Kit for predicting intestinal acute graft versus host disease after allogenic hematopoietic stem cell transplantation and application thereof

Abstract

The invention discloses a kit for predicting intestinal acute graft versus host disease after allogenic hematopoietic stem cell transplantation and application of the kit. In particular, the invention discloses an antibody composition, which comprises a CD3 antibody, a CD161 antibody, a V alpha 7.2 antibody, a CD114 antibody, an IL-17A antibody, an IL-4 antibody and a CXCR6 antibody. According to the antibody composition, the kit and the detection method disclosed by the invention, only one tube of seven antibody compositions is used, accurate and rapid detection on the allo-HSCT donor graft can be realized through one-time sample loading, and the expression proportions of MAIT cells and CD114, IL-17A, IL-4 and CXCR6 thereof in the graft are determined, so that possible intestinal aGVHD after transplantation can be effectively predicted before transplantation. The method disclosed by the invention has important guiding significance for predicting the intestinal aGVHD of a hematopoietic stem cell transplantation patient and formulating a clinical treatment scheme.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, relates to the application of cellular immunology to post-transplantation complications, and specifically relates to a kit for predicting acute intestinal graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation and its application. Background Art

[0002] Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective treatment for hematological malignancies, but acute graft-versus-host disease (aGVHD) remains the main cause of morbidity and mortality, threatening the survival of patients. Among patients receiving allo-HSCT, the incidence of aGVHD is 30-50%, of which severe aGVHD is 14%. Therefore, early warning and targeted intervention after transplantation are crucial to improve efficacy.

[0003] Mucosal-associated invariant T cells (MAIT cells) are a type of multifunctional innate immune T cells that are widely present in humans and are mainly distributed in the blood and mucosal tissues, such as the intestine, lung, and liver. MAIT cells exert their effects by recognizing MR1 proteins and their ligands, and have traditional effector and tissue maintenance functions, indicating their importance in protecting the mucosa from microbial invasion. Our previous work found that in myeloablative allo-HSCT, the reconstruction of recipient MAIT cells mainly depends on the number of MAIT cells in the graft, and the number of MAIT cells in the graft is associated with a low incidence of intestinal aGVHD after transplantation. The anti-intestinal GVHD effect of MAIT cells may be activated by interaction with intestinal flora, triggering tissue repair function, and jointly inhibiting the occurrence of intestinal GVHD by exerting a certain immunosuppressive effect. MAIT cells in adult blood express a variety of chemokine receptors, such as CCR5, CCR6, CXCR6, and CCR9, which enable MAIT cells to be recruited from the circulation system to inflamed tissues and play a key role in anti-intestinal aGVHD and tissue repair. Therefore, the tissue migration ability and absolute number of MAIT cells in tissues are key factors for their mucosal protection or anti-intestinal aGVHD effects.

[0004] In view of this, researching and developing new prediction methods for intestinal aGVHD and advancing the prediction and intervention of intestinal aGVHD to before transplantation will have important guiding significance for the prediction of intestinal aGVHD and the formulation of clinical treatment plans for hematopoietic stem cell transplantation patients, and will have broad clinical application value for further improving transplantation efficacy, enhancing patients' quality of life and transplantation success rate. Summary of the invention

[0005] One of the purposes of the present invention is to provide an antibody composition, a kit and its application for predicting intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (allo-HSCT) before allogeneic hematopoietic stem cell transplantation; the technical problems to be solved by the present invention are not limited to the described technical subjects, and those skilled in the art can clearly understand other technical subjects not mentioned in this article through the following description.

[0006] To achieve the above objectives, the present invention first provides an antibody composition for predicting intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, wherein the antibody composition comprises CD3 antibody, CD161 antibody, Vα7.2 antibody, CD114 antibody, IL-17A antibody, IL-4 antibody and CXCR6 antibody.

[0007] Each antibody in the antibody composition may be a monoclonal antibody.

[0008] Furthermore, each antibody in the antibody composition may be an antibody labeled with a fluorescent dye.

[0009] The fluorescent dyes include, but are not limited to, fluorescein isothiocyanate (FITC), allophycocyanin (APC), allophycocyanin-cyanidin 7 (APC-CY7), APC-H7, phycoerythrin (PE), phycoerythrin-Texas Red (PE-TR, ECD), phycoerythrin-cyanidin 5 (PE-CY5), phycoerythrin-cyanidin 7 (PE-CY7), PerCP / Cy5.5, carboxyfluorescein diacetate succinimidyl ester (CFSE), green fluorescent protein (GFP), Hoechst 33342), propidium iodide (PI), 4',6-diamidino-2-phenylindole (DAPI), 7-aminoactinomycin D (7-AAD), PB dye (PacificBlue), aminomethylcoumarin acetate (AMCA), BV480, BV785, BV510, BV711, BV421, BV605, V500 (BDHorizon V500), and Alexa 700 (Alexa Fluor 700, AF700).

