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Application of TNFSF15 protein as lymphocyte immunopotentiator and activation method of TNFSF15 protein

An immunopotentiator and lymphocyte technology, applied in the field of medicine, to achieve significant effects of promoting anti-tumor immune response, inhibiting tumor growth, and activating T cell killing

Pending Publication Date: 2021-11-19
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the regulation of B cells and T cells to exert anti-tumor effects by TNFSF15

Method used

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  • Application of TNFSF15 protein as lymphocyte immunopotentiator and activation method of TNFSF15 protein
  • Application of TNFSF15 protein as lymphocyte immunopotentiator and activation method of TNFSF15 protein
  • Application of TNFSF15 protein as lymphocyte immunopotentiator and activation method of TNFSF15 protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 TNFSF15 can promote the proliferation and activation of lymphocytes

[0045] Add the isolated spleen lymphocytes to a 6-well plate, 5×10 per well 6 3 cells, 3 mL medium (RPMI 1640 containing 15% FBS, 1% penicillin-streptomycin mixed solution (100X) (Penicillin-Streptomycin Solution) double antibody). The cells were stimulated with 3 μg / mL TNFSF15, and a corresponding volume of buffer was added to the control group as Vehicle. The proliferation (cyclin B1 gene ccnb1; mitotic checkpoint serine / threonine protein kinase BUB1 gene bub1) and activation (Icos, Icosl and Fasl genes) of lymphocytes were detected by qPCR. Flow cytometry analysis, the results are as follows figure 1 As shown, it was shown that TNFSF15 stimulation significantly promoted the increase of mRNA expression of lymphocyte proliferation genes ccnb1 and bub1, and lymphocyte immunity-promoting genes fasl, icos and icosl.

Embodiment 2

[0046] Example 2 TNFSF15 promotes the proliferation and activation of B cells through NF-κB

[0047] Add the isolated spleen lymphocytes to a 6-well plate, 5×10 per well 6 Mixed cancer cells 2×10 5 Each, 3mL culture medium (2mL RPMI 1640+15% FBS containing 15% FBS, 1% double antibody, high glucose DMEM containing 1% double antibody). 3 μg / mLTNFSF15 stimulated the cells, and in the control group, a corresponding volume of buffer was added as Vehicle.

[0048] The cancer cells (LLC) used for co-cultivation were incubated with CFSE at 37° C. for 10 minutes before mixing lymphocytes, and 40% FBS was added to ice bath for 10 minutes to terminate the reaction. Add PBS to wash twice, discard the supernatant, and resuspend the cells in the medium.

[0049] Inhibitors NF-κB (1 μM) and PI3K (1 μM) stimulated immune cells for 1 h in advance, and then added cancer cells containing 3 μg / mL TNFSF15 or Vehicle.

[0050] ICOSL neutralizing antibody (10μg / mL), pre-incubated lymphocytes at ...

Embodiment 3

[0054] Example 3 TNFSF15 activates T cells through ICOSL and PI3K to exert tumor killing effect

[0055] Add the isolated spleen lymphocytes to a 6-well plate, 5×10 per well 6 Mixed cancer cells 2×10 5 Each, 3mL culture medium (2mL RPMI 1640+15% FBS containing 15% FBS, 1% double antibody, high glucose DMEM containing 1% double antibody). 3 μg / mLTNFSF15 stimulated the cells, and in the control group, a corresponding volume of buffer was added as Vehicle.

[0056] The cancer cells (LLC) used for co-cultivation were incubated with CFSE at 37° C. for 10 minutes before mixing lymphocytes, and 40% FBS was added to ice bath for 10 minutes to terminate the reaction. Add PBS to wash twice, discard the supernatant, and resuspend the cells in the medium.

[0057] Inhibitors NF-κB (1 μM) and PI3K (1 μM) stimulated immune cells for 1 h in advance, and then added cancer cells containing 3 μg / mL TNFSF15 or Vehicle.

[0058] ICOSL neutralizing antibody (10μg / mL), pre-incubated lymphocytes...

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Abstract

The invention discloses application of a TNFSF15 protein as a lymphocyte immunopotentiator and an activation method of the TNFSF15 protein, belonging to the technical field of medicines. According to the invention, the TNFSF15 protein can be used as the lymphocyte immunopotentiator to promote lymphocyte immune cells such as B cells and CD8<+>T cells to infiltrate tumors, promote anti-tumor immune response and inhibit tumor growth; the TNFSF15 also promotes proliferation of B cells and activates expression of ICOSL by activating an NF-kappa B signal, so the CD8<+>T cells are promoted to secrete GZB so as to play a tumor killing role; and meanwhile, a PI3K signal channel participates in TNFSF15 to promote CD8<+>T cells to secrete the GZB. The TNFSF15 protein provided by the invention has obvious effects on in-vitro amplification and T cell killing activation of immunotherapy, and provides more possibilities for immunotherapy of tumors.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to the application of TNFSF15 protein as a lymphocyte immune enhancer and its activation method. Background technique [0002] The occurrence, development, and metastasis of tumors are complex processes regulated by many factors. Therefore, the treatment of tumors has become a great problem. Among tumor treatment methods, immunotherapy is favored in tumor treatment because of its significant advantages in high cure efficiency, no obvious side effects, good tolerance of patients, and promotion of the recovery of the body's immune system. Antibodies in immunotherapy often play an anti-tumor role in serum, but all antibodies produced by B cells can change the function of their antigen targets on cancer cells, and regulate tumor cells through the presentation and cross-presentation of tumor antigens by dendritic cells. Activates the complement cascade to aid in NK cell-mediated tumor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/19A61P37/04A61P35/00C12N5/0781C12N5/0783C12N5/077
CPCC12N5/0635C12N5/0636C12N5/0669A61K38/191A61P37/04A61P35/00C12N2501/50
Inventor 张强哲李鲁远韩秋菊
Owner NANKAI UNIV
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