Decoding the mechanisms of chimeric antigen receptor (CAR) T cell-mediated killing of tumors: insights from granzyme and Fas inhibition.
Decoding the mechanisms of chimeric antigen receptor (CAR) T cell-mediated killing of tumors: insights from granzyme and Fas inhibition.
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DOI：
10.1038/s41419-024-06461-8
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发表时间：
2024-02-02
影响因子：
9
通讯作者：
Varadarajan, Navin
中科院分区：
文献类型：
--
作者：
Montalvo, Melisa J.;Bandey, Irfan N.;Rezvan, Ali;Wu, Kwan-Ling;Saeedi, Arash;Kulkarni, Rohan;Li, Yongshuai;An, Xingyue;Sefat, K. M. Samiur Rahman;Varadarajan, Navin
嵌合抗原受体（CAR）T细胞在癌症治疗中显示出希望，但其作用机制尚不清楚。解码单个T细胞使用的机制可以帮助提高T细胞的功效，同时还可以识别T细胞失败导致肿瘤逃逸的机制。在这里，我们使用了一套检测方法，包括细胞-细胞相互作用的动态单细胞成像，荧光报告分子的动态成像，以直接跟踪肿瘤细胞中的细胞毒素活性，以及患者输注产品上的scRNA-seq，以研究单个CAR T细胞在杀死肿瘤细胞中使用的细胞毒性机制。令人惊讶的是，颗粒酶B（GZMB）抑制剂、蛋白酶抑制剂-9（PI 9）的过表达不改变由CD 19特异性CAR T细胞介导的针对白血病细胞系NALM 6或卵巢癌细胞系SkOV 3-CD 19的细胞毒性。我们设计并验证了报告基因以直接测定肿瘤细胞中T细胞递送的GZMB活性，并证实虽然PI 9过表达在分子水平上抑制GZMB活性，但这不足以影响CAR T细胞介导的杀伤的动力学或幅度。改变由CAR T细胞介导的细胞毒性需要联合抑制肿瘤细胞特异性的多种途径：（a）B细胞系如NALM 6、Raji和Daudi对GZMB和颗粒酶A（GZMA）的联合抑制敏感;而（B）实体瘤靶标如SkOV 3-CD 19和A375-CD 19（黑素瘤）对GZMB和Fas配体的联合抑制敏感。我们通过检查Tisa-cel和Axi-cel输注产品的scRNA-seq谱实现了这些发现的翻译相关性，并以T细胞亚群依赖性方式在单细胞水平上显示了GZMB和GZMA表达之间的显著相关性。我们的研究结果强调了冗余在CAR T细胞杀伤机制中的重要性，以及这种冗余对有效T细胞的重要性。
Chimeric antigen receptor (CAR) T cell show promise in cancer treatments, but their mechanism of action is not well understood. Decoding the mechanisms used by individual T cells can help improve the efficacy of T cells while also identifying mechanisms of T cell failure leading to tumor escape. Here, we used a suite of assays including dynamic single-cell imaging of cell-cell interactions, dynamic imaging of fluorescent reporters to directly track cytotoxin activity in tumor cells, and scRNA-seq on patient infusion products to investigate the cytotoxic mechanisms used by individual CAR T cells in killing tumor cells. We show that surprisingly, overexpression of the Granzyme B (GZMB) inhibitor, protease inhibitor-9 (PI9), does not alter the cytotoxicity mediated by CD19-specific CAR T cells against either the leukemic cell line, NALM6; or the ovarian cancer cell line, SkOV3-CD19. We designed and validated reporters to directly assay T cell delivered GZMB activity in tumor cells and confirmed that while PI9 overexpression inhibits GZMB activity at the molecular level, this is not sufficient to impact the kinetics or magnitude of killing mediated by the CAR T cells. Altering cytotoxicity mediated by CAR T cells required combined inhibition of multiple pathways that are tumor cell specific: (a) B-cell lines like NALM6, Raji and Daudi were sensitive to combined GZMB and granzyme A (GZMA) inhibition; whereas (b) solid tumor targets like SkOV3-CD19 and A375-CD19 (melanoma) were sensitive to combined GZMB and Fas ligand inhibition. We realized the translational relevance of these findings by examining the scRNA-seq profiles of Tisa-cel and Axi-cel infusion products and show a significant correlation between GZMB and GZMA expression at the single-cell level in a T cell subset-dependent manner. Our findings highlight the importance of the redundancy in killing mechanisms of CAR T cells and how this redundancy is important for efficacious T cells.
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