[0010] Furthermore, the fluorescent dye labels of the CD3 antibody, CD161 antibody, Vα7.2 antibody, CD114 antibody, IL-17A antibody, IL-4 antibody and CXCR6 antibody may be PE-CY7, APC, PE, BV480, BV785, BV510 and BV711, respectively.

[0011] The name of the CD3 antibody labeled with PE-CY7 can be PE-CY7-CD3; the name of the CD161 antibody labeled with APC can be APC-CD161; the name of the Vα7.2 antibody labeled with PE can be PE-Vα7.2; the name of the CD114 antibody labeled with BV480 can be BV480-CD114; the name of the IL-17A antibody labeled with BV785 can be BV785-IL-17A; the name of the IL-4 antibody labeled with BV510 can be BV510-IL-4; the name of the CXCR6 antibody labeled with BV711 can be BV711-CXCR6.

[0012] The antibody composition of the present invention may include antibodies PE-CY7-CD3, APC-CD161, PE-Vα7.2, BV480-CD114, BV785-IL-17A, BV510-IL-4 and BV711-CXCR6.

[0013] The present invention also provides a kit, which comprises any antibody composition described herein.

[0014] The kit has at least one of the following uses: (1) Used to predict intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation; (2) Used to detect CD3, CD161, Vα7.2, CD114, IL-17A, IL-4 and CXCR6; (3) Used to detect the proportions of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells, and CXCR6+MAIT cells.

[0015] Furthermore, the kit may also include a red blood cell lysis solution and / or a fixation and permeabilization solution.

[0016] Furthermore, the kit may also include a membrane permeabilization buffer and / or a cell washing solution.

[0017] The red blood cell lysis solution, fixation permeabilization solution, permeabilization buffer and cell washing solution are all conventional reagents in the art and can be commercially obtained. For example, in one or more embodiments of the present invention, the red blood cell lysis solution is BD Lysing solution, the fixation permeabilization solution is ebioscience fixation permeabilization solution, the permeabilization buffer is 5×permeablization buffer, and the cell washing solution is PBS buffer.

[0018] Furthermore, the kit may be a flow cytometry analysis kit.

[0019] The test sample of the kit can be a peripheral hematopoietic stem cell transplant sample of an allogeneic hematopoietic stem cell transplant donor.

[0020] The various reagent components of the kit may be present in separate containers, or may be pre-combined in whole or in part into a reagent mixture.

[0021] The components of the kit may be provided in the form of a solution, such as an aqueous solution. In the case of being in the form of an aqueous solution, the concentration or content of these components can be easily determined by those skilled in the art according to different needs. For example, for storage purposes, the concentration of the components may be present in a higher form, and when in working state or use, the concentration may be reduced to the working concentration by diluting the above-mentioned higher concentration solution.

[0022] The present invention also provides the use of any of the antibody compositions described herein in any of the following: A1) Use in the preparation of a product for predicting acute graft-versus-host disease in the intestine after allogeneic hematopoietic stem cell transplantation; A2) Use in the preparation of products for detecting CD3, CD161, Vα7.2, CD114, IL-17A, IL-4 and CXCR6; A3) Use in the preparation of a product for detecting the proportion of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells.

[0023] The products may include reagents, kits and detection devices but are not limited thereto.

[0024] Furthermore, the application includes detection and / or analysis by flow cytometry using the antibody composition described herein.

[0025] Furthermore, the application may include predicting intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation based on the ratio of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells.

[0026] Furthermore, the proportions of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells can be detected by detecting the expression of CD3, CD161, Vα7.2, CD114, IL-17A, IL-4 and CXCR6.

[0027] Furthermore, the application may include the following steps: B1) Obtaining peripheral blood stem cell grafts from allogeneic hematopoietic stem cell transplantation donors; B2) adding the stem cell transplant and the antibody composition described herein into a flow cytometry tube and incubating in the dark; B3) Add red blood cell lysis buffer and fixation and permeabilization buffer, wash and centrifuge to obtain the sample to be tested; B4) Detect the sample on a flow cytometer to obtain the test result.

