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Composition, method and application thereof for obtaining follicular regulatory T cells in vitro

A composition and regulatory technology, applied in the direction of biochemical equipment and methods, animal cells, vertebrate cells, etc.

Active Publication Date: 2020-11-24
ZHONGSHAN HOSPITAL FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, baicalin has not been found to promote Foxp3 + Related reports on Tfr differentiation

Method used

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  • Composition, method and application thereof for obtaining follicular regulatory T cells in vitro
  • Composition, method and application thereof for obtaining follicular regulatory T cells in vitro
  • Composition, method and application thereof for obtaining follicular regulatory T cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Materials

[0033] MRL / lpr lupus mice: purchased from Shanghai Slack Experimental Animal Co., Ltd.

[0034] anti-CD3 / CD28 activated magnetic beads: purchased from Peprotech.

[0035] TGF-β: purchased from Peprotech, Cat. No. AF-100-21C.

[0036] IL-2: purchased from Peprotech Company, article number AF-212-12.

[0037] Baicalin: purchased from Tongtian Biology, dissolved in DMSO and prepared into solutions with different concentrations of 10 μM, 20 μM and 40 μM.

[0038]2. Methods and results

[0039] 1. Baicalin combined with cytokines promotes the proliferation of Tfr cells:

[0040] Mononuclear cells were sorted from the spleen of MRL / lpr lupus mice, and naive T cells were sorted by immunomagnetic beads and cultured in vitro. On day 0, the sorted 2×10 6 naive CD4 + T cells were resuspended in a 24-well plate, the culture medium was RPMI1640 medium containing 10% fetal bovine serum, and anti-CD3 / CD28 activated magnetic beads (25 μl, the final concentration o...

Embodiment 2

[0048] To investigate the effects of different induction conditions on the proliferation of Tfr cells and the differentiation of naive T cells into Tfr.

[0049] 1. Materials

[0050] MRL / lpr lupus mice: purchased from Shanghai Slack Experimental Animal Co., Ltd.

[0051] anti-CD3 / CD28 activated magnetic beads: purchased from Peprotech.

[0052] TGF-β: purchased from Peprotech, Cat. No. AF-100-21C.

[0053] IL-2: purchased from Peprotech Company, article number AF-212-12.

[0054] Baicalin: purchased from Tongtian Biology, dissolved in DMSO and prepared into a solution of corresponding concentration.

[0055] 2. Method

[0056] 1. Group settings

[0057] The groups are shown in Table 1.

[0058] Table 1 Experimental grouping

[0059] group Final concentrations of anti-CD3 antibody, anti-CD28 antibody, TGF-β, IL-2 and baicalin Experimental group 1 50ng / ml, 50ng / ml, 5ng / ml, 30U / ml, 40μM Experimental group 2 60ng / ml, 30ng / ml, 6ng / ml, 15U / ml, 60μM ...

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PUM

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Abstract

The invention relates to a composition and a method conducive to acquiring follicular regulatory T cells in vitro and an application of the composition. The invention proves that differentiation and number proliferation of in-vitro Tfr (follicular regulatory T cells) can be effectively induced by the aid of irritation of TGF (transforming growth factor)-beta, IL (interleukin)-2, anti-CD3 antibodies, anti-CD28 antibodies and baicalin, and expression of Foxp3 in the Tfr is facilitated. The Tfr cells amplified by the method can be used for treating autoimmune diseases and chronic inflammatory diseases, the autoimmune diseases include lupus erythematosus, dermatomyositis, vasculitides, sicca syndrome, scleroderma, rheumatoid arthritis, ankylosing spondylitis, disseminated sclerosis and autoimmune hepatitis, and the chronic inflammatory diseases include diabetes, coronary heart diseases, hyperlipemia, eczema, vitiligos, atopic dermatitis, lichen planus and the like.

Description

technical field [0001] The present invention relates to inducing differentiation to obtain follicular regulatory T cells in vitro, in particular to a composition, method and application for obtaining follicular regulatory T cells in vitro. Background technique [0002] Follicular regulatory T cells (Tfr), surface marker CD4 + CXCR5 + , the transcription factors are Foxp3 and BCL-6, which are a group of T cells with immune regulation function existing in the germinal center and peripheral blood. Tfr derived from Foxp3 + In natural regulatory T cells (nTreg), Tfr can exert contact inhibition through CTLA-4 on the cell membrane surface, and can also stimulate follicular helper T cells and B cells through secreted cytokines IL-10, TGF-β or perforin. Cells play an immunosuppressive role, inhibit the formation of germinal centers, autoantibody secretion, and control the hyperactivity of humoral immunity; Tfr can also inhibit the proliferation and secretion of other effector T c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0637C12N2500/30C12N2501/2302C12N2501/51C12N2501/515
Inventor 杨骥李明
Owner ZHONGSHAN HOSPITAL FUDAN UNIV
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