Søndergaard Jonas Nørskov, Tulyeu Janyerkye, Priest David, Sakaguchi Shimon, Wing James B
Human Immunology Team, Center for Infectious Disease Education and Research (CiDER), The University of Osaka, Suita, Japan.
Laboratory of Experimental Immunology, WPI-IFReC, The University of Osaka, Suita, Japan.
Bio Protoc. 2025 Aug 20;15(16):e5424. doi: 10.21769/BioProtoc.5424.
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.
调节性T细胞（Tregs）对于通过控制其他免疫细胞的激活和扩增来维持免疫平衡至关重要。传统的抑制试验通常依赖于共培养纯化的细胞群体，这限制了多重表型分析和生理相关性。本方案描述了一种用于在外周血单核细胞（PBMC）系统中分析Treg介导的抑制作用的高维单细胞试验。首先通过细胞分选分离Tregs，然后以确定的比例重新引入自体PBMC中。使用一个包含52个标记的质谱流式细胞术（CyTOF）面板来量化多个免疫亚群中的细胞分裂和表型反应。这种方法允许对Treg功能进行综合分析，对患者分析和药物评估具有广泛的兼容性。关键特性 • 在单细胞分辨率下量化自体PBMC中Treg介导的抑制作用。 • 使用包含52个标记的CyTOF面板对多个免疫亚群进行高维表型分析和增殖跟踪。 • 通过在复杂的PBMC环境中评估抑制作用来维持生理相关性。 • 与患者来源的样本和药物扰动实验兼容，用于转化免疫学应用。
