Anti-Dll4 Ab induces the expansion of thymus-derived nT_reg cells in the periphery upon immune stimulation. (A) T_reg cell expansion upon anti-Dll4 versus control Ab treatment in spleens of nonimmunized or CFA/OVA-immunized normal or thymectomized C57BL/6 mice. Fold increase in percentages (top) or absolute numbers (Abs nb; bottom) are shown. (B) Expansion of splenic T_reg cells in percentages (top) or absolute numbers (bottom) after treatment with anti-Dll4 or control Ab in WT or thymectomized C57BL/6 mice upon CFA/OVA immunization. (C) FoxP3/CD25 expression in CD4⁺ cells of CD45.2⁺ recipient or of CD45.1⁺CD4⁺FoxP3⁻CD25⁻ injected (donor) cells in C57BL/6 mice upon treatment with control or anti-Dll4 Ab for 21 d. (D) Percentages of proliferating (Ki67⁺DAPI⁺) T_reg cells in thymus of anti-Dll4– or control (Ctr) Ab–treated C57BL/6 mice at day 7 after start of treatment. (E) Expression of Helios and GITR (left) and pStat5 (day 14; middle) on thymic T_reg cells upon anti-Dll4 Ab treatment in C57BL/6 mice. T_eff cell are shown as a negative control for pStat5 expression. The gate of analysis was set with isotype control. (right) Detection of total Stat5 (tStat5) and pStat5 protein in total thymocytes and purified thymic T_reg cells upon 7 d of anti-Dll4 Ab treatment. mm, molecular mass. A–E show pooled data from two independent experiments with five mice per group. Error bars represent the mean ± SD. *, P < 0.05; ***, P < 0.001.