Figure 8. DEL-1 increases FOXP3E2 expression and the suppressive capacity of human iTregs.

(A–E) Human Tconv cells were stimulated with anti-CD3/anti-CD28 (0.1 beads/cell) in the presence of DEL-1–Fc or Fc control for 36 hours. (A and C) Representative FACS plots of FOXP3E2⁺ cells (A) or FOXP3⁺ cells (C) in CD4⁺CD25⁺ cells. (B and D) Percentage (left) and MFI (right) of FOXP3E2⁺ cells (B) or FOXP3⁺ cells (D) in CD4⁺CD25⁺ cells (n = 17 from 13 [A and B] or 11 [C and D] independent experiments). (E) Relative mRNA expression of RUNX1 (up) (n = 8 from 8 independent experiments) and CBFB (bottom) (n = 7 from 7 independent experiments) measured at 24 hours of Tconv cell stimulation during iTreg generation. (F–I) CFSE-labeled CD4⁺ T cells were cultured for 72 hours with anti-CD3/anti-CD28 (0.2 beads/cell) alone or in the presence of FACS-isolated DEL-1–Fc–iTregs or Fc control–iTregs (F and G), or long-term-cultured (10 days) DEL-1–Fc–iTregs or Fc control–iTregs (H and I). Representative FACS plots of proliferation of CFSE-labeled CD4⁺ T cells. Numbers in plots indicate percentage of CFSE dilution in CD4⁺ T cells cultured alone (top left); numbers above bracketed lines indicate percentage of CFSE dilution in CD4⁺ T cells cultured with either FACS-isolated DEL-1–Fc–iTregs or Fc control–treated iTregs (F), or long-term-cultured (10 days) DEL-1–Fc–iTregs or Fc control–iTregs (H). Percentage of iTreg suppression in the above conditions (G, n = 29 from 5 independent experiments; I, n = 12 from 4 independent experiments). (J) Methylation of CpG island of FOXP3 CNS2 evaluated in DEL-1–Fc–iTregs or Fc control–iTregs cultured for 10 days in the presence anti-CD3/anti-CD28 (0.1 beads/cell) and IL-2 (50 IU/mL) (n = 6 from 4 independent experiments). (B, D, E, and J) Lines connect paired data for each individual. (G and I) Data are means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 vs. Fc control (B, D, E, G, I, and J) by 2-tailed, paired Wilcoxon’s test (B, D, E, G, and I) or 2-tailed, paired Student’s t test (J).