Donor- and recipient-expanded Tregs significantly diminished GVHD severity. (A-H) Recipient BALB/c mice were pretreated with TL1A-Ig+IL-2_LD. An aHSCT (B6 → BALB/c) was performed on day 0 (n = 3 mice per group) after conditioning on day −1. TR colonic LP lymphocytes were assessed at day +4 (A) and +10 (M) after aHSCT. The frequencies (A) and absolute numbers (B) of FoxP3⁺RORγt⁻/CD4⁺ T cells, FoxP3⁺RORγt⁺/CD4⁺ T cells, total Tregs, and TH17/CD4⁺ T cells at day +4 are shown. The frequencies of CD4⁺, CD8⁺, and FoxP3⁺/CD4⁺ T cells from colon LP (C-E) and spleen cells (F-H) are shown. (I-J) Pretreated BALB/c recipients of B6 aHSCT exhibited improved appearance (day +28 after aHSCT) and significantly improved clinical scores, depicting reduced GVHD scores in both donor and recipient TL1A-Ig+IL-2_LD–treated groups compared with the untreated group; n = 8 for panel J. (K-L) TL1A-Ig+IL-2_LD–pretreated recipients demonstrated a preponderance of recipient Tregs in the colon and spleen. Donor (Kb⁺) and recipient (Kd⁺) Treg frequencies on day +5 after aHSCT (B6 → BALB/c) in the spleen (K) and colon (L) were analyzed. TL1A-Ig+IL-2_LD pretreatment (n = 8 mice per group) resulted in a majority and almost exclusively Tregs of recipient origin in both the spleen and colon, respectively. Notably, at day +5 after aHSCT, in contrast to the spleen (K), recipient Tregs in the colon (L) still represented most of the Treg population (L) after transplant with Treg-expanded donor cells; n = 5 for panels K-L. (M) The frequencies of CD4⁺FoxP3⁺/CD4⁺ T cells, at day +10 are shown. Data represent combined data from 3 independent B6→BALB/c transplant experiments (n = 6-9 per group). Data represent the mean ± SEM, with ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001 defining significance levels. DP, double positive (FoxP3+RORγt+); SP, single positive (FoxP3+RORγt-).