Method for in vitro induction and amplification of human antigen nonspecific regulatory T cell

A non-specific and regulatory technology, applied in the field of biomedicine, can solve the problems of unstable phenotype and regulation, high cost of T cells, low induction efficiency, etc. type and regulation function to stabilize the effect

Inactive Publication Date: 2017-08-22
TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with antigen-specific Treg, antigen-nonspecific Treg has wider application indications, but the biggest problem in the current existing technology is that the induction of regulatory T cells in vitro is expensive, time-consuming, low in induction efficiency, phenotype and regulation. Unstable etc.

Method used

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  • Method for in vitro induction and amplification of human antigen nonspecific regulatory T cell
  • Method for in vitro induction and amplification of human antigen nonspecific regulatory T cell
  • Method for in vitro induction and amplification of human antigen nonspecific regulatory T cell

Examples

Experimental program
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Effect test

Embodiment 1

[0045] This example provides a method for inducing and expanding human antigen non-specific regulatory T cells in vitro, and the flow chart of the method is shown in the attached figure 1 As shown, the specific steps include:

[0046] 1: Recruit voluntary subjects, draw 20ml of peripheral blood, add an equal volume of normal saline to dilute and mix well, use human Ficoll lymphocyte separation medium, separate peripheral blood mononuclear cells (ie PBMC) by density gradient centrifugation, separate get 5.6×10 7 a PBMC;

[0047] Take 2.4×10 7 PBMC for For the sorting of T cells, the PBMCs were sequentially mixed with human naive T cells (i.e. T) Incubation with biotin-labeled antibody complexes from the sorting kit, avidin magnetic beads, and magnetic field purification to remove non-CD4 + T cells were incubated with magnetic beads coated with anti-CD25 antibody and purified by magnetic field to remove CD25 + T cells, resulting in CD4 + CD25 - CD45RA + T cells. Get ...

Embodiment 2

[0053] This example provides a method for inducing and expanding human antigen non-specific regulatory T cells in vitro, and the flow chart of the method is shown in the attached figure 1 Shown, concrete steps are basically the same as embodiment 1, difference is:

[0054] Step 2: Take 4.99×10 6 CD4 + CD25 - CD45RA +T cells were resuspended in 8ml of AMI-V CTS medium containing 10% autologous serum, added 3 times the amount of anti-CD3 / CD28 magnetic beads and rhIL-2 at a final concentration of 2000U / ml, and placed at 37°C with 5% In an incubator with a saturated humidity of carbon dioxide and 95% air, the culture was stimulated for 6 days, and rhIL-2 was supplemented every 3 days during the culture process, so that the final concentration of rhIL-2 was 2000 U / ml. The remaining cells were detected by flow cytometry for the expression of molecules such as CD27, CD95, CD45RA, GITR, CD127, CD25, Foxp3, and CTLA-4.

[0055] 3: After the induction, the magnetic field removes th...

Embodiment 3

[0058] This example provides a method for inducing and expanding human antigen non-specific regulatory T cells in vitro, and the flow chart of the method is shown in the attached figure 1 Shown, concrete steps are basically the same as embodiment 1, difference is:

[0059] Step 2: Take 2.9×10 6 CD4 + CD25 - CD45RA + T cells were resuspended in 4ml of AMI-VCTS medium containing 5% autologous serum, adding 1 times the amount of anti-CD3 / CD28 magnetic beads and rhIL-2 at a final concentration of 500U / ml, and placed at 37°C with 5% carbon dioxide and 95% air saturated humidity incubator, stimulated culture for 12 days, and added rhIL-2 every 3 days during the culture process, so that the final concentration of rhIL-2 was 500U / ml. The remaining cells were detected by flow cytometry for the expression of molecules such as CD27, CD95, CD45RA, GITR, CD127, CD25, Foxp3, and CTLA-4.

[0060] 3: After the induction, the magnetic field removes the anti-CD3 / CD28 magnetic beads, counts...

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Abstract

The invention provides a method for in vitro induction and amplification of a human antigen nonspecific regulatory T cell. A human peripheral blood CD4<+> T cell with an abundant source is separated, and a CD4<+>CD25<-> T cell is induced and amplified into a CD4<+>CD25<+>CD127<dim> antigen nonspecific regulatory T cell with high purity and high regulation efficiency within a short period through a simple induction method meeting the Clinical Good Manufacturing Practice (GMP). An enough therapeutic dose of Treg can be obtained through induced cultivation of a week (6-7d), the phenotype and regulation function of the induced Treg are stable, furthermore, the incidence rate of events, such as cell activity reduction and microbial contamination due to long-term culture in vitro is greatly reduced, and the quality control method is specific and fast and is very suitable for clinical application.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for inducing and expanding human antigen non-specific regulatory T cells in vitro. Background technique [0002] Cell, tissue or organ transplantation is the main treatment for severe organ dysfunction or end-stage failure. In most cases, the organ donor (donor) and the transplant recipient (recipient) are individuals with different genetic backgrounds. Therefore, acute and chronic immune rejection will inevitably occur, and this is the main reason affecting the effect of transplantation treatment. The main body that mediates immune rejection is the effector cells in the recipient. They activate, proliferate and secrete a large number of effector molecules after recognizing foreign graft antigens, which directly and / or indirectly lead to graft damage. At present, immunosuppressive drugs need to be used to To control the function of effector cells, however, long-term...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0637C12N2501/2302C12N2501/51C12N2501/515
Inventor 陈刚王璐
Owner TONGJI HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI TECH
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