Figure 1. Clonal distribution of intraepithelial CD4⁺ T cells follows single-cell trajectories.

(A-F) Tomato⁺ (library 1) and Tomato⁻ (library 2) CD4⁺ T cells from the intestinal epithelium (IE) of a iFoxp3^Tomato mouse were sorted for scRNAseq. Cells clustered into 8 (C_0–7) populations, including sub-clusters (3a, 5a) and visualized by UMAP. Cluster names correspond to colors as indicated. (A) Slingshot pseudotime trajectory of Tomato⁺ (top) and Tomato⁻ (bottom) cells on the UMAP. CD4⁺CD8αα⁺-specific slingshot lineages (black lines) depicted in differentiation order. White numbers indicate clusters. (B) Cells ordered along diffusion component 1, separated by clusters of Tomato⁺ (TP, top) and Tomato⁻ (TN, bottom) cells. (C) RNA velocity analysis vectors (arrows) of Tomato⁺ (top) and Tomato⁻ (bottom) cells on the UMAP. (D) Diversity 50 (D50) estimate based on paired αβTCRs. (E) Circos plot of paired αβTCR CDR3 sequences; clones ordered clockwise in decreasing size. Links denote clonal sharing between populations of adjacent clusters; clonal expansions of less than 10 cells in purple and at least 10 cells in green among Tomato⁺ (left) and Tomato⁻ (right) cells. (F) Top expanded clones per Tomato⁺ (TP, red and blue) and Tomato⁻ (TN, orange and green) cells. Dashed line marks top limit of CD4⁺CD8αα⁺ cluster 2. N=1 mouse, 1,294 sequenced cells. See also Figure S1, Table S1, S2.