*Purpose: As a subset of T lymphocytes, CD8+ T lymphocytes play a vital role in the body’s immune system.CD8+ T lymphocytes are responsible for the clearance of target cells through direct killing, such as cells infected with viruses, malignant transformed cells and allogeneic cells. And, it is also involved in the occurrence of transplant rejection. Activated CD8 + T lymphocyte-mediated granulocytosis is considered to be the most significant pathway for killing “alien cells”. The granzyme of the serine protease family member is the main granule component that enters the target cell after immunological recognition. Among them, Granzyme B (GrB) is extremely fast and effective and rich in content, which can induce cell death in multiple pathways.Detection of CD8 + T lymphocyte activation can help us understand its mechanism.

*Methods: Fluorescence spectrometry has been widely used in analytical chemistry, especially in biological analysis because of its high sensitivity and selectivity. Most biological molecules have no fluorescence or weak fluorescence and low detection sensitivity. In order to make them highly sensitive, people use strong fluorescence labeling reagents or fluorescence generation reagents to label the substances which are to be tested. Meanwhile covalent or non-covalent binding substances with high fluorescence intensity are produced, which greatly increases the detection rate. In recent years, with the development of molecular imaging, fluorescence imaging has become one of the most important method to monitor biomolecules and biological processes in living cells and living organisms, and fluorescent probes with excellent performance are the material basis for imaging analysis.

*Results: Compared with the blue or green fluorophore, the deep red or near-infrared fluorophore has stronger tissue penetration and lower spontaneous fluorescence, which is more suitable for bioimaging. Therefore, this study designed a novel near-infrared small molecular probe that can be specifically recognized and cleaved by granzyme B, using fluorescence microscope, which is the main effector of cytotoxic CD8+ T lymphocytes— Granzyme B is imaged in living cell.

*Conclusions: Acute rejection is mainly caused by cytotoxic T lymphocyte-mediated cytotoxicity, and many literatures indicate that the expression levels of perforin and granzyme b increase in the early stages of acute rejection. Therefore, granzyme b can be used as an early predictor of acute rejection, and we designed a fluorescent probe to trace granzyme b, which can more directly and dynamically observe the role of ctl activation in transplant rejection.

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