Abstract 3188: Functional interrogation of human T cells using the accessible Beacon DiscoveryTM optofluidic system

Abstract Adoptive cell therapies (ACTs), such as chimeric antigen receptor therapy (CAR-T) and T-cell receptor therapy (TCR-T), involve the extraction, ex vivo study, genetic engineering, and reimplantation of live T cells. These therapies have proved invaluable in our ongoing battle against cancer, with over 1,500 active clinical trials. Despite their promise, cell-based therapies are uniquely challenging to discover, validate, and translate, requiring deeper characterization compared to small molecules or other therapies. Researchers’ success at each stage depends on accessible tools that enable the measure of viability, variability, persistence, and functionality of thousands of single cells. To this end, we have implemented Bruker’s Opto® T Cell Profiling workflow on the new Beacon Discovery system and investigated on-instrument the resulting cytotoxicity, cytokine secretion, and surface marker expression for single human MART-1 T cells interacting with T cell activating beads (TABs) and antigen-presenting live cells (APCs). The BeaconTM optofluidic single-cell analysis system provides multi-dimensional characterization and recovery of live cells using Opto-Electronic Positioning (OEP) to sort live cells into NanoPens. With applications in immune cell profiling (such as T cell profiling), antibody discovery, and cell line development, these platforms are utilized by top pharma companies and backed by 30+ published studies. These same capabilities have been enabled in the more accessible Beacon Discovery system. The Beacon Discovery has integrated fluidic handling, single cell sorting, culture and cell incubation, fluorescent microscopy, and immunoassay capabilities, all in a benchtop automated, user-friendly platform. In parallel experiments on the Beacon Discovery, single human T cells were paired with TABs or APCs, where they were cultured and assayed over 2 days. When paired with APCs, target cell cytotoxicity measurements were performed via continuous perfusion of a caspase-3 substrate and time-lapse fluorescent imaging, enabling differentiation of fast-killing and slow-killing T cells. We also assessed cytokine secretion upon activation by co-loading 3 customizable cytokine capture beads in each NanoPen. Comparing T cells paired with TABs or APCs, we observed differing levels of expression for TNF-α, IL-2, and IFN-γ. We also utilized the Beacon Discovery to detect the presence of surface markers (CD4/CD8) on the T cells during co-culture with TABs or APCs. These functional measures, combined with cell recovery for sequencing, are ideal for accelerating ACT development. In summary, the Beacon Discovery optofluidic system is an approachable, easy-to-use instrument that offers an intuitive user interface and powerful data analysis and visualization software, empowering labs of all sizes to revolutionize their single-cell functional analysis. Citation Format: Patrick N. Ingram, Qiong Gao, Leqian Liu, Julie Perreau, Ritika Kukreja, Or Gadish, Brandon R. Bruhn, Minha Park, Edward Han, Alireza Salmanzadeh. Functional interrogation of human T cells using the accessible Beacon DiscoveryTM optofluidic system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3188.