Quantitative detection and characterization of antigen-specific T cells are crucial to our understanding of immune responses as well as the development of new immunotherapies. Herein, we report a spatiotemporally resolved method for the detection and quantification of cell–cell interactions via Photocatalytic proXimity CELl Labeling (PhoXCELL). The biocompatible photosensitizer dibromofluorescein (DBF) was leveraged and optimized as a nongenetic alternative of enzymatic approaches for efficient generation of singlet oxygen upon photoirradiation (520 nm) on the cell surface, which allowed the subsequent labeling of nearby oxidized proteins with primary aliphatic amine-based probes. We demonstrated that DBF-functionalized dendritic cells (DCs) could spatiotemporally label interacting T cells in immune synapses via rapid photoirradiation with quantitatively discriminated interaction strength, which revealed distinct gene signatures for T cells that strongly interact with antigen-pulsed DCs. Furthermore, we employed PhoXCELL to simultaneously detect tumor antigen-specific CD8+ as well as CD4+ T cells from tumor-infiltrating lymphocytes and draining lymph nodes in murine tumor models, enabling PhoXCELL as a powerful platform to identify antigen-specific T cells in T cell receptor (TCR)-relevant personal immunotherapy.

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FucoID is an intercellular proximity labeling technique for studying cell-cell interactions (CCIs) via fucosyltransferase (FT)–meditated fucosyl-biotinylation, which has been applied to probe antigen-specific dendritic cell (DC)–T cell interactions. In this system, bait cells of interest with cell surface–anchored FT are used to capture the interacting prey cells by transferring a biotin-modified substrate to prey cells. Here, we leveraged FucoID to study CCIs directed by different molecular pairs, e.g., programmed cell death protein-1(PD-1)/programmed cell death protein-ligand-1 (PD-L1), and identify unknown or little studied CCIs, e.g., the interaction of DCs and B cells. To expand the application of FucoID to complex systems, we also synthesized site-specific antibody-based FT conjugate, which substantially improves the ability of FucoID to probe molecular signatures of specific CCI when cells of interest (bait cells) cannot be purified, e.g., in clinical samples. Collectively, these studies demonstrate the general applicability of FucoID to study unknown CCIs in complex systems at a molecular resolution.

Mitochondria are an important subcellular organelle involved in many cellular activities. Therefore, it is very important to monitor the concentration of various substances in mitochondria. In this work, we constructed...