Flow Cytometric Detection of Human Regulatory T Cells

Abstract

Tregs are absolutely required for the maintenance of self tolerance in mouse and man. Major abnormalities in Treg number or function cause rare but fatal syndromes with autoimmune, allergic and inflammatory features. Whether subtle Treg abnormalities contribute to the pathogenesis of sporadic autoimmune, allergic and immunoinflammatory diseases in man remains controversial. Robust methods for identifying and isolating human Tregs in patients and healthy controls are essential if we are to understand their role in these increasingly common diseases. We have outlined below a flow cytometric technique to detect and isolate the entire human Treg population based on expression of CD4, CD25, and CD127. Use of a number of additional antibodies for defining subsets within the Treg compartment is described. For analysis, anti-Foxp3 can be added to the cocktail, but the necessity for fixation and permeabilisation may reduce the signal from other antibodies.

Higher proportions of circulating FOXP3+ and CTLA-4+ regulatory T cells are associated with lower fractions of memory CD4+ T cells in infants

“…We found that intracellular double-staining for FOXP3 and CTLA-4 leads to suboptimal gating strategies, as the two intracellular markers influence each other. Accordingly, it was demonstrated recently that staining for more than one intracellular marker will quench other fluorochromes used [37]. Thus, in present study, data obtained from double-stained samples were not comparable with those obtained from samples stained with FOXP3 or CTLA-4 alone.…”

Chronically HIV-1 Infected Patients Exhibit Low Frequencies of CD25+ Regulatory T Cells

“…Several studies demonstrated that low or negative CD127 expression is an excellent biomarker of Treg, particularly in combination with CD25 or Foxp3. In fact, CD127 and CD25 stain allows human Treg enrichment for in vitro functional studies [23, 24]. So far, the combination of CD25, CD127 and Foxp3 expression is considered the best way to distinguish between human Treg and CD4 + Tcon [23].…”

IL6 gene allele-specific C/EBPα-binding activity affects the development of HBV infection through modulation of Th17/Treg balance

“…Treg was defined as CD3 + CD4 + CD25 + CD127 − . 52 At least 0.2 million cells were acquired and analysis using FACSDiva software (BD Biosciences).…”