Figure 3. Id3^lo Treg cells are highly suppressive.

(a) Heatmap of genes encoding for transcriptional regulators that are differentially (> 4-fold) expressed in splenic Id3^hi compared to Id3^lo Treg cells (CD4⁺CD25⁺CD45RB^lo) of naive Id3^GFP/+ mice. RNA was obtained from 4 independent experiments. (b) Difference in transcript abundance in Id3^hi versus Id3^lo Treg cells plotted against that in Prdm1^- versus Prdm1⁺ Treg cells [35]. Colors indicate transcripts significantly up-regulated more than 2-fold in Id3^lo (blue) or Id3^hi (red) Treg cells. The r-value of Spearman correlation is indicated. (c, d) Heatmaps of genes encoding for (c) cytokines, chemokines and their receptors and (d) genes encoding for molecules involved in Treg cell function that are differentially (> 4-fold) expressed in splenic Id3^hi compared to Id3^lo Treg cells (CD4⁺CD25⁺CD45RB^lo) of naive Id3^GFP/+ mice. RNA was obtained from 4 independent isolations. (e, f) 10⁵ CFSE labeled CD4⁺ effector T (Teff) cells (Thy1.1) were co-cultured under Th1, Th2 or Th17 cell polarizing conditions with sorted CD44^hiId3^hi (blue) or CD44^hiId3^lo (orange) Treg cells (CD4⁺CD25^hi) from IL2/IL2mAb treated Id3^GFP/+ mice (Thy1.2). (e) Representative CFSE fluorescence of Teff cells (Thy1.1⁺) cultured at the indicated Treg/Teff cell ratios and quantification of the percentage of divided Teff cells. Mean ± SEM from 3 independent experiments are shown. ^*p < 0.05; ns = not significant (paired Student’s t test). (f) Flow cytometric quantification of the percentage of Teff cells (Thy1.1⁺) expressing IFNγ, IL13 or IL17A after restimulation with PMA/Iono for 4 hours. Mean ± SEM from 3 independent experiments (Th1, Th17) or mean ± SEM from several wells of 1 representative experiment (Th2) are shown. ^*p < 0.05; ^**p < 0.01; ns = not significant (Student’s t test).