Abstract
Regulatory T cells (Tregs) are essential for maintaining immune balance by controlling the activation and expansion of other immune cells. Conventional suppression assays often rely on co-culturing purified cell populations, which limits multiplexed phenotyping and physiological relevance. This protocol describes a high-dimensional, single-cell assay for profiling Treg-mediated suppression within a peripheral blood mononuclear cell (PBMC) system. Tregs are first isolated by cell sorting and then reintroduced into autologous PBMCs at defined ratios. A 52-marker mass cytometry (CyTOF) panel is used to quantify cell division and phenotypic responses across multiple immune subsets. This approach allows for integrated analysis of Treg function with broad compatibility for patient profiling and drug evaluation. Key features • Quantifies Treg-mediated suppression in autologous PBMCs at single-cell resolution. • Enables high-dimensional phenotyping and proliferation tracking across multiple immune subsets using a 52-marker CyTOF panel. • Maintains physiological relevance by assessing suppression in a complex PBMC environment. • Compatible with patient-derived samples and drug perturbation experiments for translational immunology applications.