[0028] Furthermore, the application may include the following steps: D1) Obtaining peripheral blood stem cell grafts from allogeneic hematopoietic stem cell transplantation donors; D2) adding the stem cell transplant and antibodies PE-CY7-CD3, APC-CD161, PE-Vα7.2, BV480-CD114 and BV711-CXCR6 (cell surface flow cytometry detection antibodies) into a flow cytometry tube and incubating in the dark; D3) Add red blood cell lysis buffer to dissolve red blood cells; D4) Add fixation and permeabilization solution and permeabilization buffer to fix and permeabilize the membrane; D5) Add antibodies BV785-IL-17A and BV510-IL-4 (intracellular flow cytometry detection antibodies); D6) Centrifuge and resuspend to obtain the sample to be tested; D7) Detect the sample on the flow cytometer to obtain the test results.

[0029] The detection results may include: the expression of CD3, CD161, Vα7.2, CD114, IL-17A, IL-4 and CXCR6, and the proportions of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells.

[0030] In the above application, after B4) or D7), the following steps may also be included: C1) obtaining the proportions of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells according to the test results, and scoring using the median value of each cell proportion; C2) Prediction of intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation based on the scoring results.

[0031] Furthermore, the method of scoring using the median value of each cell ratio can be: each cell ratio (i.e., MAIT cell ratio, CD114+MAIT cell ratio, IL-17A+MAIT cell ratio, IL-4+MAIT cell ratio, and CXCR6+MAIT cell ratio) is scored as 0 or 1 according to whether it is less than or greater than or equal to its median value. A cell ratio less than its median value is scored as 0; a cell ratio greater than or equal to its median value is scored as 1.

[0032] Furthermore, the median value of the MAIT cell ratio may be 2.9%, the median value of the CD114+MAIT cell ratio may be 0.03%, the median value of the IL-4+MAIT cell ratio may be 0.008%, the median value of the IL-17A+MAIT cell ratio may be 0.03%, and the median value of the CXCR6+MAIT cell ratio may be 1.52%.

[0033] Furthermore, the proportions of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells can all be proportions of CD3+T cells.

[0034] Furthermore, when the score is ≤2, the incidence of intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation is predicted to be high, and when the score is >2, the incidence of intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation is predicted to be low.

[0035] The present invention also provides a device for predicting acute intestinal graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, the device comprising a data receiving module, a data processing module and an output module; the data receiving module is used to receive the proportion data of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells in the peripheral blood stem cell transplant of the allogeneic hematopoietic stem cell transplantation donor; the data processing module is used to predict acute intestinal graft-versus-host disease after allogeneic hematopoietic stem cell transplantation according to the proportion data; and the output module is used to display the prediction result.

[0036] Furthermore, the data processing module can score according to the ratio of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells, combined with the median value of each cell ratio, and predict intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation based on the scoring results.

[0037] Furthermore, the data processing module can score 0 or 1 according to whether each cell ratio (i.e., MAIT cell ratio, CD114+MAIT cell ratio, IL-17A+MAIT cell ratio, IL-4+MAIT cell ratio, and CXCR6+MAIT cell ratio) is less than or greater than or equal to its median value. A cell ratio less than its median value is scored as 0; a cell ratio greater than or equal to its median value is scored as 1.

[0038] Furthermore, the device may also include a computer-readable storage medium, which can be used to store proportion data of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells in the peripheral blood stem cell transplant of an allogeneic hematopoietic stem cell transplant donor.

[0039] After extensive and in-depth research, the inventors of the present invention obtained, for the first time, an antibody composition for predicting acute intestinal graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation, a fluorescent labeling combination of the corresponding antibodies, a kit, and a detection method. Since there are several fluoresceins available for each monoclonal antibody, when different fluoresceins are selected for labeling, the specificity and sensitivity of detecting the proportion and phenotype of MAIT cells in the graft may be different, and when the seven monoclonal antibodies are labeled with different combinations of fluoresceins, it is unknown whether the flow cytometer can effectively and accurately detect consistent expression results. The inventors of the present invention further explored and optimized the fluorescent labeling combination of the seven monoclonal antibodies through a lot of creative work, and only one tube of the seven antibody compositions was used to achieve accurate and rapid detection of the donor graft through one loading, thereby determining the MAIT cells in the graft and their CD114, IL-17A, IL-4, and CXCR6 expression ratios. The antibody composition, kit and detection method of the present invention can effectively predict intestinal aGVHD that may occur after transplantation before transplantation, provide a new management tool for the prevention and intervention of intestinal aGVHD in clinic, and have important guiding significance for the prediction of intestinal aGVHD and the formulation of clinical treatment plans for hematopoietic stem cell transplantation patients.

