PDF(Pubmed)
Abstract：
Granzyme B (GZMB) is a key enzyme released by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells to induce apoptosis in target cells. We designed a novel fluorogenic biosensor which is able to assess GZMB activity in a specific and sensitive manner. This cleavage-responsive sensor for T cell activity level (CRSTAL) is based on a fluorescent protein that is only activated upon cleavage by GZMB or caspase-8. CRSTAL was tested in stable cell lines and demonstrated a strong and long-lasting fluorescence signal upon induction with GZMB. It can detect GZMB activity not only by overexpression of GZMB in target cells but also following transfer of GZMB and perforin from effector cells during cytotoxicity. This feature has significant implications for cancer immunotherapy, particularly in monitoring the efficacy of chimeric antigen receptor (CAR)-T cells. CAR-T cells are a promising therapy option for various cancer types, but monitoring their activity in vivo is challenging. The development of biosensors like CRSTAL provides a valuable tool for monitoring of CAR-T cell activity. In summary, CRSTAL is a highly sensitive biosensor that can detect GZMB activity in target cells, providing a means for evaluating the cytotoxic activity of immune cells and monitoring T cell activity in real time.
摘要：
颗粒酶B（GZMB）是细胞毒性T淋巴细胞（CTL）和自然杀伤（NK）细胞释放的一种关键酶，可诱导靶细胞凋亡。我们设计了一种新型荧光生物传感器，能够以特异性和灵敏的方式评估GZMB活性。这种用于T细胞活性水平（CRSTAL）的切割响应传感器基于荧光蛋白，该荧光蛋白仅在GZMB或caspase-8切割后才被激活。在稳定的细胞系中测试CRSTAL,并且在用GZMB诱导时显示强且持久的荧光信号。它不仅可以通过靶细胞中GZMB的过表达来检测GZMB活性，而且可以在细胞毒性过程中从效应细胞转移GZMB和穿孔素后检测GZMB活性。这一特征对癌症免疫疗法具有重要意义。特别是在监测嵌合抗原受体（CAR）-T细胞的功效。CAR-T细胞是各种癌症类型的有前途的治疗选择，但是监测它们在体内的活动是具有挑战性的。像CRSTAL这样的生物传感器的开发为监测CAR-T细胞活性提供了有价值的工具。总之,CRSTAL是一种高灵敏度的生物传感器，可以检测靶细胞中的GZMB活性，提供了一种评估免疫细胞的细胞毒活性和实时监测T细胞活性的方法。
