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Method for inducing in-vitro expansion of CD8<+> regulatory T cells by immunosuppressants

An in vitro amplification and immunosuppression technology, applied in the field of immunology, can solve the problems of difficult clinical application, long amplification time, difficulty in meeting GMP requirements, etc., and achieve the effect of great clinical application potential

Inactive Publication Date: 2016-08-10
SHANGHAI BLOOD CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problem with this method of in vitro amplification is that the amplification time is relatively long, and the amplified CD8 high Tregs need to be sorted by flow cytometry, which is difficult to meet GMP requirements
[0007] Although antigen-specific Treg cells have strong immune response specificity and do not affect the normal anti-pathogen and anti-tumor functions of the body, most autoimmune diseases have not yet found specific antigens, and they cannot be obtained in time during many treatments. Donor APC or waiting for a long time for in vitro expansion, therefore, the widespread clinical application of antigen-specific Treg cells is still difficult to achieve

Method used

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  • Method for inducing in-vitro expansion of CD8&lt;+&gt; regulatory T cells by immunosuppressants
  • Method for inducing in-vitro expansion of CD8&lt;+&gt; regulatory T cells by immunosuppressants
  • Method for inducing in-vitro expansion of CD8&lt;+&gt; regulatory T cells by immunosuppressants

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Embodiment 1

[0033] Material:

[0034] Healthy human mononuclear cells (PBMC); AB blood type human serum; lymphocyte separation medium (Ficoll); 1640 culture medium; phosphate buffer saline (PBS); fetal bovine serum (FBS); human CD8 + Cell sorting kit; Anti-CD3 / CD28 monoclonal antibody coated immunostimulatory magnetic beads; Recombinant human interleukin 2 (IL-2); Transforming growth factor 1 (TGF-β1); Rapamycin (RAPA ).

[0035] method:

[0036] 1. Human CD8 + Isolation and purification of T cells (sterile environment)

[0037] 1.1 Separation of PBMC cells from buffy coat blood by Ficoll density gradient centrifugation

[0038] Centrifuge a unit of buffy coat blood at 3500rpm for 15 minutes; absorb the concentrated buffy coat after centrifugation, dilute it with the original plasma, mix it and slowly resuspend it on Ficoll, and suck out the buffy coat after density gradient centrifugation; add PBS to wash to obtain PBMC cells .

[0039] 1.2 Using human CD8 + T cell sorting kit for...

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Abstract

The invention relates a method for inducing in-vitro expansion of CD8<+> regulatory T cells by immunosuppressants. According to the method, expansion of CD8<+> regulatory T cells is jointly induced by TGF-beta and RAPA (rapamycin). The invention further provides a CD8<+> Treg (regulatory T cell) with an immunosuppression function and application of the CD8<+> Treg with the immunosuppression function. The method has the advantages that the TGF-beta and the RAPA are added into a polycloning in-vitro expansion system of the CD8<+> regulatory T cells for the first time, and a great number of CD8<+> Tregs with the remarkable immunosuppression function are obtained successfully; as multi-round expansion is conducted, the CD8<+> Tregs can increase exponentially so as to meet the demands of clinical cell treatment. The CD8<+> Tregs can be kept stable in the aspects of activity, purity, phenotype, nullipotency, suppression function and the like and can be used for treating various autoimmune diseases through adoptive infusion, thereby being huge in clinical application potential.

Description

technical field [0001] The invention relates to the technical field of immunology, in particular, an immunosuppressant-induced regulatory CD8 + Methods for in vitro expansion of T cells. Background technique [0002] In recent years, CD8 + Regulatory T cells (Tregs) have become a hotspot in the study of the Treg cell family because of their powerful immunosuppressive function, and based on CD8 + Cell therapy of Tregs has also attracted much attention as an emerging potential treatment. [0003] Studies have found that, with the traditional CD4 + Tregs are similar, CD8 + Tregs not only continuously express Foxp3, the signature functional transcription factor of Treg cells, but also express CD103, CD25, CD44, GITR, CD62L, FasL, CTLA-4 and other molecules. In 2006, Uss E. et al. found that CD103, a member of the integrin family, is a CD8 + A functional molecule of Tregs mainly mediates the mutual adhesion between cells and cells and between cells and extracellular matrix....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P19/02A61P29/00A61P19/04A61P37/02A61P3/10A61P13/12
CPCC12N5/0637A61K35/17C12N2501/15C12N2501/2302C12N2501/51C12N2501/515C12N2501/999
Inventor 杨洁范华骅孙娟杨懿铭谢如锋蒋雪玉刘李栋
Owner SHANGHAI BLOOD CENT
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