Figure 1. Identification of Tregs.

PBMCs were stained ex vivo for CD3, CD4, CD25, Foxp3, CD152, and CD127. Lymphocytes were gated and dead cells excluded. (A) The Foxp3 cell gate was set to exclude Foxp3^– and CD25^– events within CD3⁺CD4⁺ cell populations. CD152⁺CD127^– events were measured in CD25⁺Foxp3^+lo (52%) and CD25⁺Foxp3^+hi cells (95%) and in (B) CD25^neg (4%), CD25^int (5%), and CD25^hi cells (63%). Levels of CD152, CD127, and Foxp3 staining were measured in CD25^neg, CD25^int, and CD25^hi populations using selected gates. (C) CD25^hi cells were 68.9% CD152⁺, 14.3% CD127^–, and 80.7% Foxp3⁺, compared with respective values of 13.2%, 89.9%, and 3.7% for CD25^int cells and 8.6%, 79.8%, and 0.6% for CD25^neg cells. Results are from 1 representative of 3 normal human controls tested in 4 independent experiments. (D) Agarose gel with DNA stained with ethidium bromide. Expression of Foxp3 in CD4⁺CD25⁺ T cells compared with PBMCs or CD4⁺CD25^– T cells, normalized for RNA input using β-actin primers (gel representative of 3 different experiments).