Figure 5. Helios⁺ fetal induced T_reg cells have increased FOXP3 expression.

Sorted fetal and adult naïve T cells were stimulated with αCD3/αCD28/αCD2 tetramers and IL-2 in the absence or presence of TGF-β over 1, 3 or 5 days, and analyzed by flow cytometry. The number of biological replicates for each time point and stimulation condition are specified in table S8.

(A) Representative flow cytometry plots show T_reg induction in the presence of IL-2 and TGF-β for fetal (top) and adult (bottom) naïve T cells respectively gated on live, CD4⁺ T cells.

(B) Quantification of percentage of Helios⁺FOXP3⁺ iT_reg cells gated in (A) for adult (AN) and fetal naïve (FN) T cells stimulated in the presence or absence of TGFβ. Statistics calculated using 2-way ANOVA with Tukey’s honest significant difference post-test, ***p<0.001. Error bars denote mean ± s.d.

(C) Representative flow cytometry plots shown for one fetal sample stimulated with IL-2 and TGF-β at day 1. (D) Quantification of the proportion of fetal T_reg cells in the Helios⁺ or Helios^- population as gated in (C) over 1, 3 and 5 days in the absence (left) or presence (right) of TGF-β.

(E) Quantification of FOXP3 mean fluorescence intensity for Helios⁺ and Helios^- iT_reg cells as gated in (C) across all time points in the absence (left) or presence (right) of TGF-β. Statistics for (D,E) were calculated by two-sided Wilcoxon signed-rank test, ***p<0.001, **p<0.01, *p<0.05. All boxplots show median (center line), interquartile range (box) and tenth and ninetieth percentiles (whiskers)