[0040] Beneficial effects of the present invention: The kit of the present invention can be used to quickly and accurately detect the proportion of MAIT cells and the expression of their functional factors in donor grafts, providing a reliable new method for routine and batch testing in clinical laboratories. Existing aGVHD prediction methods, such as ST2 and REG3α kits, have been used in clinical practice, but have obvious limitations. These kits require the collection of patient serum specimens before the onset of aGVHD and are tested by ELISA. However, because the onset time of aGVHD is unpredictable, multiple sampling is required, and it is difficult to grasp the best sampling time. In addition, the test results are easily affected by multiple factors, the operation is complicated, the cost is high, there is a lack of unified prediction values, and the prediction efficiency is not absolute.

[0041] In contrast, the antibody composition and kit of the present invention are based on flow cytometry, and can predict intestinal aGVHD in advance before transplantation through a single sampling. It is easy to operate, cost-controlled, and the prediction effect is significantly better than traditional methods, and has higher clinical application value. This detection method can be used as an effective indicator for predicting intestinal aGVHD after allogeneic hematopoietic stem cell transplantation (allo-HSCT), providing important guidance for patient prognosis judgment and the formulation of clinical treatment plans.

[0042] Definition of terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0043] The term "flow cytometry (FCM)" generally refers to the technology of multi-parameter quantitative analysis and sorting of single cells or particles in a flowing state by measuring the fluorescence, light scattering and light absorption of cells or particles. In flow cytometry, the cells or particles to be tested are generally made into a suspension before detection, and then fluorescently stained to prepare the test sample. When the test sample passes through the detection area, it is irradiated with a laser beam to generate a light signal, which is converted into an electrical signal by a photoelectric converter and transmitted to a computer, and displayed in the form of a graph (such as a histogram and a scatter plot) on a computer analysis system. Flow cytometry can complete a variety of cell detection tasks, such as analysis of cell surface antigens or determination of cell subpopulation ratios, cell apoptosis detection, cell cycle analysis, etc.

[0044] The term "fluorescent dye" generally refers to a substance that can emit light of another wavelength after absorbing light of a certain wavelength. Generally, the wavelength of light emitted by a fluorescent dye is longer than the light absorbed. Fluorescent dyes can be used alone or in combination as composite fluorescent dyes. Fluorescent dyes may include, but are not limited to, small molecules, proteins (or peptides), organic polymers, and quantum dots. In the present invention, "fluorescent dye" and "fluorescein" have the same meaning and can be used interchangeably.

[0045] The term "label" in the present invention is understood to mean that the antibody is combined, conjugated, labeled, connected or attached to a fluorescent dye. The antibody can be covalently or non-covalently labeled. For example, the antibody can be labeled by an amino group (including a lysine side chain and an amino terminus). Those skilled in the art can routinely prepare fluorescent dye-labeled antibodies without excessive experimentation. Fluorescent dye-labeled antibodies can be prepared according to any method known in the art, or commercially available fluorescent dye-labeled antibodies can be directly used.

[0046] The term "erythrocyte lysing solution (also known as hemolytic agent)" generally refers to a reagent for removing red blood cells, including any substance that can dissolve red blood cells in the blood. It uses the hypotonic principle to break up red blood cells in blood or bone marrow specimens. When blood or bone marrow samples are detected by flow cytometry, the interference caused by red blood cells can be eliminated to achieve accurate analysis of target cell surface antigens. The red blood cell lysing solution can be selected from NH 4 Cl, KHCO 3 EDTA-Na 2 Or one or a combination of two or more of Tris. The red blood cell lysate can be prepared by methods known in the art, or can be directly commercially available.

[0047] The term "fixation and permeabilization solution" generally refers to the reagent used to fix and permeabilize cell suspensions in flow cytometry. The fixation and permeabilization solution allows antibodies to enter cells smoothly and preserves the cell morphology intact, which can reduce nonspecific staining in experiments.

[0048] The term "median" is also called "median or median", which generally refers to the number in the middle position of a set of data arranged in order, representing a value in a sample, population or probability distribution, which can divide the set of values ​​into two equal upper and lower parts. For a finite set of numbers, all the observed values ​​can be sorted and the one in the middle can be found as the median. If there are an even number of observed values, the average of the two middle values ​​is taken as the median. In the present invention, the median refers to the median percentage of MAIT cells, CD114+ MAIT cells, IL-17A+ MAIT cells, IL-4+ MAIT cells and CXCR6+ MAIT cells in CD3+ T cells in peripheral stem cell transplants.

