Fig. 2: GPR174-deficient Tregs improve blood flow recovery after HLI.

a Gpr174 mRNA expression in B cells, CD8⁺ T cells, CD4⁺Foxp3⁻ T cells, CD4⁺Foxp3^GFP Tregs, ILC2s, neutrophils, and macrophages isolated from non-ischemic and ischemic muscle in WT mice (n = 4). b Representative immunofluorescent images of GPR174 (red), Foxp3 (green), and DAPI (blue) in CD4⁺Foxp3^GFP Tregs isolated from non-ischemic and ischemic muscle in WT mice. Scale bar, 10 μm. c Representative images of necrotic limb 7 days after Tregs adoptive transfer. d, e Representative Laser Doppler images and quantification of hindlimb blood perfusion in Rag1^−/− mice upon WT or GPR174-deficient Tregs (n = 6 for PBS → Rag1^−/− mice; n = 7 for wild-type Tregs→Rag1^−/− mice; n = 7 for GPR174-deficient Tregs→Rag1^−/− mice). f Representative immunofluorescent images of CD31(red) (upper panels) and αSMA (green) (lower panels) and DAPI (blue) staining in ischemic muscle cross sections. Scale bar, 50 μm. g Quantification of CD31 (left) and lumen perimeter (right) in Rag1^−/− mice upon WT or GPR174-deficient Tregs (n = 6 for PBS → Rag1^−/− mice: 37 arteries from 6 mice; n = 7 for wild-type Tregs→Rag1^−/− mice: 46 arteries from 7 mice; n = 7 for GPR174-deficient Tregs→Rag1^−/− mice: 47 arteries from 7 mice). h Scheme of mouse aortic endothelial cell tube formation and aortic ring assay. i, j Representative images of capillary-like structures and quantification of branch points and total tube length in mouse aortic endothelial cells cocultured with Tregs isolated from WT or Gpr174^−/Y mice for 16 h (n = 5). Scale bar, 200 μm. k, l Representative images and quantification of vascular sprouting of vessel segments cocultured with Tregs isolated from WT or Gpr174^−/Y mice for 5 days (n = 6). Scale bar, 200 μm. For all statistical plots, the data are presented as mean ± SD. Two-tailed unpaired t-tests in (a, j, l). Two-way repeated measures ANOVA with Sidak’s multiple comparisons test in (e). One-way ANOVA with Bonferroni multiple comparisons test in (g). Source data are provided as a Source Data file.