Fig 2: Lack of IEC-intrinsic MHC class II has no effect on the epithelial transcriptome but controls the variability of microbial communities(A) Ileum-derived IECs stained for H2-A show knockout of H2-A on IECs in IEC?MHCII mice. n = 11 WT and n = 8 IEC?MHCII. A Student’s t test was used.(B) Volcano plot of RNA expression levels from sorted ileal IECs shows that only the targeted gene (H2-Ab1) is significantly changed (after FDR correction; 13,481 genes tested) in IEC?MHCII mice. The increased Mt1 is an artifact that reflects Cre expression, due to a small fragment of this gene left on the Villin:Cre expression construct.(C and D) Principal coordinates plots from Bray-Curtis (C) or weighted-UniFrac (D) metrics of fecal microbiota from mice housed by genotype (2 cages per genotype, 3–4 mice per cage). Permanova p values are shown with 9,999 permutations.(E) Weighted-UniFrac distances from fecal microbiota of male mice individually housed post-weaning. n = 6WT and 5 IEC?MHCII, except at week 5 when only 3WT samples had enough quality sequences for analysis. Two-way ANOVA with Holm-Sidak multiple testing correction was used.(F) Principal coordinates plot of ileal microbiota from Bray-Curtis distances along with Anosim results with cage and genotype factors as the independent variables.(G) Relative abundance of 2 taxa significantly different between genotypes in the ileum when separately housed. Both taxa initially identified with ANCOM (see main text) at the level of ASVs (2 each) were plotted as means of the ASVs summarized to their best taxonomic classification with significance between groups on plots shown with two-way ANOVA with Tukey’s correction for multiple testing.(H) Ileal microbiota alpha-diversity metrics. A Kruskal-Wallis test with Dunn’s correction was used.

Fig 3: MNPs can acquire MHC class II from IECs in vivo(A) The proportion of mT+mG+ MNP subsets in the siLP. Cells subsets are pregated on Live, Dump- (CD3, CD19, NK1.1, Ly6G), CD45+, and MHC class II+. Representative flow plots are shown for CD11c+ macrophages and DCs. Two-way ANOVA with Holm-Sidak correction was used.(B) Representative image panel of cells from imaging flow cytometry analysis of mT+mG+ MNPs. Cells are Live, Dump- (CD3, IgM), CD45+, and MHC class II+. No CD11c or CD11b subsetting due to reduced channel availability on imaging cytometry platform was used.(C) A cartoon schematic of mixed MHC class II bone-marrow transfer experiments.(D) The percentage of host MHC class II (H2-Ab) positive donor cells (HLA-DR) in the siLP. Cells are CD11c- P3/P4 macrophages gated as in previous figures. A representative plot (pregated on CD45+, Dump-, CD11b+/CD11c+, and CX3CR1+/CD64+ cells) is shown, along with an overlay of the H2-Ab isotype control demonstrating the MHC class II-haplotype specificity of the staining.

Fig 4: MNPs can acquire H2-A from IECs to induce T cell responses(A) Violin plots of H2-Ab1 transcripts from scRNA-seq data in the siLP show knockout of H2-Ab1 in IECs but normal expression in other known MHC class II+ cells types.(B) Results from in vitro association of MODEK (H2k haplotype) cells and H2-Ab1-/- C57BL/6 bone-marrow-derived MNPs. Data are expressed as a fold change of H2-Ak signal over the background from the average of MNPs incubated without MODEK cells. Two-way ANOVA with Holm-Sidak’s multiple comparison test within cell type to no MODEK control was used.(C) As in (B) (quantifying H2-Ak) but using MNPs sorted directly from the tissue of H2-Ab1-/- C57BL/6 mice. Ordinary one-way ANOVA with Tukey’s multiple comparison test was used.(D) The proportion of CFSE-labeled OT-II cells that underwent 1 or more divisions and were Foxp3+ after incubating with OVA-pulsed macrophages sorted directly from the tissue of WT or IEC?MHCII mice. Representative flow plots illustrate the gating strategy for assessing proliferating OT-II cells. Data are representative of 2 experiments; a Student’s t test was used.