[0049] The term "prediction" in the present invention may refer to determining whether a subject or patient is at risk of developing intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. For example, predicting and assessing the likelihood of developing intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation before allogeneic hematopoietic stem cell transplantation (i.e., assessing the risk of future onset). BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1G-CSF mobilization enhances the hyporesponsiveness characteristics of MAIT cells.

[0051] Figure 2 G-CSF-CSF3R interaction mediates MAIT cell function.

[0052] Figure 3 The immunosuppression and tissue repair of G-MAIT cells are beneficial for resistance to intestinal aGVHD.

[0053] Figure 4 The chemotactic heterogeneity of G-MAIT cells affects the occurrence of intestinal aGVHD.

[0054] Figure 5 The proportion and functional molecules of MAIT cells in the graft can predict the occurrence of intestinal aGVHD.

[0055] Figure 6 A flow cytometry-based prediction panel for intestinal aGVHD. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0057] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0058] The red blood cell lysis solution BD Lsying solution in the following examples was purchased from BD Biosciences, catalog number 349202.

[0059] The fixed permeabilization buffer and 5× permeablization buffer kit in the following examples were purchased from BD Biosciences, catalog number 562574.

[0060] The antibodies in the following examples can all be directly purchased commercially. The sources of the antibodies in the examples of the present invention are shown in Table 1.

[0061] Example 1: Antibody composition for predicting acute intestinal graft-versus-host disease after allogeneic hematopoietic stem cell transplantation After in-depth research, the inventors of the present invention have developed an antibody composition for detecting the expression of MAIT cells and their related functional factors CD114, IL-17A, IL-4 and CXCR6 in donor grafts. The antibody composition includes the following fluorescently labeled monoclonal antibodies: PE-CY7-CD3, APC-CD161, PE-Vα7.2, BV480-CD114, BV785-IL-17A, BV510-IL-4 and BV711-CXCR6. Detailed information on each monoclonal antibody is shown in Table 1.

[0062]

[0063] Example 2: Application of antibody composition in prediction of intestinal aGVHD This example uses the antibody composition of the present invention to predict intestinal aGVHD based on flow cytometry, and the specific steps are as follows: 1. Reagent preparation (1) Prepare 10× PBS buffer with a pH of 7.2-7.4 and dilute it 10 times with distilled water to make 1× PBS buffer; (2) Take the red blood cell lysis solution BD Lysing Solution (10×) and dilute it 10 times with distilled water to obtain 1× BD Lysing Solution; (3) Prepare eBioscience fixation and permeabilization solution in a ratio of 1:3; (4) Prepare 5× permeablization buffer and dilute it 5-fold to 1× buffer (i.e., 1× permeablization buffer).

[0064] 2. Collection of transplant specimens Collect 20 μl of peripheral stem cells from healthy donors who have been treated with G-CSF in clinical practice and put them into EP tubes.

[0065] 3. Fluorescent labeling of cell surface antibodies on transplant specimens (1) Mix the whole blood and count the white blood cells; (2) Take a flow cytometry tube and add about 10 μl of the transplant (white blood cell count> 1×10 6 ) to the flow tube; (3) Add 5 μl PE-CY7-CD3, 5 μl APC-CD161, 5 μl PE-Vα7.2, 5 μl BV480-CD114 and 5 μl BV711-CXCR6 of the corresponding cell surface antibodies, mix gently, and incubate at room temperature in the dark for 20 min; (4) Add 2 ml of 1× BD Lysing solution, mix well, and keep away from light at room temperature for 8 min to lyse red blood cells; (5) Centrifuge at 1500 rpm for 5 min and remove the supernatant; (6) Add 2 ml of 1× PBS to resuspend the cells, then centrifuge at 1500 rpm for 5 min, remove the supernatant, and harvest the cell pellet.

[0066] 4. Fluorescent labeling of intracellular antibodies (1) Resuspend the cell pellet obtained in step 3 by flicking, add 1 ml of the prepared eBioscience fixation and permeabilization buffer, and vortex for 30 seconds; (2) Incubate at 4°C in the dark for 30 min; (3) Add 2 ml of 1× permeabilization buffer and centrifuge at 1500 rpm for 5 min. (4) Discard the supernatant, flick to resuspend, add 3 μl of intracellular antibody BV785-IL-17A and 3 μl of BV510-IL-4, and incubate at room temperature for 20 min; (5) Add 2 ml of 1× permeabilization buffer and centrifuge at 1500 rpm for 5 min. (6) Discard the supernatant and add 100 μl 1× permeabilization buffer to resuspend the sample to prepare for testing. (7) If there are any particles visible to the naked eye before loading, they must be filtered out before loading.

