Department of Pathology, Immunology and Laboratory Medicine, Rutgers University-New Jersey Medical School, Newark, NJ, United States; Center for Immunity and Inflammation, New Jersey Medical School, Rutgers-The State University of New Jersey, Newark, NJ, United States.
Department of Pathology, Immunology and Laboratory Medicine, Rutgers University-New Jersey Medical School, Newark, NJ, United States; Center for Immunity and Inflammation, New Jersey Medical School, Rutgers-The State University of New Jersey, Newark, NJ, United States.
Methods Cell Biol. 2023;173:155-171. doi: 10.1016/bs.mcb.2022.07.009. Epub 2022 Sep 29.
Chimeric antigen receptor (CAR)-modified cell therapy is an effective therapy that harnesses the power of the human immune system by re-engineering immune cells that specifically kill tumor cells with tumor antigen specificity. Key to the effective elimination of tumor cells is the establishment of the immunological synapse (IS) between CAR-modified immune cells and their susceptible tumors. For functional activity, CAR-modified cells must form an effective IS to kill tumor cells specifically. The formation of the CAR-specific IS requires the coordination of many cellular processes including reorganization of the cytoskeletal structure, polarization of lytic granules, accumulation of tumor antigen, and phosphorylation of key signaling molecules within the IS. Visualization and assessment of the CAR IS quality can reveal much about the molecular mechanisms that underlie the efficacy of various CAR-modified immune cells. This chapter provides a standardized method of assessing the IS quality by quantifying the tumor antigen (defining the CAR IS formation), cytoskeleton (key component of CAR IS structure), and various molecules of interest involved in the IS formation (key molecular mechanism signatures of CAR IS function) using immunofluorescence on the glass-supported planar lipid bilayer, with a focus on tumor antigen only in this study. We provide specific insights and helpful tips for reagent and sample preparation, assay design, and machine learning (ML)-based data analysis. The protocol described in this chapter will provide a valuable tool to visualize and assess the IS quality of various CAR-modified immune cells.
嵌合抗原受体 (CAR)-修饰细胞疗法是一种有效的治疗方法，通过对具有肿瘤抗原特异性的免疫细胞进行工程改造，利用人体免疫系统的力量特异性地杀死肿瘤细胞。有效消除肿瘤细胞的关键是在 CAR 修饰的免疫细胞与其易感肿瘤之间建立免疫突触 (IS)。为了发挥功能活性，CAR 修饰的细胞必须形成有效的 IS 来特异性地杀死肿瘤细胞。CAR 特异性 IS 的形成需要许多细胞过程的协调，包括细胞骨架结构的重排、溶酶体的极化、肿瘤抗原的积累以及 IS 内关键信号分子的磷酸化。CAR IS 质量的可视化和评估可以揭示各种 CAR 修饰免疫细胞的功效所基于的分子机制。本章提供了一种通过在玻璃支持的平面脂质双层上进行免疫荧光染色，定量肿瘤抗原（定义 CAR IS 的形成）、细胞骨架（CAR IS 结构的关键组成部分）和参与 IS 形成的各种感兴趣分子（CAR IS 功能的关键分子机制特征），评估 IS 质量的标准化方法，本研究仅关注肿瘤抗原。我们为试剂和样品制备、测定设计以及基于机器学习 (ML) 的数据分析提供了具体的见解和有用的提示。本章中描述的方案将为可视化和评估各种 CAR 修饰免疫细胞的 IS 质量提供有价值的工具。
