oIL-2 selectively expands oIL-2Rβ transduced Tregs in vitro and enhances Treg suppressive function. (A) Representative flow cytometry plot displaying CD25⁺Foxp3⁺ Treg expression (left) and frequency (right) in NT, tEGFR, and oIL-2Rβ Tregs (right). (B) Representative histogram plots of tEGFR expression (left) and frequency (right) in NT, tEGFR Tregs, and oIL-2Rβ Tregs. (C) Heatmap and hierarchical clustering of the differential gene expression from fTregs, tEGFR Tregs, and oIL-2Rβ Tregs. Genes related with activation and Treg suppression are highlighted. Expression for each gene is scaled across single rows. (D) Heatmap displaying mean fluorescence intensity (MFI) of Treg markers CTLA-4, CD25, ICOS, Lag3, and Ki67 in Tregs freshly harvested (day 0 [D0]) or tEGFR Tregs and oIL-2Rβ Tregs on day 7 after harvest. (E) Absolute number of Tregs after 4-day stimulation with WT (1000 IU/mL) or oIL-2 (100 000 IU/mL). Data are representative of 4 independent experiments. (F) Percent suppression of CD4⁺ and CD8⁺ T cells after coculture with Tregs at 1:12 Treg:Tcon ratio. (G) Representative histograms show dilution of proliferation dye on CD4⁺ T cells after coculture with Tregs at 1:12 Treg:Tcon ratio. Data are representative of 3 independent experiments. Student t test with Bonferroni correction for multiple comparisons was used for statistical analysis. Error bars indicate the standard deviation (SD) of the mean. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. CTV, CellTrace Violet; ns, no significance.