[0067] 5. On-machine testing: The samples to be tested are processed and tested according to the operating requirements of the flow cytometer to obtain the data of specific fluorescent-labeled transplant cells. The expression ratio of MAIT cells and their CD114, IL-17A, IL-4 and CXCR6 in the transplant samples is determined with the help of instrument statistics and analysis software to form a scoring system as the main basis for predicting intestinal aGVHD.

[0068] 6. Experimental results The peripheral blood stem cell grafts of allo-HSCT donors collected from Peking University People's Hospital from January 1, 2024 to August 1, 2024 were studied using the methods in steps 1-5 above. The enrolled donors were excluded from the cases of secondary transplantation of their recipients. The enrolled healthy donors were required to complete the pre-transplantation examination and meet the requirements as allo-HSCT donors. Case selection and sample collection were approved by the Ethics Committee of Peking University People's Hospital (Ethics No. 2023PHB363-001) and were carried out with the consent of the donors or legal guardians.

[0069] All donors were given granulocyte colony-stimulating factor (G-CSF, 5 μg / kg / d, for 5 consecutive days) to mobilize bone marrow (G-BM) and peripheral blood (G-PB) before transplantation. The mononuclear cell count (MNC) was greater than 6×10 8 / kg. On the day of transplantation, unprocessed bone marrow (collected on day 4 after G-CSF) and peripheral blood stem cells (PBSCs, collected on day 5 after G-CSF) were infused into the recipients. All patients received G-PB allogeneic transplantation only. All recipients in this study received myeloablative conditioning regimen.

[0070] A total of 83 transplant donors and their matched recipients were enrolled in the clinical cohort of this study. The clinical characteristics of the recipients and donors are shown in Table 2 .

[0071]

[0072] Among the 83 transplanted patients, 78.3% (65 / 83) received haploid transplantation. The incidence of aGVHD was 24.1% (20 / 83), including 12% (10 / 83) of skin aGVHD, 9.6% (8 / 83) of intestinal aGVHD, and 16.9% (14 / 83) of stage II-IV aGVHD.

[0073] To better characterize MAIT cells in G-CSF-mobilized grafts (G-MAIT), we collected PB samples from four healthy donors before and 5 days after G-CSF treatment. MAIT cells were isolated by fluorescence-activated cell sorting (FACS) for subsequent scRNA-seq analysis ( Figure 1 A). After strict quality control, a total of 80,709 MAIT cells were obtained and further divided into 14 clusters ( Figure 1 B).

[0074] The functions of MAIT cells in immunosuppression, cytotoxicity, and tissue repair have been extensively explored. Our analysis showed that G-MAIT cells showed higher expression of immunosuppressive and tissue repair features, and lower expression of cytotoxic features ( Figure 1C), indicating enhanced immune tolerance and tissue repair capabilities. In addition, analysis of the G-CSF-related signaling pathway gene set showed ( Figure 1 D), cluster 12 had the highest G-CSF score and was significantly increased after G-CSF mobilization. Cluster 12 was also the only cell population expressing the G-CSF receptor CSF3R. These results suggest that the enhanced immunosuppressive and tissue repair properties of G-MAIT may be induced by the interaction of G-CSF with CSF3R expressed by cluster 12.

[0075] Gene set enrichment analysis (GSEA) of G-MAIT cell cluster 12 showed that the IL-17 and TNF signaling pathways, which are beneficial to tissue repair function, were significantly enriched ( Figure 2 A). At the same time, the downregulation of the graft-versus-host disease signaling pathway ( Figure 2 A), further supporting the possibility that G-MAIT cells mediate anti-GVHD effects through G-CSF-CSF3R interaction of cluster12.

