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Abstract Although tens of immune synapse (IS) protein species are known, there remain many unknown IS-localized protein species. Understanding the proteome of the IS between a target cell and a lymphocyte is crucial for advancing immuno-oncology. The low abundance of ISs and the lack of a definitive enrichment marker have limited efficient proteomic profiling. Here, we used Microscoop™, an innovative system that integrates microscopy, machine learning, and targeted photo-biotinylation to enable precise and spatially specific enrichment of IS proteins, thereby facilitating proteomic discovery of the IS. We utilized Raji B cells as antigen-presenting cells (APCs) and induced IS formation with Jurkat T cells. The system first employed immunofluorescence imaging of CD3, a common IS marker in Jurkat T cells, and used a convolutional neural network-based deep learning algorithm to train the recognition of ISs from CMTPX-stained Raji B cells. Our automated system then successfully achieved spatially-targeted biotin-tagging of proteins at ISs through thousands of fields of view of imaging, deep learning-driven pattern generation, and photochemical labeling. Subsequent streptavidin pull-down and mass spectrometry analysis enabled the identification of IS-specific proteins. Remarkably, our spatial proteomic approach led to the isolation and identification of hundreds of different species at the IS interface, including proteins associated with key components of T-cell receptor (TCR) signaling pathways such as the TCR/CD3 complex, Src and Tec family tyrosine kinases, and pivotal NF-kB signaling proteins. Beyond these, we also identified a significant enrichment of proteins not previously associated with the IS. Our study not only illuminates previously unknown aspects of immune regulation at the IS interface, but also shedded light on proteins potentially important for immuno-oncology. These insights have significant implications for cancer research, particularly in understanding and manipulating immune responses for therapeutic purposes. Citation Format: Weng-Man Chong, Cecile Li, Chun-Kai Huang, Chantal Hoi Yin Cheung, Hsiao-Jen Chang, Chia-Wen Chung, Jung-Chi Liao. Identification of the novel immune synapse-localized proteome for immuno-oncology using Microscoop-induced targeted photo-biotinylation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 1043.
Chong et al. (Fri,) studied this question.