A 50-marker mass cytometry panel to expand analysis of the functional breadth of human immune cells

Human immune single-cell proteomic functional profiling has historically been performed with a limited number of inflammatory and/or cytotoxic readouts, capturing only a fraction of the complex orchestra of factors that comprise immune responses. Given the rising global crisis of chronic inflammation and the lack of clinically available treatment options, there is an urgent need to gain insight into the cell subsets that exhibit anti-inflammatory functional profiles and elucidate the mechanisms regulating these effector capacities. To address this, we developed a 50-marker CyTOF panel that enables unprecedented functional fingerprinting of human T cells, NK cells, monocytes, and B cells, detecting 24 intracellular targets. Healthy donor PBMCs were stimulated ex vivo and stained with this panel; from T cells, cytokines associated with the hallmark Type 1 (IFN-γ, TNF-α), Type 2 (IL-4, IL-5, and IL-13), and Type 17 (IL-17A, IL-17F) functional lineages were detected, as well as the chemokines MIP-1-α, MIP1-β, and IL-8 and the cell repair factor amphiregulin; from monocytes, IL-1β, IL-35, and IL-8 were detected. To ascertain if some of the cytokines less commonly included in Intracellular Cytokine Staining (ICS) panels were produced in response to physiological TCR stimulation via viral peptides, we measured the T cell response to a CMV-EBV-Flu (CEF) pool; in addition to TNF-α, IFN-γ, and IL-2, we also found that individual T cells produced additional cytokines with IFN-γ and TNF-α, such as amphiregulin, MIP-1α, IL-13, and IL-4. This mass cytometry panel provides an exceptionally broad and deeply resolved view of the functional diversity of human immune cells, surpassing, to our knowledge, the capabilities of previously reported approaches. Due to minimal signal overlap, CyTOF enables flexible panel customization, allowing markers and metal tags to be readily expanded or modified. Based on its resolution and adaptability, we anticipate that this panel and its derivatives will enable the discovery of novel immunomodulatory mechanisms for therapeutic intervention.