嵌合抗原受体（CAR）T细胞疗法是一种工程细胞疗法，其中T细胞被分离并遗传修饰，以含有对肿瘤细胞抗原具有特异性的人工合成CAR。当抗原结合时，CAR T细胞将启动信号级连反应，导致相关肿瘤细胞的溶解。细胞因子释放综合征（CRS）是与CAR T细胞疗法关联的主要毒性问题，并且在当前可用的商业产品中仍然是一个突出的安全问题。CRS是由CAR T细胞与内源性单核细胞和巨噬细胞的相互作用驱动的，这可能导致免疫细胞过度活化，并且使某些细胞因子达到超生理水平。在进行人类试验之前，应在临床前模型中评估任何给定CAR构建体引发的毒性潜力。虽然有适用于此目的的活体小鼠模型，但这些常常是复杂的异种移植模型，只有少数中心有供应。因此，有必要开发一种体外测定方法，以测量CAR T细胞引发细胞因子释放综合征的潜力。本文介绍的测定方法是评估任何给定CAR构建体通过肿瘤细胞和单核细胞相互作用产生潜在的细胞因子释放综合征的倾向的临床前工具。该文章提供了详细的靶细胞制备步骤以及从外周血单个核细胞（PBMCs）体内制备单核细胞（monocytes）的步骤，以及在96孔板中播种三种细胞类型的步骤以及通过酶联免疫吸附测定（ELISA）或多重贡献珠法分析产生的细胞因子的收集与分析。© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. 基本方案1：K562靶细胞的制备基本方案2：从同种PBMCs中分离单核细胞基本方案3：在96孔板中播种CAR T细胞、单核细胞和K562细胞基本方案4：通过单细胞因子ELISA分析共培养上清液备选方案：通过多重细胞因子珠法分析共培养上清液。© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

Chimeric antigen receptor (CAR) T cell therapy is an engineered cell therapy where T cells are isolated and genetically modified to contain a synthetic CAR with specificity to a tumor cell antigen. Upon antigen binding, the CAR T cell will initiate signaling cascades that result in lysis of the associated tumor cell. Cytokine release syndrome (CRS) is the primary toxicity associated with CAR T cell therapy and remains a prominent safety issue with currently available commercial products. CRS is driven by interaction of the CAR T cells with endogenous monocytes and macrophages, which can lead to immune cell overactivation and an increase in certain cytokines to supraphysiological levels. Identifying the potential of any given CAR construct to drive toxicities in vivo should be assessed in preclinical models prior to human trials. While there are in vivo mouse models available for this purpose, these are often complex xenograft models available in few centers. Thus, there is a need to develop an in vitro assay for measuring the CRS potential of CAR T cells. The assay described here is a preclinical tool for assessing the propensity of any given CAR construct to produce potentially CRS-driving cytokines following tumor cell and monocyte interactions. This article provides a detailed protocol for target cell preparation and isolation of monocytes from peripheral blood mononuclear cells (PBMCs) autologous to the CAR T cells, as well as protocols for seeding the three cell types in a co-culture assay and collecting/analyzing the cytokines produced via an ELISA or multiplex bead array. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of K562 target cells Basic Protocol 2: Isolation of monocytes from autologous PBMCs Basic Protocol 3: Seeding of CAR T cells, monocytes, and K562 cells in 96-well plates Basic Protocol 4: Analysis of co-culture supernatants by single-cytokine ELISA Alternate Protocol: Analysis of co-culture supernatants by multiplex cytokine bead array.© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.