Tu149 - A Novel 50-marker Intracellular Staining Panel Enables Unrivaled Detection of Functional Diversity Among Human Immune Cell Subsets

Abstract Text: The inflammatory functions of immune cells are well studied in clinical and translational immunology. However, the active anti-inflammatory and immunosuppressive axes have been grossly understudied; this gap negatively impacts discovery of therapeutic targets and drug development. Mass cytometry, an orthogonal platform for multidimensional single-cell analysis, avoids limitations of fluorescence-based cytometry such as signal spillover, autofluorescence and spectral unmixing data reduction. Understanding that mass cytometry would prove superior for detection of intracellular analytes and be ideal for use in downstream multivariate data analyses, we developed a 50-marker CyTOF™ panel with comprehensive profiling of immune subsets and 20-plus intracellular cytokines spanning major functional lineages. PBMC were stimulated using multiple conditions, stained and acquired on the CyTOF XT system. Datasets were analyzed using PhenoGraph clustering and visualized with opt-SNE to determine cellular diversity. Our findings showed a diverse array of functional signatures, including clear detection of IL-10- and IL-13-producing cells, and further revealed surprising co-expression patterns with the pro-inflammatory cytokine IFN. More significantly, we were able to detect expression of several secretory analytes not easily or previously identified by fluorescence cytometry, including the immunosuppressive cytokine TGF-. The CyTOF panel provides the widest visualization of functional diversity of human immune cells ever measured to our knowledge, and achieves this without additional controls, complicated panel optimization that occurs with large fluorescent panels or sacrificing signal resolution. Our work reveals novel immune regulatory mechanisms that may spur new avenues for significantly improving treatment of chronic inflammation and augmentation of vaccine responses.