Abstract 2191: A 50-marker mass cytometry intracellular cytokine staining panel: unprecedented resolution enables unrivaled detection of functional diversity present among human immune cell subsets

Abstract Understanding mechanisms of immune evasion to therapeutics or during responses to cancer, autoimmunity and infectious diseases requires high-dimensional functional profiling at the single-cell level. Measurement of functional immune cell signatures that include both inflammation and immunosuppression provides key insights into several facets of cancer research and therapy. If single-cell detection of cytokines spanning many cell lineages was possible, 1) determining mechanisms underlying success/failure of checkpoint blockade, 2) defining immunosuppressive activity of the tumor-resident cell subsets and 3) identifying immunological biomarkers that predict clinical outcomes would be within reach. We used CyTOF™ technology to achieve this goal of high-parameter cross-functional profiling to avoid the limitations of fluorescence-based cytometry, such as signal spillover, autofluorescence, compensation errors and spectral unmixing complications. A 50-marker CyTOF panel that achieves comprehensive profiling of immune subpopulations with detection of 20-plus intracellular cytokines spanning Th1, Th2, Th17 and Treg lineages was developed. PBMC were cultured with an array of stimulation conditions, stained and acquired on the CyTOF XT system. Datasets were analyzed using PhenoGraph clustering and visualized with opt-SNE to determine cellular functional diversity. Our findings show clear detection of IL-5-, IL-10- and IL-13 producing cells, found in surprising co-expression patterns with pro-inflammatory cytokines such as IFNγ. More significantly, we were able to detect expression of a number of secretory analytes that are historically difficult to identify by flow cytometry at a single-cell level, including the immunosuppressive cytokine TGF-β. Mass cytometry, in concert with this new expanded cytokine panel, provides a far wider lens of visualization of the functional diversity of human immune cells than has been achieved previously. We predict that through the use of this panel, novel immune regulatory mechanisms that abate/prevent cellular responses in tumors will be revealed. In sum, our findings indicate that the CyTOF XT platform is well positioned as a catalyst for seminal discoveries in immune profiling to drive therapeutic design and advanced disease monitoring in cancer. Citation Format: Laura Polanco,1 Michael J. Cohen,2 Erika L. Smith-Mahoney,1 Lauren Tracey,2 Christina Loh,2 Amedeo J. Cappione,2 Anna C. Belkina,1 David King,2 Jennifer E. Snyder-Cappione1. A 50-marker mass cytometry intracellular cytokine staining panel: unprecedented resolution enables unrivaled detection of functional diversity present among human immune cell subsets [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 2191.