[0076] We further verified the effect of G-CSF on MAIT cells by flow cytometry. Since IFN-γ, IL-4, and IL-17A are effector molecules of Th1, Th2, and Th17 cells, respectively, we detected their expression in MAIT cells in G-PBSCs. The analysis found that compared with IFN-γ and IL-17A, the expression of IL-4 in MAIT cells in G-PBSCs was significantly upregulated ( Figure 2 B), indicating that G-CSF induces MAIT cells to polarize toward type 2 and promotes immune tolerance, which is consistent with the transcriptome results. Flow cytometry analysis also confirmed that a small number of MAIT cells expressing CD114 (encoding CSF3R) increased significantly after G-CSF induction ( Figure 2 C, P=0.048). To determine whether CD114 expression of G-MAIT cells is associated with the occurrence of aGVHD, we further verified this in a clinical cohort of G-PBSCs from donors. According to the occurrence of aGVHD in the recipients, the corresponding G-PBSCs were divided into three groups: no aGVHD, intestinal aGVHD, and skin aGVHD. Compared with the other two groups, the CD114 expression of MAIT cells in the G-PBSCs in the intestinal aGVHD group was lower ( Figure 2 C), further indicating that CD114(CSF3R) in G-MAIT cells is involved in the occurrence of intestinal aGVHD.

[0077] We further explored the relationship between the transcriptional activity of G-MAIT cells and intestinal aGVHD. Compared with the intestinal aGVHD group, the expression of CSF3R and its related target genes in MAIT cells of the non-intestinal aGVHD group was higher ( Figure 3 A, and higher scores for immunosuppression and tissue repair features ( Figure 3 B), showing stronger migration and retention characteristics ( Figure 3 C). These results suggest that in addition to their immunosuppressive effects, the enhanced tissue repair, migration, and residence capabilities of G-MAIT cells also play a vital role in maintaining intestinal mucosal integrity and preventing intestinal aGVHD. Key markers such as CD69, IL-17A, IL-4, and IFN-γ are associated with tissue residence, repair, immune tolerance, and cytotoxic effects. We detected these markers in the G-PBSCs clinical cohort by flow cytometry. The expression of IL-4 and IL-17A in the intestinal aGVHD group was significantly lower than that in the other two groups, while there were no significant differences in other markers ( Figure 3 These results further suggest that immunosuppression and tissue repair are key to the anti-intestinal aGVHD effects of G-MAIT cells.

[0078] The tissue repair function of circulating MAIT cells depends on chemotaxis mediated by chemokine receptors. The results showed that the chemokine signaling pathway was downregulated in the intestinal aGVHD group ( Figure 4 A). To further investigate the chemotactic function of MAIT cells, we analyzed the differences in chemokine receptor expression between the group without intestinal aGVHD and the group with intestinal aGVHD. The results showed that the former had higher expression of CCR1, CCR5, CCR7, CXCR3, and CXCR6, while the latter had increased expression of CCR2 and CCR6 ( Figure 4 B). At the protein level, the expression of CXCR6 and CCR7 was significantly downregulated in the intestinal aGVHD group compared with the non-aGVHD and skin aGVHD groups ( Figure 4 C), while the expression of other receptors did not change significantly ( Figure 4 C). In summary, the above results indicate that CXCR6 may mediate the migration of MAIT cells in the transplant to the intestine, enabling them to play an anti-intestinal aGVHD role.

[0079] Combining sc-RNA-sq analysis of MAIT cells before and after G-CSF mobilization, as well as in vitro and in vivo experimental validation, we determined that the gene set composed of CSF3R, IL17A, IL4, and CXCR6 may play an important role in the mechanism of intestinal aGVHD. The gene set signature score showed that the group without intestinal aGVHD showed a higher gene set signature ( Figure 5 A), indicating that this gene combination can predict the occurrence of intestinal aGVHD.

[0080] We further validated the clinical potential of these findings and found that intestinal aGVHD was associated with a low proportion of G-MAIT cells in G-PBSCs, whereas skin aGVHD was not associated with the proportion of G-MAIT cells ( Figure 5 B-5C). This result is consistent with our previous work and further confirms that the proportion of MAIT cells in G-PBSCs can specifically predict the occurrence of intestinal aGVHD. In addition, we detected another non-traditional CD161-expressing T cell population, γδ T cells, and found that there was no correlation between their proportion and intestinal aGVHD ( Figure 5 B).

