Fig. 3

Coreceptor and costimulation blockade-induced transplant tolerance is predominantly mediated by intragraft proliferation of CD4⁺ Treg. a Biaxial scatter plots by t-SNE analysis showing single-cell transcriptomic clustering of ~ 1000 CD4⁺hCD2⁻ (T_H) or ~ 1000 CD4⁺hCD2⁺ (T_R) cells purified from rejecting and tolerated grafts, respectively. Cells were colored individually according to their initial expression of CD4 and hCD2 during FACS sorting. b t-SNE analysis showing single-cell transcriptomic clustering of ~ 1000 CD4⁺hCD2⁺ Treg cells during transplant rejection and tolerance, respectively. Cells were further subgrouped into specific clusters (C1-4) and colored individually according to expression patterns of specific marker genes and spatial proximity in the biaxial plot. c From selected pathways determined by GO functional annotations in terms of biological processes of each cluster (Additional file 1: Tables S4–S6), upregulated genes with an average expression level > 0.05 were displayed by the heatmap. d Scran analysis showing cell cycle phase classifications. e Immunostaining and f quantifications of Ki67⁺FOXP3⁺ cells among total FOXP3⁺ cells in rejecting and tolerated grafts. Inserts denote magnified Ki67⁺FOXP3⁺ cells. Scale bars in e: 50 μm and inserts: 10 μm. n = 6 per group, **P < 0.01. Abbreviations: R-T_H, CD4⁺hCD2⁻ Th of rejecting grafts; R-T_R, CD4⁺hCD2⁺ Treg of rejecting grafts; T-T_H, CD4⁺hCD2⁻ Th of tolerated grafts; and T-T_R, CD4⁺hCD2⁺ Treg of tolerated grafts