Search

CN-122005786-A - Application of CD74 positive regulatory T cells in treatment of graft versus host disease

CN122005786ACN 122005786 ACN122005786 ACN 122005786ACN-122005786-A

Abstract

The invention relates to the technical field of cellular immunotherapy and discloses an application of CD74 positive regulatory T cells in treating graft-versus-host disease, wherein the CD74 positive regulatory T cells are proportionally composed of 85-95% of CD4+CD25+CD127-phenotype regulatory T cells in a sorted and purified cell population, wherein a CD74 high-expression subgroup accounts for 60-75% of the total amount of the regulatory T cells, and the cell subgroup functional immune molecule combination comprises CTLA4, FOXP3, TIGIT and TNFRSF18, wherein the CD74 positive regulatory T cells are subjected to preventive infusion by first using the CD74 positive regulatory T cells, are subjected to repeated therapeutic infusion when early aGVHD symptoms appear by single infusion on the day of transplantation, and are subjected to graded treatment to inhibit pathological immune response and promote immune tolerance once a week, and are subjected to 1X10 6 cells/receptor each time.

Inventors

  • XU YANG
  • LEI LEI
  • Lv Fulian
  • CHEN YIYIN
  • WU DEPEI
  • HU BO
  • LIN DANDAN
  • SHEN YING
  • QI LIJUAN
  • HU SHUHONG

Assignees

  • 苏州大学附属第一医院

Dates

Publication Date
20260512
Application Date
20260407

Claims (10)

