FOXP3⁺Hel-FL and FOXP3⁺Hel-Δ3B differentially mediate CD4⁺and CD8⁺eTreg suppression of T-cell proliferation. Labeled autologous target Tconv cells were cocultured at a 1:1 ratio with each eTreg cell strain or empty vector control cells with no stimulation or stimulation with anti-CD3 and anti-CD28 coated beads. After 96 hours, proliferation of target cells (CD19^– eFluor670⁺) was assayed via flow cytometry. (A) Percent suppression for each eTreg cell strain. Cells were plated as follows: 5 × 10⁴ target Tconvs alone, 5 × 10⁴ target Tconvs + 5 × 10⁴ empty vector control cells, 5 × 10⁴ target Tconvs + 5 × 10⁴ FOXP3 eTregs, 5 × 10⁴ target Tconvs + 5 × 10⁴ FOXP3⁺Hel-FL eTregs, or 5 × 10⁴ target Tconvs + 5 × 10⁴ FOXP3+ Hel-Δ3B eTregs. Tregs were either both CD4⁺ and CD8⁺ (n = 5 for each condition), CD4⁺ only (n = 7), or CD8⁺ only (n = 6). T cells from 4 different donors were used. Negative percent suppression was plotted as 0% suppression. (B) Representative dot plots of responder cell proliferation 96 hours after coculture with eTregs or empty vector control. *P ≤ .05 in each comparison based on a 1-tailed Wilcoxon test. ND, not detectable; ns, not statistically significant.