Figure 3. MyD88 and TRIF signalling are necessary for MHC-II expression by IEC in the ileum pre-transplant.

(A) Mixed Lymphocyte Reaction (MLR) of CFSE-labelled sort-purified HY-reactive Marilyn CD4⁺ T cells co-cultured with sort-purified 7AAD^neg EpCAM⁺ CD45^neg IEC from the small intestine of irradiated male (HY-positive) or female (HY-negative) mice, analysed at d7. Data are representative of 3 replicate experiments (n = 3 – 4). (B) Epithelial cells from the small intestine (7AAD^negVillin⁺CD45^neg) of naïve VillinCre^posRosa26-YFP mice, those 4d post-TBI or 3d post-BMT were sort-purified and processed for RNAseq gene expression analysis (n = 4 – 5 per group). (C) First and second principal component projections. (D) Clustered heat map of the top 50 gene-sets that significantly differentially increased at FDR < 0.05 between groups of samples analysed using ssGSEA. (E) Representative flow plots and MFI of MHC-II expression in the epithelial fractions from naïve B6.WT and B6.Myd88^−/−Trif^−/− mice. Data combined from 2 replicate experiments (n = 7 per group). (F) Representative confocal images of MHC-II expression on ileum before and 24h after TBI with quantification normalized to naïve WT mice (n = 3 per group). (G-H) Male B6.WT and B6.Myd88^−/−Trif^−/− mice were transplanted with Marilyn^luc+ T cells and analyzed on d7. WT female recipients were utilized as negative controls (n = 4). (G) Representative BLI images and signal quantification in small intestine, colon and spleen are shown. (H) Marilyn ^luc+ T cell quantification and T-bet expression in mLN and spleen. (n = 5 – 8 per male group combined from 2 replicate experiments). Statistical analysis by unpaired t-test (A), Mann-Whitney U test (E,G,H) or ANOVA (F) (mean ± SEM). *P < 0.05, ** P < 0.01, *** P < 0.001. See also Figure S2 -4.