αDR3 induces the selective expansion of functional Tregs. (A) T cells were isolated from mAb-treated C57BL/6 (αDR3 vs isotype) mice and stained for FoxP3. Frequencies of FoxP3⁺ Tregs in CD4⁺ T cells (left) and total Tregs numbers per C57BL/6 donor mice (right) treated with αDR3 vs isotype control mAb. Tregs number/donor = (the percentage of FoxP3⁺ of live cells) × (live cell counts) / (n of donor mice). The pooled 24-T cells isolations were analyzed (****P < .0001). (B) T cells from αDR3 or isotype mAb-treated C57BL/6 mice were stained for CD4, CD8, CD44, CD62L, FoxP3, and Ki67. SPADE analysis was performed and the representative results are shown. Node colors were scaled to FoxP3 (upper) and Ki67 (lower) expression levels. Naïve (CD62L⁺CD44^–), central memory (CD62L⁺CD44⁺), and effector memory (CD62L^–CD44⁺) phenotypes were defined by the expression of CD44 and CD62L. Note that proliferating Ki67⁺FoxP3⁺CD4⁺ Tregs were observed in T cells from αDR3-treated mice (arrow). (C-D) C57BL/6-FoxP3.Luc.DTR-4 mice were treated with αDR3 or isotype mAb (n = 5/group). (C) BLI signals from Tregs were quantitated and compared on days 4, 7, 11, and 18 by multiple Student t tests with Holm-Sidak correction (***P < .001, ****P < .0001). (D) Images at indicated days after treatment. Representative results of more than 2 experiments are displayed. (E) CD4⁺CD25⁺ Tregs were isolated from αDR3 or isotype mAb–treated C57BL/6 mice and plated at various ratios with freshly isolated T cells (2 × 10⁵/well) from nontreated C57BL/6 mice and γ-irradiated splenocytes from Balb/c mice (4 × 10⁵/well). [³H] Thymidine incorporation was measured and compared by multiple Student t tests using Holm-Sidak correction (*P < .05, **P < .01). The representative data of 2 independent experiments are shown.