细胞生物学
生物
T细胞
流式细胞术
免疫系统
T细胞受体
细胞毒性T细胞
人口
获得性免疫系统
抗原提呈细胞
外周血单个核细胞
抗原
免疫学
作者
Katherine Pohida,Camille Lake,Debra Yee,Andrew Snow
出处
期刊:Bio-protocol
[Bio-Protocol]
日期:2022-01-01
卷期号:12 (4)
标识
DOI:10.21769/bioprotoc.4326
摘要
When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immune system relies upon clonal expansion of antigen-specific T cell populations to exercise acquired effector and cytotoxic functions to clear it. However, T cell expansion must be modulated to effectively combat the perceived threat without inducing excessive collateral damage to host tissues. Restimulation-induced cell death (RICD) is an apoptotic program triggered in activated T cells when an abundance of antigen and IL-2 are present, imposing a negative feedback mechanism that constrains the growing T cell population. This autoregulatory process can be detected via increases in caspase activation, Annexin V binding, and loss of mitochondrial membrane potential. However, simple changes in T cell viability through flow cytometric analysis can reliably measure RICD sensitivity in response to T-cell receptor (TCR) restimulation. This protocol describes the in vitro polyclonal activation, expansion, and restimulation of human primary T cells isolated from donor peripheral blood mononuclear cells (PBMC). This simple procedure allows for accurate quantification of RICD via flow cytometry. We also describe strategies for interrogating the role of specific proteins and pathways that may alter RICD sensitivity. This straightforward protocol provides a quick and dependable tool to track RICD sensitivity in culture over time while probing critical factors that control the magnitude and longevity of an adaptive immune response. Graphic abstract: In-vitro simulation of restimulation-induced cell death in activated human T cells.
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