Application of autocrine VEGFB to T cell metabolism and functions as well as tumor immunity treatment

A technology of VEGF-B and cells, which is applied in the field of biomedicine, can solve the problems of limited understanding of antioxidant VEGF-B, and achieve the effect of promoting intake and enhancing effect

Active Publication Date: 2019-10-15
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Nevertheless, knowledge of the...

Method used

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  • Application of autocrine VEGFB to T cell metabolism and functions as well as tumor immunity treatment
  • Application of autocrine VEGFB to T cell metabolism and functions as well as tumor immunity treatment
  • Application of autocrine VEGFB to T cell metabolism and functions as well as tumor immunity treatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1, the way of activating T lymphocytes in vitro

[0053] Isolation and purification of CD4 from mouse splenocytes + and CD8 + T cells. via CD4 + / CD44 - / CD62L + and CD8 + / CD44 - / CD62L + Antigen marker screening CD4 + and CD8 + T cells, EasySep mouse CD4 + or CD8 + T cell isolation kit or MoFloAstrios (BeckMan) sorting.

[0054]For activation and proliferation of non-antigen-specific T cells, CD3 (5 μg / ml) and CD28 (2 μg / ml) antibodies were activated for 72 hours. The T cell activation medium used was RPMI 1640+10% serum+100 units / ml of penicillin / streptomycin+2 millimolar of L-glutamine+55 micromolar of β-mercaptoethanol). Antigen-specific T cell activation, CD8 + OT-1naiveT cells were isolated from whole splenocytes and then treated with OVA 257-264 Peptide (1 μg / ml, SIINFEKL, Sigma) and IL-2 (100 units / ml, PeproTech) activation for 3 days.

[0055] CD4+ Activated by Cytokine-Induced Antibodies to CD3 and CD28 in Mice T cells were diffe...

Embodiment 2

[0056] Embodiment 2, flow cytometry

[0057] Using fluorescently labeled antibodies (Biolegend, eBioscience and BD Pharmingen), red mitochondrial tracking dye (Invitrogen), mitochondrial reactive oxygen species detection dye (Invitrogen), BODIPY FL C16 (Invitrogen), DCFH-DA (Biyuntian), TMRM (Invitrogen), JC1 (Invitrogen), Annexin V / PI (ebioscience) and other staining were performed according to the manufacturer's instructions. OVA-specific CD8+ T cells were stained with H2-Kb OVA257-264 MHC peptide tetramer (MBL). Cells were incubated with CD16 / CD32 purified from mouse (#101302, BioLegend) at 4°C for 30 minutes before membrane staining. Intracellular cytokine staining, cells were treated with brefeldin A (BD, Biosciences) for 2.5 hours. Cell Fixation / Cell Wall Fixation / Permeabilization Kit (#554714, BD, Biosciences) was used for intracellular VEGF-B, IFN-γ, IL-4, IL-17 staining. For intracellular staining of VEGF-B, use an antibody against VEGF-B (AF-590, 1:2000, R&D Syste...

Embodiment 3

[0062] Embodiment 3, real-time fluorescent quantitative PCR (qRT-PCR)

[0063] The expression of transcripts was detected by real-time fluorescent quantitative PCR (qRT-PCR). The qRT-PCR reaction was performed using Light Cycler480 system (Roche, Applied Science, Germany). Actb (β-actin) was used as an internal reference. See Table 2 for primer sequences.

[0064] Table 2.qRT-PCR Primers

[0065]

[0066]

[0067]

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Abstract

The invention belongs to the field of biologic medicines, and particularly relates to an application of autocrine VEGF-B to T cell metabolism and functions as well as tumor immunity treatment. For thefirst time, the invention discloses that blood vessel endothelium growth factors B(VEGF-B) are metabolism adjusting and controlling factors selectively secreted by different subtypes of activated T cells. Through antibodies, or peptide activated T cells, and/or excitants which can promote biologic synthesis of mitochondria, or reagents for adjusting and controlling ROS/AP-1 signals pertinent to the mitochondria, the activated T cells can be induced by a ROS/AP-1 signal channel to secrete VEGF-B, so that fatty acid ingestion, apoptosis resistance, tumor resistance activity and the like of theT cells can be promoted. The metabolism of T lymphocytes is programmed through tumor microenvironment, and the autocrine VEGF-B has great application prospects in tumor immunity treatment.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to the application of autocrine VEGF-B in promoting T cell metabolism and function and in tumor immunotherapy. Background technique [0002] Under conditions such as viral infection, inflammation, and tumors, naive T lymphocytes can proliferate and differentiate into effector and memory T cell subsets after being activated by antigen-presenting cells to fight against factors that threaten the body. During this process, activated T lymphocytes can produce a large number of signaling molecules, such as cytokines, growth factors, etc., and regulate cell morphology, proliferation, migration, and differentiation in an autocrine or paracrine mode; at the same time, physiological stimuli are considered to be Key factors affecting T lymphocyte metabolism and function. Studies have shown that glucose metabolism is critical for INF-γ secretion by CD8+ effector T cells; therapeutic fecal...

Claims

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Application Information

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IPC IPC(8): A61K38/18A61K45/00A61K39/395A61K38/38A61K31/4184A61P35/00A61P37/06A61P1/00A61P1/04A61P37/02A61P29/00A61P19/02A61P25/28C12N5/0783C12N15/11
CPCA61K38/1866A61K45/00A61K39/395A61K38/38A61K31/4184A61P35/00A61P37/06A61P1/00A61P1/04A61P37/02A61P29/00A61P19/02A61P25/28C12N5/0636C12N2501/165
Inventor 程金科贺兼理王田实郑铨
Owner SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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