BACKGROUND: Thymus selected CD4(+)CD25(bright) 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 CD25(bright) T-cells. CD4(dim)CD25(bright) 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.
BACKGROUND: Thymus selected CD4(+)CD25(bright) 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 CD4CD25 dot-plot, the CD4 MFI of Treg for 152 participants was calculated to be 0.83 +/- 0.043*MFI of CD25(bright) T-cells. CD4(dim)CD25(bright) 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.
Authors: Laure Vallotton; Karine Hadaya; Jean-Pierre Venetz; Leo H Buehler; Donatella Ciuffreda; Ghaleb Nseir; Laura Codarri; Jean Villard; Giuseppe Pantaleo; Manuel Pascual Journal: Clin J Am Soc Nephrol Date: 2011-07-14 Impact factor: 8.237