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Methods for induction of antigen-specific regulatory t cells

A specific and regulatory technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve problems that are prone to serious infections and affect the quality of life

Inactive Publication Date: 2015-03-11
IMCYSE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Non-specific immunosuppressive therapies known in the art are often prone to serious infections and other serious outcomes that severely impact quality of life

Method used

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  • Methods for induction of antigen-specific regulatory t cells
  • Methods for induction of antigen-specific regulatory t cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0123] Example 1. Induction of apoptosis in vitro

[0124] Antigen Presenting Cells (APCs) Prepared from C57BL / 6 Mice and Loaded with Peptides Covering Class II Restricted T-Cell Epitopes of Autoantigens Involved in Experimental Autoimmune Encephalomyelitis (EAE) for Multiple Sclerosis Model .

[0125] Therefore, with the sequence VGW YRSPFSRVV The myelin oligodendrocyte glycoprotein (MOG) peptide of HLYR [SEQ ID.NO: 1], corresponding to amino acid residues 37-52 of the MOG protein, was used to load the cells.

[0126] This peptide contains dominant T cell epitopes. The P1 position, the first amino acid anchored to the MHC class II groove, is Y40 (P1-P9 sequences are underlined).

[0127] Cytolytic CD4+ T cells (cCD4+ T cells) were obtained from the spleen of animals immunized four times with aluminum hydroxide and 50 μg of the peptide of SEQ ID. NO: 1, wherein the amino-terminal 3 amino acids are replaced by the sequence CGPC, generating the sequence CGPC Peptide of YR...

Embodiment 2

[0129] Example 2. Isolation of apoptosomes

[0130] The supernatant of apoptotic cells obtained in Example 1 was collected and subjected to two centrifugation steps (500×g, 5 minutes) to remove cells. Then, the supernatant was filtered through a 1.2 μM hydrophilic syringe filter. After centrifugation at 100,000 xg for 30 minutes, the apoptotic bodies contained in the pellet were harvested and used for cell experiments.

[0131] Alternatively, apoptotic cells and apoptosomes can be separated by affinity using antibodies directed against cell surface components expressed as a result of apoptosis. Examples of these are antithrombin antibodies. In a preferred preparation step, anti-thrombin antibodies are covalently coupled to magnetic microbeads. After incubation for 1 hour at 20°C with gentle shaking, the beads remained on the magnet. Then, apoptosomes were recovered by elution with a weakly acidic buffer.

[0132] These methods are known in the art. (Schiller et al. (2008...

Embodiment 3

[0133] Example 3. Generation or Obtaining of Immature Dendritic Cells (iDC)

[0134] Bone marrow progenitor cells were obtained from the upper and lower patella. B and T lymphocytes were removed using magnetic reduction using CD19 and CD90 microbeads, respectively. Negative isolates containing CD19-CD90-iDC progenitor cells were resuspended in serum-free medium containing 500 U / ml recombinant GM-CSF and seeded (3x10 6 cells / ml) on tissue culture plates and maintained at 37°C. Cells were washed every other day for 6 days to avoid disrupting aggregates. On day 6, iDC aggregates were removed, washed and added to new plates. On day 7, cells were harvested and used for analysis.

[0135] These methods are known in the art. See eg, Inaba et al. (2009) Curr. Prot. immunol. 1(86), Section 3.7. Unit pp. 10-12.

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Abstract

The present invention relates to methods to elicit immature antigen-presenting cells loaded with apoptotic cells or apoptotic bodies. The present invention also relates to methods of obtaining antigen-specific regulatory T cells in vitro or in vivo. Cells loaded with apoptotic bodies / cells and regulatory T cells are obtainable by inducing apoptosis of antigen-presenting cells by cytolytic CD4+ T cells. The cells are used for suppressing or preventing diseases such as autoimmune diseases, graft rejection and allergic diseases, and medicaments related thereto. Further disclosed are the use of antigen-specific regulatory T cells for suppressing or preventing diseases such as autoimmune diseases, graft rejection and allergic diseases, and medicaments related thereto. Further disclosed are populations of antigen-specific regulatory T cells obtained by said method.

Description

field of invention [0001] The present invention relates to methods for obtaining antigen-specific regulatory T cells and their use as medicines for the treatment of conditions such as autoimmune diseases, allergic diseases or transplant rejection. Background of the invention [0002] Regulatory T cells (Treg), especially those expressing the transcriptional receptor Foxp3 (forkhead box P3), are required to maintain normal immune homeostasis. In the absence of such cells, autoimmunity develops rapidly, with clinical manifestations such as diabetes and other autoimmune diseases (see Sakaguchi et al. (2012) Nature Med. 18, 54-58). Foxp3+ Treg are actively selected in the thymus and constitute the population of cells found in peripheral blood that stably express Foxp3. The threshold for selecting cells in the thymus following allogeneic recognition of self-peptides presented by thymic epithelial cells allowed Foxp3+ cells to be found with a significant affinity in peripheral bl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P37/02A61P37/08A61P37/06A61P29/00A61K39/00
CPCA61K39/0008C12N2502/1121C12N5/0637A61K39/001C12N5/0639C12N2502/1114A61K35/17C07K14/70539A61P29/00A61P37/02A61P37/06A61P37/08A61K2035/122
Inventor J-M·圣莱美
Owner IMCYSE
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