Application of autocrine vegfb in T cell metabolism and function and tumor immunotherapy
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A VEGF-B, 1. VEGF-B technology, applied in the field of biomedicine, can solve problems such as limited knowledge of antioxidant VEGF-B, and achieve the effect of promoting intake and enhancing effect
Active Publication Date: 2022-02-25
SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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Nevertheless, knowledge of the antioxidant VEGF-B is still limited
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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...
[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
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Abstract
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. The present invention discloses for the first time that vascular endothelial growth factor B (VEGF-B) is a metabolic regulation factor selectively secreted by different subtypes of activated T cells. The present invention activates T cells through antibodies or peptides, and / or uses agonists capable of promoting mitochondrial biosynthesis, or reagents that regulate mitochondria-related ROS / AP-1 signaling, and can induce activated T cells through the ROS / AP-1 signaling pathway. Cells secrete VEGF-B, which promotes fatty acid uptake, anti-apoptosis, anti-tumor activity, etc. of T cells. The invention programs the metabolism of T lymphocytes through the tumor microenvironment, and has broad application prospects in tumor immunotherapy.
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...
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