Fig. 7: Roles of LTα₃ and TNF in sustaining B cells in ileitis.

a, scRNA-seq of sorted live, CD45⁺ mesenteric cells from three patients with CD with matched noninflamed and inflamed draining mesenteric samples. The dotplot depicts average expression of genes in the TNF/LTα/LTβ family along with their receptors. A UMAP plot of mesenteric immune cells is depicted in Extended Data Fig. 5b,c. b, Distribution of TNF⁺CD45⁺ cells analyzed with no ex vivo stimulation after staining permeabilized cells in cell suspensions of the ileum, mesentery or draining mLN of BM chimeric mice for TNF. TNF⁺ cells were gated and then divided into cell types that were TNF⁺ to assess proportional contribution of each population to the fraction of TNF⁺ cells in the tissue. This does not account for amount of TNF produced per cell or the possibility that some TNF is secreted and some is membrane-anchored. The same three mice were studied across the different tissue sites. c,d, UMAP projections of scRNA-seq on mLN cells that had migrated from the ileum in the previous 20 h. Feature plots (c) depict key genes identifying T cells (Cd3e), B cells (Ighd), DCs (Fscn1) and expression patterns of Tnf, Lta and Ltb. Cluster names assigned on the basis of marker genes are labeled in d, with distinct UMAP projections depicting profiles obtained in WT or TNF^ΔARE/+ chimeras treated with WT or anti-LTα₃ neutralizing mAb. e, Dotplot depicting average expression of genes in the TNF/LTα/LTβ family along with their receptors and other genes that were impacted by anti-LTα₃ treatment in either of the two B cell clusters or in CD4⁺ T cells. f–i, Mixed BM chimeras wherein WT recipient mice received μMT-TNF^ΔARE/+ BM at 90% and 10% marrow from CD19^Cre/+× Td^tomatofl/fl mice or CD19^Cre/+× TNF^fl/fl× Td^tomatofl/fl mice to resupply the B cell compartment with these respective genotypes and to mark CD19^Cre active cells with Td^Tomato expression. f, Percentage of CD19⁺ B cells in the blood 10 weeks after BMT to assess CD45.2⁺ donor engraftment. g, Weekly percent weight change in chimeras that had received μMT-TNF^ΔARE/+/CD19^cre/+Td^tomato/+ mixed BM (n = 5) or μMT-TNF^ΔARE/+/CD19^cre/+Td^tomato/+TNF^fl/fl (n = 5) B cells. Data were normalized to mouse weight 2 weeks post-BMT. Statistical analysis was carried out using two-way ANOVA with Tukey’s multiple comparisons post hoc test, showing that the curves were not statistically distinct. h,i, Percentage of CD19⁺ in the blood (h) or ileum (i) that were Td^tomato+ 32 weeks after BMT (one experiment μMT-TNF^ΔARE/+/CD19^cre/+Td^tomato/+ (n = 5 at 10 weeks, n = 3 at 32 weeks), μMT-TNF^ΔARE/+/CD19^cre/+Td^tomato/+TNF^fl/fl (n = 5)). Flow cytometry gating strategies in Supplementary Figs. 1, 2 and 5. Graphs depict mean ± s.e.m. Two-tailed Mann–Whitney test was applied for statistical analyses in f, h and i.

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