α-GalCer-lipo administration promotes both Tcon-derived Tregs and adoptively transferred Treg proliferation. WT BALB/c mice (CD45.2⁺H2^d+) received BM cell transplants from WT C57BL/6 donor mice (CD45.2⁺H2^b+) and transferred Tcons from CD45.1⁺ C57BL/6 and Tregs from CD45.2⁺Foxp3^GFP+ C57BL/6 donor mice. Spleen, liver, pLNs, and mLNs were recovered from the recipient mice on day 10 after BMT. A single-cell suspension was analyzed by flow cytometry. (A) Representative plots of CD4⁺ gated splenocytes. x-axis shows Foxp3 expression detected by intracellular staining; y-axis shows Foxp3^GFP expression. CD45.2 gated cells are shown in red; CD45.1 gated cells are shown in blue. Lower left box indicates Tcons; lower right box indicates Tcon-derived Tregs; upper right box indicates transferred Tregs. (B) Mean ± standard deviation (SD) of the proportion of Foxp3⁺/total CD4⁺ T cells in the indicated organ. Open bar indicates CD45.1⁺ Tcon-derived Tregs; filled bar indicates CD45.2⁺Foxp3^GFP+ transferred Tregs. (C) Absolute number of total CD4⁺Foxp3⁺ Tregs in the spleen. (D) Proportion among whole T cells (above) and absolute numbers (bottom) of CD4⁺ Tregs (including Tcon derived and transferred), CD4⁺Foxp3⁻ Tcons, and CD8⁺ Tcons in the spleen. Mean ± SD from pooled data of 2 independent experiments. P values calculated by Student t test for 2-group comparison and ordinary 1-way analysis of variance with Holm-Sidak correction. (E) Uniform manifold approximation and projection (UMAP) plot for T cells from concatenated data set of 1 representative experiment. CD4⁺Foxp3⁻ Tcons shown in orange; CD8⁺ Tcons shown in green; CD45.1⁺ Tcon-derived Tregs shown in blue; Foxp3^GFP+ transferred Tregs shown in red. (F) UMAP plots gated on 1 representative mouse in each group. *P < .05, **P < .01, ***P < .001, ****P < .0001.