(A) Schematic of in vivo model for mixed chimerism induction. After 3.3 Gy TBI, CD45.2⁺Thy1.1⁺H2^d+ BALB/c mice received Luc⁺CD45.1⁺Thy1.1⁺H2^b+ C57BL/6 BMCs (15 × 10⁶) plus CD45.2⁺Thy1.2⁺H2^d+Foxp3^GFP+ Tregs (1 × 10⁶) that were transduced with oIL-2Rβ (oTreg: transduction efficiency was about 30%). All mice were treated with anti-CD40L (0.3 mg i.p. on d0) followed by i.p. administration of PBS (n = 7), WT IL-2 MSA 25,000 IU/d (n = 7), or ortho IL-2 MSA 25,000 IU/d (n = 7) for 6 days. Spleen, blood, and peripheral lymph nodes were recovered and analyzed by flow cytometry on d6. (B–D) Ortho IL-2 administration increases the population (B) and expression levels of CD25 and ICOS (C and D) on tRFP-positive fraction (brown gate), but not on tRFP-negative fraction (black) of oTregs in the spleen. (E) Box plots showing relative cell number normalized by PBS control mice in Thy1.2⁺Foxp3^GFP+ with (tRFP⁺ oTreg. top) or without (tRFP^– oTreg, middle) IL-2Rβ expression, and Thy1.1⁺ T cells (host T cells, bottom). (F and G) Ortho IL-2 administration increases oTreg population without affecting Thy1.1⁺ T cells. Representative pseudocolor plots of CD45.2⁺ splenocytes (F) and box plots (G). (H) The percentage of oTregs in CD45.2⁺CD4⁺ T cells in total body blood collected from the inferior vena cava and peripheral lymph nodes. Left, representative pseudocolor plots in blood. Right, comparison between PBS- and ortho IL-2–treated mice. Pooled data from 2 independent experiments including 3 to 4 mice per group per each experiment. *P < 0.05; **P < 0.01; ***P < 0.001, between indicated groups calculated by Mann-Whitney U test for 2-group comparison, Dunn’s Kruskal-Wallis test for multiple group comparison. TBI, total body irradiation; CD40L, CD40 ligand.