Figure 2. Naive fetal T cells have an increased propensity to differentiate into Tregs and exhibit enhanced signaling through the TGF-β pathway.

a. Flow cytometry analysis of staining for intracellular FoxP3 and surface CD25 on fetal and adult CD4⁺ T cells stimulated with 0.8 μg/mL α-CD3. Graph shows percentage of FoxP3⁺CD25⁺ cells within the CD4⁺ population following stimulation across a range of α-CD3 concentrations. b. Flow cytometry analysis of staining for intracellular FoxP3 on fetal and adult FoxP3⁺CD4⁺ T cells following stimulation as above. Graph shows FoxP3 MFI within the FoxP3⁺CD4⁺ population following stimulation. c. Flow cytometry analysis of staining for intracellular FoxP3 and surface CD25 on fetal and adult CD4⁺ T cells stimulated with 0.8 μg/mL anti-CD3 in the presence of 8 ng TGF-β. Graph shows percentage of FoxP3⁺CD25⁺ cells within the CD4⁺ population following stimulation across a range of TGF-β concentrations. d. Flow cytometry analysis of staining for intracellular FoxP3 on fetal and adult FoxP3⁺CD4⁺ T cells stimulated with 0.8 μg/mL anti-CD3 in the presence of TGF-β. Graph shows FoxP3 MFI within the FoxP3⁺CD4⁺ population following stimulation, as above. e–h. Flow cytometry analysis of staining for TGF-βRI, TGF-βRII, TGF-βRIII, and SMAD2 on fetal and adult naïve CD4⁺ T cells. Bar graphs show percentage of TGF-βRI⁺ cells and MFI of TGF-βRII, TGF-βRIII, and SMAD2 within the naïve CD4⁺ population. i. Flow cytometry analysis of staining for intracellular phosphorylated SMAD2/SMAD3 on fetal and adult naïve CD4⁺ T cells in the presence of 2 ng TGF-β. Bar graph shows phosphorylated SMAD2/SMAD3 MFI within the naïve CD4⁺ population. Panels a–d are pre-gated on cell size, live, CD3⁺CD8⁻CD4⁺ (see Supp. Fig. 1). Panels e–i are pre-gated on cell size, live, CD3⁺CD8⁻CD4⁺CD45RA⁺ (see Supp. Fig. 2). The data are representative of results from 8 fetal and 8 adult individuals in 3 independent experiments. Statistics refer to Mann-Whitney test. *** p<.001, ** p<.01, * p<.05