CD4^dimCD25^bright treg cell frequencies above a standardized gating threshold are similar in asthmatics and controls

Background: Thymus selected CD4+CD25bright natural regulatory Treg cells expressing FOXP3 may contribute to control of immune responses. No unique markers have been available to identify and characterize Treg. We present a gating strategy that allows enumeration of Treg on the basis of CD4 and CD25 and investigate whether asthmatics have fewer Treg than controls. Methods: Asthmatics and controls were selected from responses to a mailed questionnaire. CD25, CD4, HLA DR, and appropriate isotypes were recorded by flow cytometry. Results: The CD4 T cells expressing most CD25 are a separate population expressing FOXP3 and lower levels of CD4 and CD127. On a CD4 CD25 dot-plot, the CD4 MFI of Treg for 152 participants was calculated to be 0.83 ± 0.043*MFI of CD25bright T-cells. CD4 dimCD25bright T cells in a rectangular gate with a CD4 MFI ≤ 0.9 (0.83 + [2*0.043])*MFI of CD25+ T cells were enumerated and shown to be similar for controls (median 8.34%) and asthmatics (median 10.1%). HLA DR expression on Treg correlated with CD25 expression. Conclusions: A standardized two color gating method defines Treg. It may be applied in most clinical scenarios and is useful for sorting viable Treg. Asthmatics and controls have similar numbers of Treg. © 2007 International Society for Analytical Cytology.