After 3.3 Gy TBI, CD45.2⁺H2^d+ WT BALB/c mice received Luc⁺CD45.1⁺H2^b+ C57BL/6 BMCs (15 × 10⁶) plus CD45.2⁺H2^d+Foxp3^GFP+ Tregs (1 × 10⁶) that were transduced with oIL-2Rβ (oTreg: transduction efficiency was ~30%) or those expanded without viral vector (UT Tregs). All mice were treated with anti-CD40L (0.3 mg i.p. on d0), followed by i.p. administration of PBS, WT IL-2 MSA, or ortho IL-2 MSA (both IL-2, 25,000 IU/d) for 14 days. Flow cytometry analysis for PBMCs obtained from the tail vein was performed on d14. (A) Representative pseudocolor plots gated by CD45.2⁺H2k^d+CD4⁺ T cells. (B) Box plots showing the percentages of Foxp3^GFP+CD4⁺ (top) and CD8⁺CD45.2⁺ cells (bottom). (C) Box and whiskers with minimum to maximum showing percentages of H2Κ^b+CD45.1⁺ donor cells in PBMCs on d14. Horizontal dotted line indicates 1% of donor cells. ⁺⁺⁺P < 0.001, comparing the mean rank of each column to the mean rank of PBS control without Tregs (gray triangle) by Dunn’s Kruskal-Wallis multiple comparisons. (D) Representative bioluminescent images for mice that received oTregs showing distribution of engrafted BMCs. Box plots showing total photon flux on d4 (E), d10, and d28 (F). Pooled data from 2 independent experiments including 5 mice per group per each experiment. *P < 0.05; **P < 0.01; ***P < 0.001, between indicated 2 groups calculated by Dunn’s Kruskal-Wallis multiple comparisons among the 3 treatment groups or by Mann-Whitney U test between 2 groups.