  1. 1. The use of a CD74 positive regulatory T cell for the treatment of graft versus host disease, wherein the CD74 positive regulatory T cell is proportionally composed of a population of cells after sorting and purification having a proportion of regulatory T cells of the cd4+cd25+cd127-phenotype of 85-95%, wherein a subpopulation of high CD74 expression comprises 60-75% of the total amount of regulatory T cells, and wherein the subpopulations of cells are combined with a functional immune molecule.
  2. 2. Use of a CD74 positive regulatory T cell according to claim 1 for the treatment of graft versus host disease, wherein the preparation of said CD74 positive regulatory T cell comprises the steps of: Step one, raw material acquisition, namely taking spleen of a Foxp3-EGFP transgenic mouse as a starting material, and separating initial CD4+ T cells; Inducing and differentiating the initial CD4+ T cells into induced regulatory T cells in vitro; Step three, stimulation of a heterologous antigen, namely stimulation of the heterologous antigen is used for inducing high expression of CD 74; step four, cell proliferation verification, namely detecting proliferation capacity of CD74 high Treg cells; Step five, checking the inhibition function, namely detecting the immune inhibition function of the CD74 high Treg cells; Step six, target organ enrichment and clone expansion verification, namely verifying targeting and expansion capacity of CD74 high Treg cells in vivo, collecting CD74 high Treg cells subjected to function verification, and finally obtaining a CD74 positive regulatory T cell preparation after quality inspection is qualified.
  3. 3. The use of a CD74 positive regulatory T cell according to claim 2 for the treatment of graft versus host disease, wherein in step one the raw material is obtained: s1.1, killing cervical dislocation of a Foxp 3-enhanced green fluorescent protein transgenic mouse, and soaking and sterilizing the mice in 75% alcohol for 5-6 minutes; s1.2, taking out the spleen in an ultra clean bench in an aseptic manner, grinding by using a frosted glass to prepare a cell suspension, filtering by using a nylon net with 200 meshes, and centrifuging for 5-6 minutes at the rotation speed of 1400-1500rpm and the temperature of 1-4 ℃; S1.3, standing for 30-45 minutes, discarding supernatant, adding erythrocyte lysate, cracking at room temperature, stopping reaction by using RPMI-1640 culture medium, and centrifuging and washing again, wherein the centrifugal speed range is 1200-1800rpm, the centrifugal time is 5-8 minutes, the erythrocyte cracking time is 3-7 minutes, and the purity of the separated cells is more than or equal to 95%; s1.4, sorting by using a mouse initial CD4+ T cell sorting kit to obtain initial CD4+ T cells.
  4. 4. The use of a CD74 positive regulatory T cell according to claim 2 for the treatment of graft versus host disease, wherein differentiation is induced in vitro in step two: s2.1, adding 100 mu l/well of a sterile PBS solution containing 2 mu g/ml of an anti-mouse CD3 antibody and 2 mu g/ml of an anti-mouse CD28 antibody into a 96-well plate, and washing 1-2 times by using a phosphate buffer solution after coating for 1.8-2 hours at a temperature of 36-37 ℃; S2.2, inoculating the sorted initial CD4+ T cells into a 96-well plate after coating at the rate of 2X 10 5 /200 mul/well, adding 2 mug/ml of anti-mouse CD28 antibody, 20ng/ml of recombinant human interleukin-2 and 5ng/ml of recombinant mouse transforming growth factor-beta into a culture medium, and culturing for 70-72 hours under the conditions of 37 ℃ and 5% concentration of CO 2 to obtain induced regulatory T cells; S2.3, wherein the coating concentration range of the anti-CD 3 antibody is 1-3 mug/ml, the coating time is 1-3 hours, the cell inoculation density is 1-3 multiplied by 10 5 /hole, the recombinant human interleukin-2 concentration is 10-30ng/ml, the recombinant mouse transforming growth factor-beta concentration is 3-8ng/ml, and the culture time is 48-96 hours.
  5. 5. The use of a CD74 positive regulatory T cell according to claim 2 for the treatment of graft versus host disease, wherein the heterologous antigen stimulus in step three verifies that: s3.1, respectively obtaining syngeneic and allogeneic mouse spleen cells, and removing T cells by using a mouse CD90.2 positive sorting kit to prepare antigen presenting cells; s3.2, co-culturing the induction type regulatory T cells obtained in the step two and the heterologous APC for 47-48 hours according to the ratio of 1:1, and inducing high expression of CD 74; S3.3, sorting by using a flow sorter according to the expression level of the CD74 after the co-culture is finished, wherein the front 20% -30% of the fluorescence intensity of the CD74 is a CD74 high-expression subgroup, and the rear 20% -30% of the fluorescence intensity of the CD74 is a CD74 low-expression subgroup, wherein the co-culture proportion is 1:2-2:1, the co-culture time is 36-60 hours, and the sorting purity is more than or equal to 90%.
  6. 6. The use of a CD74 positive regulatory T cell according to claim 2 for the treatment of graft versus host disease, wherein in step four the cell proliferation assay: S4.1, performing cell tracing violet dye staining on the sorted CD74 high and CD74lowTreg cells, namely adjusting the cell concentration to 1X 10 6 /ml, adding the cell tracing violet dye into the cells according to 1 μl/ml, staining the cells for 25-30 minutes at 36-37 ℃, and washing the cells for 2-3 times by using phosphate buffer; S4.2, carrying out flow detection after continuous culture for 70-72 hours in a complete culture medium, and verifying that the CD74 high Treg cells have stronger proliferation capability, wherein the cell concentration range is 0.5-2X 10 6 /ml, the dye dosage is 0.5-2 mu l/ml, the dyeing time is 15-45 minutes, and the culture time is 48-96 hours.
  7. 7. The use of a CD74 positive regulatory T cell according to claim 2 for the treatment of graft versus host disease, wherein the inhibition function in step five is verified: s5.1, obtaining spleen cells of a C57BL/6 mouse, sorting CD4+CD25-conventional T cells by using a mouse CD4+CD25+ regulatory T cell sorting kit, and performing Far Red staining; s5.2, setting different ratios, and respectively co-culturing CD74 high and CD74lowTreg cells with Tconv cells; S5.3, detecting the level of FarRed after 72 hours, and the result shows that the CD74 high Treg has a stronger function of inhibiting the proliferation of conventional T cells, wherein the CO-culture temperature is 37 ℃, the concentration of CO 2 is 5%, and the detection time is 48-96 hours.
  8. 8. The use of a CD74 positive regulatory T cell according to claim 2 for the treatment of graft versus host disease, wherein the target organ enrichment and clonal expansion in step six is verified: S6.1, constructing a mouse acute graft versus host disease model, namely, receiving a lethal dose of 650cGy whole body irradiation by a BALB/c receptor mouse, and infusing bone marrow cells cleared by T cells and donor-derived initial T cells through tail veins; S6.2, cell injection, namely 5X 10 5 CD74 high Treg cells or CD74lowTreg cells are injected into the tail vein of the experimental day as a control; S6.3, taking tissue materials, namely killing the mice on the 14 th day after transplantation, systematically collecting peripheral blood, spleen, skin, liver, lung and small intestine tissues, and preparing single cell suspension; S6.4, detecting the distribution of target organs, namely detecting the proportion of donor-derived EGFP+ Treg cells in each tissue by multicolor flow cytometry, and verifying that CD74 high Treg cells are preferentially accumulated in the target organs; S6.5, cloning and amplifying the fractions, namely sorting CD74 high and CD74lowTreg cells in a target organ, extracting RNA, carrying out high-throughput sequencing on a TCR beta chain CDR3 region, adopting mixed T cell receptor analysis software and a VDJ analysis tool to analyze cloning diversity, and showing that the CD74 high Treg cells in the liver show obvious cloning advantages, and the Shannon diversity index is obviously reduced, so that the local extensive and specific cloning and amplifying are proved; S6.6, preparing a cell preparation, namely collecting the CD74 high Treg cells subjected to functional verification, and obtaining the CD74 positive regulatory T cell preparation with the activity rate of more than 90%, the CD74 expression positive rate of more than 85% and the sterility test negative after quality inspection.
  9. 9. The use of a CD74 positive regulatory T cell according to claim 2 for the treatment of graft versus host disease, wherein the CD74 positive regulatory T cell prepared according to the method of claim 2 is mixed with physiological saline or human serum albumin solution for infusion to prepare a cell infusion preparation, which is used for the purpose of treating or preventing graft versus host disease by intravenous infusion to a recipient after allogeneic hematopoietic stem cell transplantation, wherein the CD74 positive regulatory T cell is infused together with donor bone marrow cells and donor initial T cells on the day of transplantation, or is infused alone at a specific time point after transplantation.
  10. 10. The use of a CD74 positive regulatory T cell according to claim 9 for the treatment of graft versus host disease, wherein the CD74 positive regulatory T cell is administered by prophylactic infusion of the CD74 positive regulatory T cell at a single infusion on the day of transplantation at a dose of 5 x 10 5 cells/receptor, followed by repeated therapeutic infusions of the CD74 positive regulatory T cell at the onset of early aGVHD symptoms, fractionated treatment, 2-3 times per week, 1 x 10 6 cells/receptor per dose.

