Chemistry-Enabled\nIntercellular Enzymatic Labeling\nfor Monitoring the Immune Effects of Cytotoxic T Lymphocytes <i>In Vivo</i>

Monitoring the effector function of cytotoxic T lymphocytes\n(CTLs) <i>in vivo</i> remains a great challenge. Here, we\ndevelop a chemistry-enabled\nenzymatic labeling approach to evaluate the tumor-specific immune\nresponse of CTLs by precisely monitoring the interaction between CTLs\nand tumor cells. <i>Staphylococcus aureus</i> sortase A\n(SrtA) is linked to the CTL surface through bioconjugate chemistry\nand then catalyzes the transfer of fluorescent-labeled substrate,\n5-Tamra-LPETG, to CTLs. Meanwhile, the tumor cells are specifically\ndecorated with N-terminal glycine residues (G<sub>5</sub> peptide)\nthrough the inherent glycolmetabolism of cathepsin B-specific cleavable\ntriacetylated <i>N</i>-azidoacetyl-d-mannosamine\n(CB-Ac<sub>3</sub>ManNAz) and click chemistry. After the infiltration\nof engineered CTLs into the tumor tissues, the immune-synapse-mediated\nspecific interaction of CTLs and tumor cells leads to the accurate\nfluorescent labeling of tumor cells through the SrtA-catalyzed 5-Tamra-LPETG\ntransfer. Therefore, the immune effect of CTLs as well as the performance\nof immune drugs can be determined, providing a novel strategy for\npushing ahead immunotherapy.