CD4dimCD25bright Treg cell frequencies above a standardized gating threshold are similar in asthmatics and controls.
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.
Hoffmann
Hans Jürgen
HJ
Department of Respiratory Medicine, Aarhus University Hospital, Aarhus, Denmark. hjhof@as.aaa.dk
Malling
Tine M
TM
Topcu
Ayfer
A
Ryder
Lars P
LP
Nielsen
Kaspar R
KR
Varming
Kim
K
Dahl
Ronald
R
Omland
Oyvind
O
Sigsgaard
Torben
T
D000328:Adult; D000465:Algorithms; D001249:Asthma / Q000276:immunology* / Q000378:metabolism; D015704:CD4 Antigens / Q000032:analysis*; D016022:Case-Control Studies; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D051858:Forkhead Transcription Factors / Q000032:analysis; D006684:HLA-DR Antigens / Q000032:analysis; D006801:Humans; D007090:Image Interpretation, Computer-Assisted; D016130:Immunophenotyping / Q000379:methods*; D053645:Interleukin-2 Receptor alpha Subunit / Q000032:analysis*; D053699:Interleukin-7 Receptor alpha Subunit / Q000032:analysis; D008297:Male; D015203:Reproducibility of Results; D050378:T-Lymphocytes, Regulatory / Q000737:chemistry / Q000276:immunology*
D015704:CD4 Antigens; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; D006684:HLA-DR Antigens; D053645:Interleukin-2 Receptor alpha Subunit; D053699:Interleukin-7 Receptor alpha Subunit