Systematic gating for CD25^bright aTregs.

Abstract

<p>A. First, in order to specify the upper limit of CD25 staining in conventional T-cells for each sample, the ninety-ninth percentile of PE/Cy5 fluorescence intensity within CD4⁻CD45RO⁻ cells (a population containing no Tregs) was determined as gate A. CD4⁺CD25^bright aTregs in the same sample were then quantified as those CD4⁺ cells that exceeded this value at least three-fold (gate B). B–D. The CD25^bright gate (B) contains high proportions of Foxp3+ cells in samples from SLE patients, unaffected relatives and healthy control subjects. As exemplified by an active SLE patient (panel B), an unaffected relative (panel C) and a healthy control subject (panel D), the CD25^bright gate B defined as 3xMFI(gate A) regularly contained 70–90% Foxp3+ cells within CD4+ lymphocytes when additional samples were stained for the same markers as previously but now in combination with intracellular Foxp3 staining.</p