(A) Singlet live CD4 T cells were first gated on CD45RO+ (memory) cells, then FOXP3+CD25+ or FOXP3+Helios+ cells to identify memory Tregs. Right graph shows paired comparison of %FOXP3+CD25+ with %FOXP3+Helios+ memory CD4 T cells in HIV negative children, with median with interquartile range. P-value reported for Wilcoxon matched-pairs signed rank test. (B) Paired comparison of %FOXP3+CD25+ (grey) and %FOXP3+Helios+ (black) memory Treg populations in HIV-, ART-, and ART+ children. P-values for paired comparisons between %FOXP3+CD25+ and %FOXP3+Helios+ memory Tregs were calculated with Wilcoxon matched-pairs signed rank test. (C) Cells were first gated on the FOXP3+Helios+ population in CD45RO+ CD4 T cells. Within FOXP3+Helios+ memory Tregs, CD25 negative cells were identified. Aggregate %CD25 negative cells within FOXP3+Helios+ memory CD4 T cells in HIV-, ART-, and ART+ children are shown on the right. Graph shows median with interquartile range. P-value reported for two-sided Mann-Whitney test. **** indicates p<0.0001.