CD25⁺Foxp3⁺Treg after DR3 activation retain suppressive effects in TCR mediated T-cell proliferation. CD4⁺CD25⁺Foxp3⁺ Treg were isolated from αDR3 or isotype control Ab-treated mice and cultured with freshly isolated T cells (2 × 10⁵) from untreated mice. (A) The gating strategy for CD25⁺ Treg isolation from Foxp3-Luci4 mice. (B) T cells were tagged with CVT and cultured with CD3/CD28 beads, together with isolated Treg. (i) The representative plots of proliferating T cells tagged with CVT were shown. (ii-iii) The frequencies of proliferating (CVT ^dim) CD4⁺Foxp3⁻ (ii) and CD8⁺ T cells (iii) were analyzed by FACS (n = 6; *P < .05; ***P < .001). (C) MLR using freshly isolated T cells from nontreated WT-B6 albino mice and γ-irradiated splenocytes from Balb/c mice with or without isolated Treg was performed. [³H] thymidine incorporation was measured and analyzed (**P < .01; ***P < .001). The representative data of 2 independent experiments (each experiments were triplicate) are shown (**P < .01, ***P < .001).