FIGURE 1.

Manual gating approach to classify the FOXP3⁺ T cell subpopulations. The manual gating approach to identify and classify FOXP3⁺ T cell subpopulations is depicted. (A) Representative flow cytometric data showing the three subsets of FOXP3⁺ T cells in PBMCs: CD45RO⁺FOXP3^high effector Treg, CD45RO⁻FOXP3^low naive Treg, and CD45RO⁺FOXP3^low non-Treg. These subpopulations are classified in a sequential manner using the following four gates: (B) the lymphocyte gate on the window displaying FSC and SSC, (C) the CD4⁺ gate using CD4 and SSC, (D) the FOXP3⁺ gate using FOXP3 and CD45RO, and (E) the FOXP3⁺ T cell subpopulation gate using FOXP3 and CD45RO. The level of FOXP3^high and FOXP3^low cells was determined so that CD45RO⁻FOXP3^high cells were <0.2% of CD4⁺ T cells.