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Antigen-specific Treg as well as preparation method and application thereof

A specific and antigen-specific technology, applied in the field of antigen-specific Treg and its preparation, can solve the problems of inapplicability to large-scale clinical application, complex preparation process of antigen peptide, high production cost, etc., to induce long-term immune tolerance and save labor costs Compared with preparation cost and simple operation

Active Publication Date: 2021-06-11
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the preparation process of genetically engineered antigenic peptides is complicated, the cycle is long, the yield is low, and the production cost is high, which is not suitable for large-scale clinical application

Method used

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  • Antigen-specific Treg as well as preparation method and application thereof
  • Antigen-specific Treg as well as preparation method and application thereof
  • Antigen-specific Treg as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This embodiment provides a method for preparing antigen-specific Treg, which includes the following steps:

[0043] S1. On day 0, collect monocytes from the peripheral blood of recipients, culture them at 37°C for 1 hour, remove the medium, remove non-adherent cells, or sort mononuclear cells by magnetic bead sorting and flow cytometry. Nucleated cells were added to a fresh medium containing GM-CSF at a concentration of 100 ng / mL and IL-4 at a concentration of 20 ng / mL and cultured for 24 hours to obtain the first culture supernatant;

[0044] On the first day, 1 ng / mL of TGF-β1 and 20 ng / mL of IL-10 were added to the above-mentioned first culture supernatant and cultured for 24 hours to obtain the second culture supernatant;

[0045] On the second day, the tolDC cytokine cocktail was added to the second culture supernatant and cultured for 24 hours to induce the maturation of the tolDC to obtain the receptor tolDC. Among them, the tolDC cytokine cocktail includes the ...

Embodiment 2

[0052] This embodiment provides a method for preparing antigen-specific Treg, which includes the following steps:

[0053] S1. On day 0, collect monocytes from the peripheral blood of recipients, culture them at 37°C for 1 hour, remove the medium, remove non-adherent cells, or sort mononuclear cells by magnetic bead sorting and flow cytometry. Nucleated cells were added to a fresh medium containing GM-CSF at a concentration of 80 ng / mL and IL-4 at a concentration of 15 ng / mL and cultured for 24 hours to obtain the first culture supernatant;

[0054] On the first day, 0.5 ng / mL of TGF-β1 and 15 ng / mL of IL-10 were added to the above-mentioned first culture supernatant and cultured for 24 hours to obtain the second culture supernatant;

[0055] On the second day, the tolDC cytokine cocktail was added to the second culture supernatant and cultured for 24 hours to induce the maturation of the tolDC to obtain the receptor tolDC. Among them, the tolDC cytokine cocktail includes the...

Embodiment 3

[0062] This embodiment provides a method for preparing antigen-specific Treg, which includes the following steps:

[0063] S1. On the 0th day, collect the mononuclear cells from the peripheral blood of the recipient, culture them at 37°C for 1 hour, remove the medium, remove the non-adherent cells or sort out the mononuclear cells by magnetic bead sorting and flow cytometric sorting. Nucleated cells were added to a fresh medium containing GM-CSF at a concentration of 120 ng / mL and IL-4 at a concentration of 25 ng / mL and cultured for 24 hours to obtain the first culture supernatant;

[0064] On the first day, 1.5 ng / mL of TGF-β1 and 25 ng / mL of IL-10 were added to the above-mentioned first culture supernatant and cultured for 24 hours to obtain the second culture supernatant;

[0065] On the second day, the tolDC cytokine cocktail was added to the second culture supernatant and cultured for 24 hours to induce the maturation of the tolDC to obtain the receptor tolDC. Among them...

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Abstract

The invention is applicable to the technical field of biology, and provides a preparation method and application of antigen-specific Treg, and the preparation method comprises the following steps: preparing a receptor tolDC; preparing a plasmid which contains all CDS region sequences of donor MHC-I molecule mRNA and is provided with a T7 promoter, and then preparing ivt-mRNA of the donor MHC-I molecule by applying an in-vitro transcription technology; the ivt-mRNA is transfected into the receptor tolDC, and the receptor tolDC for expressing and presenting the donor MHC-I molecular antigen is obtained; and co-culturing a receptor tolDC for expressing a donor MHC-I molecular antigen and a receptor nTreg cell under the condition of containing IL-2, so as to obtain the donor MHC-I molecular antigen specificity Treg. According to the preparation method, the receptor Treg cell with donor MHC-I antigen specificity and proper TCR-MHC antigen complex affinity can be prepared.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an antigen-specific Treg and its preparation method and application. Background technique [0002] With the improvement of organ preservation methods, the development of surgical techniques and the continuous research and development of immunosuppressive drugs, organ transplantation has become an effective means of treating patients with end-stage organ failure. However, in allogeneic organ transplantation, the mismatch between recipient and donor major histocompatibility complex I (MHC-I) can cause immune rejection, leading to impaired function and even failure of the transplanted organ. Although the use of immunosuppressive drugs can reduce the incidence of acute rejection, it cannot effectively prevent the occurrence and development of chronic rejection, and the resulting functional impairment of transplanted organs greatly shortens the survival time of transplanted or...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N5/0783C12N5/0784A61K39/00A61P37/02
CPCC12N5/0637C12N5/0639C07K14/70539A61K39/001114A61P37/02C12N2502/1121C12N2501/2302C12N2501/2304C12N2501/22C12N2501/231C12N2501/15C12N2501/2301C12N2501/25C12N2501/2306C12N2501/02Y02A50/30
Inventor 李明谦吕国悦张鹏
Owner JILIN UNIV
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