Abstract：
Antigen-presenting cells (APCs) present three activating signals to T cells engaged in physical contact: 1) antigen, 2) costimulation/corepression, and 3) soluble cytokines. T cells release two kinds of effector particles in response to activation: trans-synaptic vesicles (tSVs) and supramolecular attack particles, which transfer intercellular messengers and mediate cytotoxicity, respectively. These entities are quickly internalized by APCs engaged in physical contact with T cells, making their characterization daunting. This paper presents the protocol to fabricate and use Bead-Supported Lipid Bilayers (BSLBs) as antigen-presenting cell (APC) mimetics to capture and analyze these trans-synaptic particles. Also described are the protocols for the absolute measurements of protein densities on cell surfaces, the reconstitution of BSLBs with such physiological levels, and the flow cytometry procedure for tracking synaptic particle release by T cells. This protocol can be adapted to study the effects of individual proteins, complex ligand mixtures, pathogen virulence determinants, and drugs on the effector output of T cells, including helper T cells, cytotoxic T lymphocytes, regulatory T cells, and chimeric antigen receptor-expressing T cells (CART).
摘要：
抗原呈递细胞（APC）向参与身体接触的T细胞提供三种激活信号：1）抗原，2)共刺激/共抑制，和3)可溶性细胞因子。T细胞响应激活释放两种效应粒子：跨突触小泡（tSVs）和超分子攻击粒子，传递细胞间信使并介导细胞毒性，分别。这些实体被与T细胞物理接触的APC迅速内化，使他们的表征令人生畏。本文介绍了制造和使用珠支持的脂质双层（BSLB）作为抗原呈递细胞（APC）模拟物来捕获和分析这些跨突触颗粒的方案。还描述了用于细胞表面上蛋白质密度的绝对测量的协议，具有这种生理水平的BSLB的重建，和流式细胞术程序，用于跟踪T细胞的突触颗粒释放。该协议可以适用于研究单个蛋白质的影响，复杂的配体混合物，病原体毒力决定因素，以及对T细胞效应物输出的药物，包括辅助性T细胞,细胞毒性T淋巴细胞，调节性T细胞,和表达嵌合抗原受体的T细胞(CART)。
