Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, United States of America; Cancer Biology Graduate Program, University of Michigan Medical School, Ann Arbor, MI 48109, United States of America.
Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI 48109, United States of America.
J Immunol Methods. 2022 Jul;506:113290. doi: 10.1016/j.jim.2022.113290. Epub 2022 May 27.
Immunotherapies for the treatment of cancer have spurred the development of new drugs that seek to harness the ability of T cells to recognize and kill malignant cells. There is a substantial need to evaluate how these experimental drugs influence T cell functional outputs in co-culture systems that contain cancerous cells. We describe an imaging cytometry-based platform that can simultaneously quantify activated T cells and the capacity of these T cells to kill cancer cells. Our platform was developed using the Nur77-GFP reporter system because GFP expression provides a direct readout of T cell activation that is induced by T cell antigen receptor (TCR) signaling. We combined the Nur77-GFP reporter system with a cancer cell line that displays a TCR-specific antigen and evaluated the relationship between T cell activation and cancer cell death. We demonstrate that imaging cytometry can be used to quantify the number of activated cytotoxic CD8+ T cells (CTLs) and the capacity of these CTLs to recognize and kill adherent MC38 cancer cells. We tested whether this platform could evaluate heterogenous lymphocyte populations by quantifying the proportion of antigen-specific activated T cells in co-cultures that contain unresponsive lymphocytes. The effects of a SRC family kinase inhibitor on CTL activation and MC38 cell death were also determined. Our findings demonstrate that the Nur77-GFP reporter system can be used to evaluate the effects of diverse treatment conditions on T cell-cancer co-cultures in a microtiter plate-based format by imaging cytometry. We anticipate the combined analysis of T cell activation with T cell-mediated cancer cell death can be used to rapidly assess immuno-oncology drug candidates and T cell-based therapeutics.
免疫疗法治疗癌症刺激了新药物的开发，这些药物试图利用 T 细胞识别和杀死恶性细胞的能力。在含有癌细胞的共培养系统中，评估这些实验药物如何影响 T 细胞的功能输出，具有重要意义。我们描述了一种基于成像细胞术的平台，该平台可以同时定量激活的 T 细胞和这些 T 细胞杀死癌细胞的能力。我们的平台是使用 Nur77-GFP 报告系统开发的，因为 GFP 表达提供了 T 细胞激活的直接读数，这种激活是由 T 细胞抗原受体 (TCR) 信号诱导的。我们将 Nur77-GFP 报告系统与显示 TCR 特异性抗原的癌细胞系结合，并评估了 T 细胞激活与癌细胞死亡之间的关系。我们证明成像细胞术可用于定量测定激活的细胞毒性 CD8+T 细胞 (CTL) 的数量，以及这些 CTL 识别和杀死贴壁 MC38 癌细胞的能力。我们测试了该平台是否可以通过定量测定共培养物中无反应性淋巴细胞中抗原特异性激活 T 细胞的比例来评估异质淋巴细胞群体。还确定了 SRC 家族激酶抑制剂对 CTL 激活和 MC38 细胞死亡的影响。我们的研究结果表明，Nur77-GFP 报告系统可用于通过成像细胞术评估不同治疗条件对基于微滴定板的 T 细胞-癌细胞共培养物的影响。我们预计 T 细胞激活与 T 细胞介导的癌细胞死亡的联合分析可用于快速评估免疫肿瘤学候选药物和基于 T 细胞的疗法。
