Fig. 1.

Gating strategy for six-color flow cytometric detection of recent thymic emigrant (RTE), mature naive (MN), CD31⁺, and CD31⁻ memory regulatory T cells (Tregs)/responder T cells (Tresps). At first, CD4⁺ T cells (P1) were gated by side scatter characteristics (SSC) versus fluorescence intensity of CD4 (a). Then CD4⁺CD127^low+/–FoxP3⁺ Tregs (P2) and CD4⁺CD127⁺FoxP3⁻ Tresps (P3) were gated by fluorescence intensity of FoxP3 versus CD127 (b). Ki67⁺ cells of CD4⁺CD127^low+/–FoxP3⁺ Tregs (P4) and CD4⁺CD127⁺FoxP3⁻ Tresps (P5) were gated by fluorescence activity of Ki67 versus CD45RA (c and d). The percentages of RTE Tregs/Tresps (P6, P10), MN Tregs/Tresps (P7, P11), CD31⁺ memory Tregs/Tresps (P8, P12), and CD31⁻ memory Tregs/Tresps (P9, P13) were estimated by analyzing the CD4⁺CD127^low+/–FoxP3⁺ Treg pool (e) and the CD4⁺CD127⁺FoxP3⁻ Tresp pool (f) for its fluorescence intensity of CD31 versus CD45RA. The Ki67 expression of RTE Tregs/Tresps (P14, P18), MN Tregs/Tresps (P15, P19), CD31⁺ memory Tregs/Tresps (P16, P20), and CD31⁻ memory Treg/Tresps (P17, P21) were estimated by analyzing the fluorescence intensity of FoxP3 versus Ki67, respectively (g and h)