Description

Application of CD74 positive regulatory T cells in treatment of graft versus host disease Technical Field The invention relates to the technical field of cellular immunotherapy, in particular to an application of CD74 positive regulatory T cells in treating graft versus host disease. Background Graft Versus Host Disease (GVHD) is one of the most serious and fatal complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT), caused by donor-derived immune cell attack on recipient tissue organs. Despite advances in immunosuppressive drugs, the incidence and mortality of GVHD is still high, and prolonged use of immunosuppressive agents leads to increased risk of severe infections and tumor recurrence. Regulatory T cells (tregs) play a central role in maintaining immune tolerance and suppressing excessive immune responses. In recent years, adoptive reinfusion of in vitro expanded Treg cells has become a very promising strategy for GVHD treatment. However, current Treg cell therapies face several key challenges (1) heterogeneity problem-Treg cells are a functionally heterogeneous population, not all of which have equally powerful suppressive functions. The prior art generally uses cd4+cd25+cd127-as a phenotypic marker for Treg cells for sorting and expansion, but the population is still very heterogeneous, containing sub-populations of varying functional strength, resulting in an unstable therapeutic effect. (2) The stability problem is that under the in vivo inflammation environment, part of Treg cells lose Foxp3 expression and inhibition functions and even are converted into effector T cells, so that immune response is aggravated and treatment safety is affected. (3) Targeting problems-lack of Treg cells specific to GVHD target tissue results in ineffective migration to the lesion site after cell reinfusion, limited efficacy or need of large dose reinfusion, increasing treatment cost and risk of side effects. (4) Cloning and expansion ability problems in vivo persistence and expansion ability are key to determining Treg cell efficacy and long-term tolerance. Conventional Treg cells have short survival time in vivo, limited expansion capacity, and difficulty in establishing durable immune tolerance. Therefore, there is a strong need in the art to identify a subpopulation of Treg cells with greater inhibition, greater stability and better clonal expansion capacity to enhance the efficacy and safety of adoptive cell therapy for GVHD. Disclosure of Invention Aiming at the defects of the prior art, the invention provides the application of the CD74 positive regulatory T cells in treating graft versus host disease, has the advantages of strong antigen specificity targeting capability, high immunosuppressive efficiency, good in vivo proliferation durability, high target organ enrichment efficiency and high clinical preparation standardization degree, and solves the problems of large functional heterogeneity, insufficient targeting, poor in vivo stability, weak clone expansion capability and inaccurate curative effect in the treatment of GVHD by the existing Treg cells. In order to achieve the aim, the invention provides the following technical scheme that the CD74 positive regulatory T cells are used for treating graft versus host disease, the CD74 positive regulatory T cells are proportionally composed of the following components, in a cell population after sorting and purification, the regulatory T cells with the CD4+CD25+CD127-phenotype account for 85-95%, wherein a CD74 high expression subgroup accounts for 60-75% of the total quantity of the regulatory T cells, and the cell subgroup is combined with a functional immune molecule. Preferably, the preparation of the CD74 positive regulatory T cells comprises the steps of: Step one, raw material acquisition, namely taking spleen of a Foxp3-EGFP transgenic mouse as a starting material, and separating initial CD4+ T cells; Inducing and differentiating the initial CD4+ T cells into induced regulatory T cells in vitro; Step three, stimulation of a heterologous antigen, namely stimulation of the heterologous antigen is used for inducing high expression of CD 74; step four, cell proliferation verification, namely detecting proliferation capacity of CD74 high Treg cells; Step five, checking the inhibition function, namely detecting the immune inhibition function of the CD74 high Treg cells; Step six, target organ enrichment and clone expansion verification, namely verifying targeting and expansion capacity of CD74 high Treg cells in vivo, collecting CD74 high Treg cells subjected to function verification, and finally obtaining a CD74 positive regulatory T cell preparation after quality inspection is qualified. Preferably, in the first step, the raw material is obtained: s1.1, killing cervical dislocation of a Foxp 3-enhanced green fluorescent protein transgenic mouse, and soaking and sterilizing the mice in 75% alcohol for 5-6 minutes; s1.2, takin