DN T cell transformation and multiplication method

A double-negative, cell-based technology, applied in biochemical equipment and methods, biological preparations for removing bad cells, animal cells, etc., can solve problems such as long culture time, small number of initial DNT cells, and impact on the immune function of DNT cells. Achieve short culture period, significant immunosuppressive function, prevention and treatment of autoimmune diseases

Active Publication Date: 2016-04-13
BEIJING IMMUTECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The defect of this method is: 1. initial DNT cell quantity is few (only accounts for 1~5% of T cell total number in the periphery); 7 ; 3. The

Method used

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  • DN T cell transformation and multiplication method

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Embodiment 1

[0046] Embodiment 1 Amplification and transformation method of DNT cells according to the present invention

[0047] (1) Obtain EDTA anticoagulated whole blood from C57BL / 6 mice, and separate peripheral blood mononuclear cells (PBMC) with lymphocyte separation medium. Lymph nodes and spleens of C57BL / 6 mice were separated, ground and filtered through a 70um filter, and erythrocytes were lysed with erythrocyte lysate to obtain mononuclear cells (spleen cells, Splenocytes). Depletion of CD8 in mononuclear cells by immunomagnetic bead negative selection + T cells (anti-CD8 antibody-magnetic bead sorting, to remove CD8 positive cells) and NK cells (anti-NK1.1 antibody-magnetic bead sorting, to remove NK cells), but without CD4 isolation + For T cells, there is no need to isolate natural DNT cells. Such as figure 1 As shown, the left column is the composition of initial T lymphocytes, and the right column is the composition of T lymphocytes after negative selection of CD8 and NK...

Embodiment 2

[0050] Embodiment 2 Amplification transformation method of DNT cells according to the present invention

[0051] (1) Lymph nodes and spleens of C57BL / 6 mice were separated, ground and filtered through a 70um filter, and red blood cells were lysed with red blood cell lysate to obtain mononuclear cells. Depletion of CD8 in mononuclear cells by immunomagnetic bead negative selection + T cells (anti-CD8 antibody-magnetic bead sorting, to remove CD8 positive cells) and NK cells (anti-NK1.1 antibody-magnetic bead sorting, to remove NK cells), but without CD4 isolation + For T cells, there is no need to isolate natural DNT cells.

[0052] (2) The mixed cells separated by negative selection in step (1) were stimulated to proliferate with mature dendritic cells in vitro (the ratio of mature dendritic cells to the mixed cells in step (1) was 1:4, and OX40 was added at the same time Activation antibody OX86, 100ug / ml and IL-220ng / ml. Culture selection 1640 medium + 10% fetal bovine ser...

Embodiment 3

[0054] Embodiment 3 Amplification transformation method of DNT cells according to the present invention

[0055] (1) Lymph nodes and spleens of C57BL / 6 mice were separated, ground and filtered through a 70um filter, and red blood cells were lysed with red blood cell lysate to obtain mononuclear cells. Depletion of CD8 in mononuclear cells by immunomagnetic bead negative selection + T cells (anti-CD8 antibody-magnetic bead sorting, to remove CD8 positive cells) and NK cells (anti-NK1.1 antibody-magnetic bead sorting, to remove NK cells), but without CD4 isolation + For T cells, there is no need to isolate natural DNT cells.

[0056] (2) The mixed cells separated by negative selection in step (1) are stimulated to proliferate in vitro with CD3 / CD28 antibody (the dosage of CD3 antibody is 5ug / ml, and in another embodiment the dosage is 1ug / ml; the dosage of CD28 antibody is 2ug / ml, in another embodiment, the amount used is 0.5ug / ml;), while adding the OX40 activation antibody ...

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Abstract

The invention discloses a DN T cell transformation and multiplication method which comprises the following steps: (1) extracting mononuclear cells in an initial sample, and removing CD8<+>T cells and NK cells from the mononuclear cells; (2) culturing the sample obtained in the step (1) in vitro with a culture medium containing a T cell stimulant and cell factors; adding an OX40 activated antibody or an OX40 ligand at the same time in the culturing process; (3) purifying DN T cells after 5-7 days' culture. The method can be implemented without purifying CD4<+>T cells or natural DN T cells, so that the operation difficulty is reduced, the operation efficiency is improved, and the culture period is short; the apoptosis rate of DN T cells in multiplication can be effectively reduced, and the immunosuppression function can be improved; the DN T cell yield is greatly improved as compared with that in the prior art; the obtained DN T cells have remarkable immunosuppression function, the percentage of the proliferated CD4 T cells is reduced to 2.94% as compared with that in the prior art, and the suppression ratio can reach 93%.

Description

technical field [0001] The invention relates to a method for transforming and amplifying double-negative T cells, in particular to a double-negative T cell in vitro that can effectively reduce the apoptosis of double-negative T cells during the expansion process and promote the immunosuppressive function of double-negative T cells. Transformation amplification method. Background technique [0002] Autologous immune cell therapy is to infuse the patient's own immune cells into the patient after undergoing various treatments outside the body, so as to achieve the purpose of treating the disease. Immunologists have discovered a variety of immunosuppressive cells, including the classic CD4 + CD25 + cells, NKT cells and CD4 - CD8 - T cells (Double NegativeTRegulatory Cells, abbreviated as double-negative T cells or DNT cells), etc., among which DNT cells have been confirmed in mice and human studies. host disease (GVHD) and the prevention and treatment of autoimmune diseases...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P37/06A61P37/02A61P35/00A61P31/00A61P37/08A61P1/16
CPCA61K35/17C12N5/0087C12N5/0637C12N2501/2302C12N2501/2315C12N2501/24C12N2501/25C12N2501/51C12N2501/515C12N2501/59C12N2501/998C12N2501/999C12N2502/1107C12N2502/1121
Inventor 张栋李新民赵新颜孙广永刘锴田丹田月施文孙晓静许虎峰
Owner BEIJING IMMUTECH LLC
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