Figure 3. DEL-1 directly promotes de novo Treg differentiation via its RGD motif.

(A and B) Naive splenic CD4⁺ cells were differentiated, or not, into Th17 cells under pathogenic conditions (see Methods) in the presence of DEL-1–Fc or Fc control. (A) FACS plots and (B) percentage of IL-17A⁺ cells in CD4⁺ T cells (n = 6 replicates). (C–E) Naive splenic CD4⁺ cells were differentiated, or not, into Tregs in the presence of DEL-1–Fc, DEL-1[RGE]–Fc, or Fc control (10 μg/mL). (C) FACS plots and (D) percentage of FOXP3⁺ cells in CD4⁺ T cells (n = 7 replicates). (E) Relative mRNA expression of Foxp3 in Tregs (n = 6 replicates). (F) Suppression of CFSE-labeled CD4⁺CD25^– T cell (Tconv) division by purified DEL-1–Fc−iTregs or Fc-iTregs. Numbers on x axis indicate CD4⁺CFSE⁺cell/iTreg ratio (n = 6 replicates). (G) Naive splenic CD4⁺ T cells were differentiated into Tregs in the presence of DEL-1–Fc or Fc control (10 μg/mL). CD4⁺CD25⁺ cells were sorted and restimulated for 4 days in medium containing IL-2 (40 ng/mL) and FOXP3 expression was assessed. (H and I) Naive splenic CD4⁺ cells were differentiated into Tregs. CD4⁺CD25⁺ cells were sorted and restimulated for 4 days with DEL-1–Fc or Fc control in medium containing IL-2 (40 ng/mL) without (H) or with TGF-β1 (5 ng/mL) (I), and FOXP3 expression was assessed. (J) Naive splenic CD4⁺ T cells were differentiated for 4 days to Tregs in the presence of DEL-1–Fc or Fc control (10 μg/mL). iTregs (CD4⁺CD25⁺) were sorted and evaluated for their methylation status of the Foxp3 CNS2 (n = 9 mice). All CD4⁺ T cells were isolated from WT mice. Data are means ± SD and are pooled from 2 (B and D–I) or 5 (J) independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Fc control (B and D–J) by 1-way ANOVA with Dunnett’s post-test for comparison with Fc control (B, D, and E), 2-way ANOVA with Holm-Šidák post hoc test for comparison with Fc control (F), or 2-tailed Student’s t test for comparison with Fc control (G–J). NS, not significant.