[0081] In addition to the proportion of MAIT cells, our transcriptomic data confirmed that MAIT cell heterogeneity is also a key factor affecting intestinal aGVHD. Using flow cytometry, we evaluated MAIT cells (CD3, CD161, Vα7.2) and the proportion of MAIT cells expressing these functional molecules (CD114, IL-17A, IL-4, CXCR6) and generated a prediction score for intestinal aGVHD ( Figure 6 A). The score evaluates 5 parameters: MAIT cell ratio (percentage of CD3+ T cells), CD114, CXCR6, IL-17A and IL-4. Each parameter is scored 0 or 1 depending on whether it is less than or greater than or equal to its median value, and the total score ranges from 0 to 5. Specifically: MAIT cell ratio (of CD3+ cells) ≥ 2.9%, 1 point; CD114+MAIT cell ratio ≥ 0.03%, 1 point; IL-4+MAIT cell ratio ≥ 0.008%, 1 point; IL-17A+MAIT cell ratio ≥ 0.03%, 1 point; CXCR6+MAIT cell ratio ≥ 1.52%, 1 point; The results showed that the MAIT cell score in the intestinal aGVHD group was significantly lower than that in the other two groups. In addition, the incidence of intestinal aGVHD in the low-score group (score ≤ 2) was significantly higher than that in the high-score group (score > 2), while the score had no effect on the incidence of skin aGVHD ( Figure 6 B). This score was significantly better than the MAIT cell proportion alone in predicting intestinal aGVHD ( Figure 5 C and Figure 6 B). In addition, the sensitivity and specificity of the antibody composition for predicting intestinal aGVHD reached 75% and 0.93%, respectively ( Figure 6 B).

[0082] In summary, the comprehensive evaluation of the proportion of MAIT cells (CD3, CD161, Vα7.2) and the expression of functional molecules (CD114, IL-17A, IL-4, CXCR6) based on flow cytometry can effectively predict the occurrence of intestinal aGVHD. For transplant patients, the antibody composition of the present invention has high specificity and sensitivity for predicting intestinal aGVHD, with a sensitivity of 75% and a specificity of 93%. It can be used as one of the reference information for predicting intestinal aGVHD of transplant patients and formulating clinical intervention treatment plans, and has a wide range of clinical application value.

[0083] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. An antibody composition for predicting acute intestinal graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, characterized in that: The antibody composition comprises CD3 antibody, CD161 antibody, Vα7.2 antibody, CD114 antibody, IL-17A antibody, IL-4 antibody and CXCR6 antibody.

2. The antibody composition according to claim 1, characterized in that Each antibody in the antibody composition is an antibody labeled with a fluorescent dye.

3. The antibody composition according to claim 1 or 2, characterized in that The fluorescent dye labels of the CD3 antibody, CD161 antibody, Vα7.2 antibody, CD114 antibody, IL-17A antibody, IL-4 antibody and CXCR6 antibody are PE-CY7, APC, PE, BV480, BV785, BV510 and BV711, respectively.

4. A kit, characterized in that The kit comprises the antibody composition according to any one of claims 1 to 3.

5. The kit according to claim 4, characterized in that The kit also includes a red blood cell lysis solution and / or a fixation and permeabilization solution.

6. Use of the antibody composition according to any one of claims 1 to 3 in any of the following: A1) Use in the preparation of a product for predicting acute graft-versus-host disease in the intestine after allogeneic hematopoietic stem cell transplantation; A2) Use in the preparation of products for detecting CD3, CD161, Vα7.2, CD114, IL-17A, IL-4 and CXCR6; A3) Use in the preparation of a product for detecting the proportion of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells.

7. The use according to claim 6, characterized in that: The application comprises using the antibody composition described in claim 2 or 3 to perform detection and / or analysis by flow cytometry.

8. The use according to claim 6 or 7, characterized in that: The application comprises the following steps: B1) Obtaining peripheral blood stem cell grafts from allogeneic hematopoietic stem cell transplantation donors; B2) adding the stem cell transplant and the antibody composition described in claim 2 or 3 into a flow cytometry tube and incubating in the dark; B3) Add red blood cell lysis solution and fixation and permeabilization solution, wash and centrifuge to obtain the sample to be tested; B4) Detect the sample on a flow cytometer to obtain the test result.

9. The use according to claim 8, characterized in that: After B4), the following steps are also included: C1) obtaining the proportions of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells according to the test results, and scoring using the median value of each cell proportion; C2) Prediction of intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation based on the scoring results.

10. A device for predicting acute intestinal graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, characterized in that: The device comprises a data receiving module, a data processing module and an output module; the data receiving module is used to receive the proportion data of MAIT cells, CD114+MAIT cells, IL-17A+MAIT cells, IL-4+MAIT cells and CXCR6+MAIT cells in the peripheral blood stem cell transplant of an allogeneic hematopoietic stem cell transplantation donor; the data processing module is used to predict intestinal acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation according to the proportion data; and the output module is used to display the prediction result.

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