Purification, Phenotypic and Functional Analyses of Regulatory T Cells in Autoimmunity

Regulatory T cells (Tregs) are essential in maintaining immune tolerance and preventing autoimmune responses. In this chapter, we describe methods for isolating highly purified human CD4⁺ Tregs from peripheral blood mononuclear cells and synovial fluid mononuclear cells of patients with rheumatoid arthritis, as an example of autoimmune disease. We outline the methods for flow cytometry-based cell sorting to separate two distinct Treg populations (naïve and effector), their phenotypic analysis through surface and intracellular staining, and their functional characterization using an in vitro suppression assay. Additionally, we discuss strategies for expanding and genetically engineering Tregs for potential therapeutic applications in autoimmune diseases.