Granzyme B（GZMB）是细胞毒性T淋巴细胞（CTL）和自然杀伤（NK）细胞释放的关键酶，用于诱导靶细胞凋亡。我们设计了一种新型荧光生物传感器，能够以特异且敏感的方式评估GZMB活性。这个针对T细胞活性水平的剪切响应型传感器（CRSTAL）基于一种荧光蛋白，只有在GZMB或caspase-8剪切时才会被激活。CRSTAL在稳定细胞系中进行了测试，并在GZMB诱导下展现出强而持久的荧光信号。它不仅能够检测靶细胞中GZMB的过度表达，还能够检测在细胞毒性过程中由效应细胞转移的GZMB和穿孔素。这个特性对于癌症免疫治疗具有重要意义，尤其是对嵌合抗原受体（CAR）-T细胞的疗效监测。CAR-T细胞是多种癌症类型的有希望的治疗选择，但在体内监测其活性具有挑战性。CRSTAL等生物传感器的开发为CAR-T细胞活性监测提供了有价值的工具。简而言之，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，提供了评估免疫细胞的细胞毒活性和实时监测T细胞活性的手段。

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.