Fig 2: TNFSF13 variant colonoids/organoids exhibit enhanced colonoid formation efficiency and proliferation.(A) ELISA for secreted TNFSF13 in colonoid conditioned media (n = 3 patient lines for control and VEO-IBD; n = 3 passages for variant). (B) Representative TNFSF13 RNAscope in colonoids from control and variant participants (scale bar: 50 μm; n = 3 patient lines for control; n = 3 passages for variant). (C) Costaining of TNFSF13 and FAS RNAscope probes with Ki67 antibody in colon biopsies. Arrows indicate cells accumulated outside epithelial crypts (n = 3 patients for control; n = 3 tissue blocks for variant). (D) Representative images of colonoid formation assays at day 6 after seeding (scale bar: 300 μm). (E) Quantification of newly formed colonoids by size at day 6. Each passage included 2 technical replicates (n = 4 patient lines for control and VEO-IBD; n = 4 passages for variant). (F) TNFSF13 and FAS RNAscope with E-cadherin immunostaining in WT and variant iPSC-derived colon organoids at day 7 (scale bar: 50 μm; n = 3 passages). (G) Representative colonoid formation in WT and variant iPSC-derived organoids at day 9 (scale bar: 400 μm; n = 3 passages, each with ≥ 2 technical replicates). (H) Quantification of colonoid formation rate and area at day 9. Colonoid size calculated by maximum vertical projection area. (I and J) Percentage of EdU+ cells following IgG or TNFSF13 neutralizing antibody (nTNFSF13) treatment in (I) control tissue–derived colonoids (n = 3 patient lines) or (J) WT iPSC-organoids at day 7 (n = 3 passages). 2-way ANOVA with multiple comparisons was used for (A and E); 2-tailed Student’s t test for (H–J). P values shown on graphs unless P > 0.05.

Fig 3: TNFSF13 augments the balance of apoptosis and proliferation through FAS-apoptosis pathway.(A) qPCR for ID and ECM1 in colonoids and (B) iPSC-derived colon organoids. (C) qPCR for ACAA2 and BCL2L1 in tissue-derived colonoids from control, VEO-IBD, variant, and (D) iPSC organoids from WT and variants. n = 3 lines of colonoids from 3 different patients for Control and VEO-IBD, n = 3 passages of colonoids for variants. (E) Representative immunostaining images for TUNEL and FABP2 in colonoids. Scale bar: 100 μm. (F) Quantification for ratio of TUNEL+ cells per colonoid and (G) TUNEL+FABP2+ cells in FABP2+ cells. One-way ANOVA (with multiple comparisons) was used for statistical analysis with 3 independent replicates. (H) Left: Representative flow cytometry plots for apoptotic cells in IgG, rTNFSF13 and rFASL treated Jurkat T cells. Right: Percentage of PI+AnnexinV+ (late apoptotic) and PI–AnnexinV+ (early apoptotic) populations across treatments. Three independent experimental replicates are shown. P-value shown in the bar graphs unless P > 0.05. Two-way ANOVA (with multiple comparisons) was used for statistical analysis in A–D and H and 1-way ANOVA (with multiple comparisons) for F and G.

Fig 4: Transcriptomic profiling in human colonoids.(A) UMAP showing the expression pattern of TNFSF13 and FAS in scRNA-seq data from human tissue–derived colonoids. (n = 2 lines of colonoids from 2 different patients for control and VEO-IBD, n = 2 independent passages of colonoids for variant.) Dot plot indicating the relative expression pattern of selected genes of TNFSF13 family and related receptors and enterocyte markers among annotated clusters for human colonoids scRNA-seq data. (B) Dot plot with relative expression of top 5 changed genes for each annotated cluster for scRNA-seq datasets in human colonoids. (C) Color scale indicates group with higher percentage of cells within a given cluster in each comparison. The color indicates the condition with higher percentage of a cluster in each pairwise comparison. (D) Dot plot with relative expression of selected genes of TNFSF13 family and related receptors and enterocyte markers among control, VEO-IBD and variants in human colonoids. (E) qPCR for ALDOB in colonoids on d7 after seeding. n = 3 lines of colonoids from 3 different patients for control and VEO-IBD, n = 3 passages of colonoids for variant. One-way ANOVA (with multiple comparisons) was used for statistical analysis. (F) Representative IF images for FABP2 and E-cadherin in human colonoids. White arrows denote FABP2+ cells. Scale bar: 50 μm. P values are shown on bar graphs unless P > 0.05.

Fig 5: Increased abundance of memory B cells and depletion of IgA+ plasma cells observed in TNFSF13 variant colon.(A) UMAP visualizations of scRNA-seq data for B cell and plasma cell clusters among lamina propria cells from control and variant colon biopsies. n = 1 patient for control and variant. Left: Overlay of control and variant samples; Right: Annotated cell clusters of control and variant samples. (B) Table indicates abundance (%) of B cell and PC subsets in control and variant samples from scRNA-seq data from variant and control colon biopsies. (C) Comparison of cell type abundance between samples from scRNA-seq data from variant and control colon biopsies. Color scale indicates which group has a higher percentage of cells within a given cluster. (D) Representative IMC overlay images of epithelial (white), B cell (green), and plasma cell (yellow) markers in colon from control, VEO-IBD and variant patient. Scale bar: 100 μm. Marker for B cell: CD20+; Markers for plasma cell: CD20–CD27+CD38+. n = 3 different patients for Control and VEO-IBD, n = 3 slides from different blocks for variants. (E) Boxplot showing the rate of immune cell composition quantified by calculating the proportion of specific markers in all cells at the same region (both lamina propria and epithelial cell populations). n = 3 different patients for control and VEO-IBD, n = 3 slides from different blocks for variant.