CPHEN-016: Comprehensive phenotyping of human regulatory T cells

Abstract

Peripheral immunological tolerance is mainly maintained by regulatory T (Treg) cells, a specific CD4 T cells subset that expresses the transcription factor Foxp3. Treg cells are crucial to control autoimmunity and inflammation and to limit tissue destruction arising from inflammatory responses. Loss of functions mutations in FOXP3 in humans induces a fatal autoimmune lymphoproliferative disorder, known as Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX). Specific Treg cell differentiation and activation states have been linked to several human diseases. Indeed, Treg cells play a crucial role in different diseases including colitis, multiple sclerosis, autoimmunity, and infection. Characterization of Treg cell functions and understanding the role of different Treg cell subsets are crucial to the development of novel Treg cell-specific therapeutics for inflammatory diseases. In this phenotype report, we will describe laboratory methods to effectively study and characterize human Treg cells.

Keywords: flow cytometry; human; phenotype; regulatory T cells.

Figure1:. Gating strategy for T_reg cells in human PBMCs.

(A and B) Forward and side scatter (FSC and SSC) analysis of human PBMCS. (C) CD3 versus viability Dye gated on single cells. (D) CD3 versus CD4 gated on CD3⁺ Viability Dye⁻ cells. (E) Foxp3 versus CD127 staining, gated on CD3⁺CD4⁺ cells.

Figure 2:. Treg and Teff cells phenotype in Human PBMCS.

A. Flow cytometric analysis of CD25 expression on circulating Tregs cells or T effector cells in healthy controls. (B-C), Flow cytometric analysis of Treg and Teff activation status on healthy controls PBMCs, CM=Central Memory, EM= Effector Memory. D. Flow cytometric analysis of Helios expression on circulating Treg or Teff cells in healthy controls E, Flow cytometric analysis of mucosal imprinted Treg and Teff on healthy controls PBMCs.