DR3 activation before alloantigen exposure promotes recipient-derived Treg expansion and prevented acute GVHD. (A) TCD-BM from WT B6 mice were transplanted into lethally irradiated Balb/c recipients on day 0, and T cells from B6-luc mice (1 × 10⁶/animal) were injected on day 2 after transplant to induce acute GVHD. αDR3 or isotype control Ab was injected on day 0. (B) Splenic T cells were isolated on day 7 after transplant and the frequencies of Foxp3⁺ cells in donor- or recipient-derived CD4⁺ T cells were analyzed by FACS. (i) Representative dot plot data of recipient-derived CD4⁺T cells were shown. (ii) The summary of statistical analysis were shown (n = 7; n.s. = not significant; **P < .01). (C) Recipient-derived Treg proliferation with or without D0 αDR3 treatment was assessed using lethally irradiated (9.5 Gy) Foxp3-Luci4 mice as recipients. Representative images on day 7 are shown (i). Summary of BLI (ii) (n = 10; *P < .05). (D) Donor T-cell proliferation (from B6-luc mice) in the recipient mice (WT Balb/c mice) with or without D0 αDR3 treatment was assessed by BLI. Representative images on day 7 are shown (i). Summary of BLI is shown in (ii) (n = 10; *P < .05). (E-F) GVHD score and survival curve are shown (*** P < .001; ****P < .0001).