TNFRSF25 signaling on CD4⁺FoxP3⁺ Tregs is critical for their activation and expansion. (A) TNFRSF25 expression is required for TL1A-Ig–induced Treg expansion in vivo. Frequency of CD4⁺FoxP3⁺ Tregs in the spleens at day 0 from their respective mice (BALB/c TNFRSF25^+/+, WT and BALB/c TNFRSF25^−/−, KO) with/without TL1A-Ig+IL-2_LD. (B) Loss of suppression in the absence of the expression of TNFRSF25 followed by TL1A-Ig in vivo stimulation. Number of total splenocytes at 80 hours after culture by their respective mice, TL1A-Ig+IL-2_LD treatment, and anti-CD3 mAb (1 μg). (C-D) Spleen and LN cells (1 × 10⁷) from B6-CD45.2 TNFRSF25 KO mice were transferred IV into B6-CD45.1 WT mice. After 24 hours, treatment with TL1A-Ig with/without IL-2_LD was initiated. Peripheral and mesenteric LNs were harvested on day 6. Single-cell suspensions were prepared and Treg expansion was analyzed by flow cytometry. (C) In contrast to TNFRSF25^+/+ Tregs, TNFRSF25^−/− (KO) Tregs do not respond to TL1A-Ig treatment in vivo. TL1A-Ig+IL-2_LD treatment induced a small increase in TNFRSF25^−/− Tregs. (D) In contrast to TNFRSF25^+/+ Tregs, TNFRSF25^−/− (KO) Tregs do not express Ki-67 in response to TL1A-Ig treatment in vivo. However, TL1A-Ig+IL-2_LD treatment induced a small increase in Ki-67⁺ TNFRSF25^−/− Tregs. KO, knockout.