oIL-2Rβ Treg and oIL-2 system reduces donor T-cell expansion and activation in vivo. (A) In vivo BLI data of Tcons^luc+ from representative animals at day 3 (left) and 6 (right) after transplantation. (B) Quantification of Tcons^luc+ luminescence from each mouse (p/s) per group (n = 5). (C) Absolute number of donor (H-2k^b) CD4⁺ and CD8⁺ CD45.2⁺ cells in lymphoid tissues and spleen following transplantation. RNA-seq analysis of CD8⁺ (D) and CD4⁺ (E) Tcons recovered at day 7 after HSCT from animals treated with the indicated oIL-2Rβ (untreated, black; PBS, blue; oIL-2, green; tEGFR Treg + oIL-2, red.). (D-E) PCA of the transcriptome based on the top 1000 DEGs across all samples. (F) Proportion among CD4⁺ and CD8⁺ of naïve (CD44⁻CD62L⁺), central memory (CD44⁺CD62L⁺), and effector memory T cells (CD44⁺CD62L⁻) in the spleen of animals without (aGVHD) and with oIL-2Rβ Tregs treated with PBS and oIL-2 after day 7 following allo-HSCT and treatment. (G) Mean ± SD of the MFI of ICOS in the CD4⁺CD45.2⁺ Tcons (left) and CD8⁺CD45.2⁺ Tcons (right) in the spleen. Pooled data from 3 independent experiments including 4 to 5 mice per group per each experiment. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; and ∗∗∗∗P < .001 between indicated groups. Student t test with Bonferroni correction for multiple comparisons was used for statistical analysis.