(A) Quantification of T cell subsets in allo- or isografts at d6 (n = 4), d9 (n = 5) ,and d12 (n = 5) after transplantation. (B) t-SNE representations of concatenated single-cell cytometry data from allo- and isografts across all time points. Top, colors/clusters represent distribution of indicated cell phenotype. Bottom,distribution of OX40-expressing cells. (C) Subset t-SNE plots for allo- or isografts at d6, d9, or d12. Red indicates OX40-expressing cells. Arrows indicate 2 primary clusters associated with OX40 expression. (D) Quantification of the number of OX40⁺ cells. (E) Correlation of OX40 with T cell phenotype. Color and circle size provide a graphical representation of r² correlation value. (F) Relative difference in the number of cells of each T cell phenotype between allograft and isograft on d9. The y axis represents fold change (Allo/Iso). (G) Fluorescent intensity (MFI) and frequency of OX40 expression on principle CD4 T cell subsets in allo- or isografts over time. (H) Representative biaxial cytometry plots of OX40 versus Foxp3 expression in allo- or isografts. (I) The proportion of Foxp3^–CD4⁺ (red), Foxp3⁺CD4⁺ (orange), and CD8⁺ T cell (yellow) phenotype among total graft-infiltrating T cells. Sections with shaded lines represents OX40⁺ cell frequency in each population. Each compartment represents average proportion of n = 4–5 samples for indicated time point. (J) Immunofluorescent microscopy images depicting infiltration of CD4⁺ T cells (red) and Foxp3⁺ cells (green) in areas surrounding and within allo- and isografts at d9. High-magnification images are of indicated insets. (K) Quantification of Foxp3⁺CD4⁺ (top) and total CD4⁺ (bottom) cell count within grafts per 200μm² (n = 3) on d9. *P < 0.05, **P < 0.01, ***P < 0.001 calculated with Mann-Whitney U test between allograft and isograft. All data are representative of at least 2 independent experiments.