Fig 2: 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.

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.