Tregs expand from the Tcon inoculum and are required for protection from GVHD. (A) Expression of FoxP3 and Helios in live splenic Thy1.1⁺CD4⁺ T cells. Shown are representative dot plots from 1 of 3 independent experiments. (B) Total numbers of live splenic Helios^–FoxP3⁺ and Helios⁺FoxP3⁺ Thy1.1⁺CD4⁺ T cells. Shown is 1 of 3 independent experiments. (C) Expression of CD25 and FoxP3 in live CD4⁺ T cells after intraperitoneal injection of 50 µg⋅kg⁻¹ diphtheria toxin (DT) into FoxP3^DTR C57BL/6 donor mice on 2 consecutive days. Shown are representative dot plots from 1 of 2 independent experiments. (D) Relative and (E) absolute numbers of donor Thy1.1⁺ Tregs re-isolated from BALB/c recipient mice that received either Treg-nondepleted (left bars) or Treg-depleted grafts (right bars), respectively. Population gated on live Thy1.1⁺CD4⁺ T cells. Shown are 3 animals per group from 1 of 2 independent experiments. (F) Pooled survival data from 2 independent experiments of BALB/c mice treated with CD4⁺ iNKT cells in the presence and absence of donor Tregs. Ten animals per group except irradiation control (n = 6). Error bars indicate standard error of the mean. *P ≤ .05; **P ≤ .01.