Donor Tregs expand from the graft and are required for protection from GVHD lethality. (A) Representative histograms and (B) relative and absolute numbers of Helios-expressing donor Tregs re-isolated from recipient livers at day +10. Error bars indicate SEM. Shown are 3 mice per group from 1 of 3 independent experiments. (C) FoxP3^DTR/GFP/luc C57BL/6 albino donor mice were injected IP with DT to deplete these mice of Tregs. Dot plots are gated on live TCR-β⁺CD4⁺ T cells and show representative examples of Tcons prepared from mice treated with PBS only or DT dissolved in PBS. (D) Relative and absolute numbers of donor Tregs re-isolated from spleens of BALB/c mice at day +10. Mice received either a Treg-nondepleted or Treg-depleted BM graft with or without adoptive transfer of 5.0 × 10⁴ FVB/N third-party CD4⁺ iNKT cells. TCD-BM and third-party CD4⁺ iNKT cells derive from untreated WT C57BL/6 mice. Error bars indicate SEM. Three animals per group from 1 of 3 independent experiments are shown. (E) Overall survival of BALB/c recipient mice receiving either a Treg-nondepleted or Treg-depleted graft from C57BL/6 donor mice with or without adoptive transfer of 5.0 × 10⁴ FVB/N third-party CD4⁺ iNKT cells. TCD-BM was derived from untreated WT C57BL/6 mice. Ten mice per group except irradiation control (n = 6). Pooled data from 2 independent experiments are shown. *P ≤ .05; **P ≤ .01; ***P ≤ .001.