balb / c mice were used as recipient mice. The recipient mice were infused with bone marrow cells of wild-type C57BL / 6 mice and CD4 + T cells.

[0111] The results showed that the infusion of CD4 + T cells and wild-type donor CD4 + Compared with T cells, the survival of aGVHD mice was significantly prolonged ( Fig.10 ), the experimental results further prove that targeting donor CD4 + SLC5A12 expressed by T cells can significantly alleviate the progression of aGVHD.

[0112] 14 days after transplantation, the target organs and spleen of the above aGVHD mice were collected and separated into cell suspensions. The results of flow cytometry showed that the loss of SLC5A12 significantly reduced the number of donor CD4 + The proportion of T cells in the spleen and intestine of recipient mice ( Fig.11 A, 11C), significantly reduced donor CD4 + The proportion of T cell activation ( Fig.11In addition, similar to the infusion of anti-SLC5A12 antibody, the loss of SLC5A12 significantly promoted the expression of CD4 + T cells and activated CD4 + T cell apoptosis Fig.11 FI).

[0113] Example 9

[0114] CD4 isolated from human peripheral blood samples + T cells, using Cas9 gene editing technology to target and knock down CD4 + Expression of SLC5A12 in T cells. Prepare 100 μM of Alt-RcrRNA (IDT, USA) and Alt-tracrRNA-ATTO550 (IDT, USA) with nuclease-Free Duplex Buffer (IDT, USA). Mix the above oligonucleotides in equimolar concentrations in a sterile PCR tube, heat to 95°C and keep warm for 5 minutes, and slowly cool to room temperature to prepare crRNA-tracrRNAduplex. Gently mix 3 μl crRNA-tracrRNA and 2 μl TrueCut Cas9 protein (Thermo Fisher, USA) and incubate at room temperature for at least 10 minutes. In a 96-well plate, add 200 μl / well cell culture medium and preheat. Place 1-10 6 T cells were resuspended in 20 μl of primary cell nucleofection solution (Lonza, Germany). T cells were mixed and incubated with 5 μl of RNP in PCR tubes or round-bottom 96-well plates at room temperature for 2 min. Cells were electroporated using a 4D nucleofector (Lonza, Germany) with pulse conditions of EO115 / FI115. After nucleofection, cells were transferred to 96-well plates using pre-warmed cell culture medium and cultured for 3-5 days. Alt-R TMCRISPR-Cas9 crRNA sequence: Cas9.SLC5A12-1 sequence → / AltR1 / rUrU rArCrUrUrGrG rCrUrC rGrGrA rArUrC rUrArU rGrUrU rUrUrA rGrArG rCrUrA rUrGrC rU / AltR2 / ; Cas9.SLC5A12-2 sequence → / AltR1 / rGrA rArGrA rCrCrU rCrCrU rArGrU rGrCrArCrCrC rGrUrU rUrUrArGrArG rCrUrArUrGrC rU / AltR2 / ; Cas9.SLC5A12-3 sequence → / AltR1 / rCrU rGrUrArCrArA rUrGrU rArGrA rUrGrA rCrCrG rGrUrU rUrUrA rGrArG rCrUrArUrGrC rU / AltR2 / ; Cas9.Negative control sequence → / AltR1 / rCrG rUrUrA rArUrC rGrCrGrUrArU rArArU rArCrG rGrUrU rUrUrArGrArG rCrUrArUrGrC rU / AltR2 / .

[0115] The experimental results showed that similar to the results of anti-SLC5A12 antibody, targeted knockdown of SLC5A12 expression could significantly reduce the expression of human CD4 + T cell activation level ( Fig.12 B). In addition, targeted knockdown of SLC5A12 expression significantly promoted CD4 + T cells and activated CD4 + T cell apoptosis Fig.12 The above experimental results further confirmed that SLC5A12 is a + T cells are important targets for immune regulation.

[0116] The present invention and its embodiments are described schematically above, and the description is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by it and designs a structural method and an embodiment similar to the technical solution without creativity without departing from the purpose of the invention, they shall all fall within the protection scope of the present invention.

Claims

1. Application of SLC5A12 protein, a marker of acute graft-versus-host disease, in the preparation of products for diagnosing acute graft-versus-host disease.

2. The use according to claim 1, characterized in that: The product for diagnosing acute graft-versus-host disease is a detection reagent using SLC5A12 protein as a detection target.

3. The use according to claim 1, characterized in that: The detection reagent includes a reagent for monitoring the expression level of the SLC5A12 protein according to claim 1 in the subject's acute graft-versus-host disease tissue, and comparing it with the expression level of the SLC5A12 protein in the tissue without acute graft-versus-host disease. If the expression level of the SLC5A12 protein in the acute graft-versus-host disease tissue is significantly increased, it is judged that the subject has acute graft-versus-host disease.

4. Use of SLC5A12 protein inhibitors in the preparation of drugs for preventing or treating acute graft-versus-host disease.

5. The use according to claim 4, characterized in that: SLC5A12 protein inhibitors can inhibit the expression of SLC5A12 protein or inhibit the function of SLC5A12 protein.

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