The gating strategies for identifying FOXP3+ Tregs (panel 8).

For panel 8, the strategies of the first to fifth gates were the same as in panels 5 and 6 (Figs 4B and 5A). After gating on CD3+CD4+ T cells (G6), FOXP3 vs CD127 was used to assess the proportions of CD4+FOXP3- effector cells (G7), CD4+FOXP3+Tregs (G8) and CD4+FOXP3+bright (G9) Tregs. CD25 vs CD127 were used to assess CD25+CD127-/dim Tregs. After gating on CD4+FOXP3- effector cells (G7), CD4+FOXP3+ Tregs (G8), CD4+FOXP3+bright Tregs (G9), and CD25+CD127-/dim (G10), CD45RO vs CD39, CD45RO vs CD127, and CD45RO vs CD25 were used for assessment of CD39, CD127 and CD25 expression on memory (CD45RO+) or naïve (CD45RO-) non-FOXP3 effector T cells, and CD4+FOXP3+, CD4+FOXP3+bright, or CD25+CD127-/dim Tregs. Gating on FOXP3+ (G8) and FOXP3+bright (G9) Tregs, and gating on CD25 and CD127 identified CD25+CD127-/dim, CD25+CD127+, CD25-CD127- and CD25-CD127+ on Foxp3+/brightTregs. The histogram of CD137 and CD154 was assessed on these cell types.