Extended Data Fig. 5: Therapeutic effect of uPAR-targeting CAR T cells on intestinal crypts.

a-q, Young (3 months) and old (18 months) mice were treated with 0.5x10^6 untransduced T cells (UT) or uPAR CAR T cells (m.uPAR-m.28z). Mice were harvested 6 weeks after infusion and scRNAseq was performed from whole small intestine (n=4 mice per group pooled into 2 replicates) a, UMAP visualization of small intestinal cell types in young and old mice treated with 0.5x10^6 UT or m.uPAR-m.28z cells generated by 10X chromium protocol. Colors indicate the 12 different identified populations. b, Dot plot showing the 42 signature gene expressions across the 12 cellular clusters. The size of the dots represents the proportion of cells expressing a particular marker, and the color scale indicates the mean expression levels of the markers (log1p transformed). c, Schematic for d-i where the transcriptome of old m.uPAR-m.28z-treated mice was compared to that of old UT treated animals. d, Volcano plot of differentially expressed genes between old mice treated with UT or m.uPAR-m.28z cells. x-axis displays log2 fold change while y axis is the -log10 adjusted p-value as calculated by MAST. e, UMAP visualization from pseudotime trajectory analysis of cells from UT or m.uPAR-m.28z-treated old mice. Arrows highlight predicted trajectories. Color scale represents pseudotime. f, Density plot from pseudotime trajectory analysis demonstrating density differences along pseudotime of all cells between UT or m.uPAR-m.28z-treated old mice. g, Line plot showing the expression levels of stemness signature score in UT or m.uPAR-m.28z-treated old mice along the indicated pseudotime axis. h, Dot plot showing Log2 fold change in the functional scores for the different terms across cell types of old mice treated with UT or m.uPAR-m.28z cells. i, Heatmap representing log 2FC in gene expression between old m.uPAR-m.28z and UT treated mice from h. j, Schematic of k-q where the transcriptome of young mice treated with m.uPAR-m.28z CAR T cells or UT cells for 6 weeks was compared. k, UMAP visualization of small intestinal cell types generated by 10X chromium protocol. Color scale indicates difference in localized cellular density between m.uPAR-m.28z and UT treated young mice 6 weeks after treatment. l, Fraction of cells for each of the different cell types shown in (k) in young mice treated with UT or m.uPAR-m.28z cells for 6 weeks. m, UMAP visualization of small intestinal cell types generated by 10X chromium protocol. Color scale indicates log2FC differences in stemness signature score between m.uPAR-m.28z and UT treated young mice 6 weeks after treatment. n, Split-violin plot indicates the expression level of 5 stem-related genes in the stem cells from young UT and m.uPAR-m.28z-treated mice after 6 weeks. Boxplots display median (center line) and interquartile range (box).o, Volcano plot of differentially expressed genes between young mice treated with UT or m.uPAR-m.28z cells after. x-axis displays log2 fold change while y axis is the -log10 adjusted p-value as calculated by MAST. p, Density plot from pseudotime trajectory analysis demonstrating density differences along pseudotime of all cells between UT or m.uPAR-m.28z-treated young mice after 6 weeks. q, Dot plot showing Log2 fold change in the functional scores for the different terms across Paneth, goblet, enteroendocrine and enterocytes of young mice treated with UT or m.uPAR-m.28z cells after 6 weeks. r, Experimental scheme for s-t: Young (3 months) and old (18 months) mice were treated with 0.5x10^6 UT or uPAR CAR T cells. Mice were harvested 8 weeks after infusion and organoids were generated from their intestinal crypts. 5 days after, organoids were subjected to RNA sequencing. (n=4 mice per group pooled into 2 replicates). s, Pathway analysis comparing organoids from old m.uPAR-m.28z vs old UT treated mice. Enrichment assessed against Molecular Signature Database Hallmark 2025 gene sets. Color indicates adjusted p-value, y-axis represents normalized enrichment score. t, Gene set enrichment analysis of stemness signature in organoids from old m.uPAR-m.28z vs old UT treated mice. Results of 1 independent experiment (a–t). Two-tailed Fisher test (f,p). Two-tailed Wilcoxon rank-sum test *P<0.05,**P<0.01, ***P<0.001, ****P<0.0001 (g-h,n,q). MAST method: two-sided, p-values adjusted for multiple comparisons using the Benjamini-Hochberg method (d,o). Enrichment score in GSEA was calculated using a weighted Kolmogorov-Smirnov–like test. P values two-sided, non-parametric, permutation-based approach, with false discovery rate (FDR) correction applied for multiple comparisons. (t). Illustration created with Biorender.com (c,j,r).