Extended Data Fig. 9: Characteristics of uPAR+ epithelial cells.
a, Intestinal crypts from n=4 young (3 months) and n=4 old (20 months old) mice were isolated and seeded to form organoids together with either UT or m.uPAR-m28z cells at 1:10 effector:target ratio. Levels of granzyme B 72h after co-culture between organoids and UT or m.uPAR-m28z cells (n=6 replicates per group). b-e, Aged murine intestinal organoids were edited by CRISPR-Cas9-sgRNAs targeting Plaur. b, Sanger sequencing confirming editing of the Plaur locus. c-e: organoids were cultured together with either UT or m.uPAR-m28z cells at 1:10 effector:target ratio for 72 h (n=4 replicates per group). c, Representative images of organoids after co-culture with UT or m.uPAR-m28z cells for 72 h. d, Quantification of the percentage of dead organoids per field 72h after co-culture between organoids and UT or m.uPAR-m28z cells (n=4 replicates per group). e, Levels of granzyme B 72h after co-culture between organoids and UT or m.uPAR-m28z cells (n=4 replicates per group). f, Percentage of cells expressing surface MHC-II as determined by flow cytometry on isolated intestinal crypts from old (18 months old) mice. (n=4 per group). g, Split-violin plot indicates the expression level of MHC-II signature score in non-immune PLAUR- or PLAUR+ cells from the duodenum, jejunum and ileum of young (25–30 years old) and old (65–70 years old) subjects33. Boxplots display median (center line) and interquartile range (box) (n=1 per group).h-i, 18 months old mice were administered 1mg/ml ovalbumin conjugated to Texas red by oral gavage. Intestinal crypts were isolated and dissociated 1h after administration. h, Graph depicting percentage of CD45+ EpCAM− uPAR− or uPAR+ cells positive for Texas red. (n=8 mice per group). i, Graph depicting percentage of CD45− EpCAM+ uPAR− or uPAR+ cells positive for Texas red. (n=8 mice per group). j, CD45− EpCAM+ uPAR+ cells induce CD4+ T cell proliferation in vitro. Representative FACS histograms of cell trace violet-labeled CD4+ T cells from OT-II mice that were cultured alone or with CD45− EpCAM+ uPAR− or CD45− EpCAM+ uPAR+ cells from 18 month old mice with or without 15ug/ml of OVA323-339. Values shown are mean ± s.d. (n=3 for T cells alone and T cells alone with OVA; n=4 mice per group for the other groups). Results of 1 independent experiment (a-g,j). Results of 2 independent experiment (h-i). Data are mean ± s.e.m. (a,d-f,h-i). Data are mean ± s.d (j). Two-tailed unpaired Student’s t-test (a,d-f,h-i). Two-tailed Wilcoxon rank-sum test *P<0.05,**P<0.01, ***P<0.001, ****P<0.0001 (g).
