MHC Class II Antigen Presentation by the Intestinal Epithelium Initiates Graft-versus-Host Disease and Is Influenced by the Microbiota

Abstract

Graft-versus-host disease (GVHD) in the gastrointestinal (GI) tract is the principal determinant of lethality following allogeneic bone marrow transplantation (BMT). Here, we examined the mechanisms that initiate GVHD, including the relevant antigen-presenting cells. MHC class II was expressed on intestinal epithelial cells (IECs) within the ileum at steady state but was absent from the IECs of germ-free mice. IEC-specific deletion of MHC class II prevented the initiation of lethal GVHD in the GI tract. MHC class II expression on IECs was absent from mice deficient in the TLR adaptors MyD88 and TRIF and required IFNγ secretion by lamina propria lymphocytes. IFNγ responses are characteristically driven by IL-12 secretion from myeloid cells. Antibiotic-mediated depletion of the microbiota inhibited IL-12/23p40 production by ileal macrophages. IL-12/23p40 neutralization prevented MHC class II upregulation on IECs and initiation of lethal GVHD in the GI tract. Thus, MHC class II expression by IECs in the ileum initiates lethal GVHD, and blockade of IL-12/23p40 may represent a readily translatable therapeutic strategy.

Keywords: MHC class II; antigen presentation; antigen-presenting cells; dendritic cells; graft-versus-host disease; ileum; interferon-gamma; interleukin-12; intestinal epithelial cells; microbiome; toll-like receptors; type 1 innate lymphoid cells.

Figure 1.. IEC in the ileum express MHC-II and MHC-II expression increases after TBI.

(A, B) MHC-II expression in the small (pooled jejunum and ileum) and large intestine was analyzed in naïve and transplanted mice. Representative flow plots and enumeration are shown. (A) Lethally irradiated VillinCre^posRosa26-YFP mice were transplanted with BM and T cells from BALB/c mice and analysed on day 3 (d3) post-transplant. Data combined from 3 replicate experiments (n = 9 per group). (B) Irradiated B6D2F1 mice were transplanted with BM and T cells from B6.WT mice and analysed on d4. Data combined from 2 replicate experiments (n = 4 – 5 per group). (C) MHC-II expression (MFI) was quantified on IEC (EpCAM⁺ CD45^neg) of the jejunum, ileum and colon from naïve B6.WT mice, combined from 2 replicate experiments (n = 10 per group). (D) Naïve B6.I-A^bβ-GFP mice, 24 hours (24h) after TBI or d3 after BMT with BM and T cells from BALB/c mice as in (A). Immunofluorescence (green = MHC-II-GFP; yellow = MHC-II (anti-IA/IE Ab staining); red = CD45; blue = DAPI). Representative images and standardized enumeration combined from 3 replicate experiments (n = 5 – 6 per group). (E) Immunofluorescence staining of colonic and ileal tissue of naive B6 mice and at 2d post-TBI. Bidirectional arrows indicate the colon’s inner mucous layer which is free from bacteria. [Muc2 = green, eubacterial probe (Eub338) = red, DAPI = blue, scale bar = 50 μm]. Statistical analysis by unpaired t-test per organ (A), Mann-Whitney U test per organ (B) or Kruskal-Wallis test (C, D) (mean ± SEM). *P < 0.05, ** P < 0.01, *** P < 0.001. See also Figure S1.

Figure 2.. The intestinal microbiota is required for MHC-II expression on IEC pre-transplant.

(A-C) (A) Representative histograms of MHC-II on ileal IEC (EpCAM⁺CD45^neg) purified from specific pathogen free (SPF) and germ free (GF) mice before (naïve) and 24h after TBI, (B) MHC-II MFI on IEC and splenic DC (CD45⁺CD3^negCD19^negNK1.1^negLy6C^negLy6G^negCD11c⁺CD64^neg) (n = 6 per group from 3 experiments), (C) Confocal images from the ileum. (D, E) (D) Representative histograms of MHC-II on IEC in the ileum of mice treated with antibiotic water or control water for 2 weeks and analyzed before (naïve) and 24h after TBI, (E) MHC-II MFI on IEC and splenic DC (n = 4 – 5 per group from 2 replicate experiments). (F, G) B6.WT and B6.Il17ra^−/− mice were housed separately or cohoused for 6 weeks. (F) Representative histograms of MHC-II on IEC and (G) frequency of MHC-II⁺ IEC (n = 8 – 10 per group combined from 2 replicate experiments). (H) Cohoused or individually housed B6.WT mice were transplanted with CD4⁺ T cells from BALB/c^luc+ mice. Quantification and representative images of d7 bioluminescence (BLI) data (allogeneic CD4⁺ T cell expansion) combined from 2 replicate experiments (n = 9 – 10 per group) are shown. Statistical analysis by unpaired t-test (H), ANOVA (ileum and spleen in B, ileum in E and G) and Kruskal-Wallis test (spleen in E) (mean ± SEM). *P < 0.05, ** P < 0.01, *** P < 0.001, ****P < 0.0001. See also Figure S1.

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.

Figure 4.. IFNγ drives MHC-II expression on IEC.

Representative (A) IFNγR and (B) MHC-II expression on IEC (7AAD^negEpCAM⁺CD45^neg) from naïve and 24h post-TBI, B6.WT and B6.Ifngr^−/− mice. Quantified data shown (right) is combined from 3 replicate experiments (n = 6 per group). (C) Fluorescence images of ileum from B6.Ifngr^−/− (naïve and 24h post-TBI) and B6.WT (naïve) mice. Representative images and quantification from 2 replicate experiments. (D) Representative MHC-II expression on IEC from naïve B6.WT, B6.Rag ^−/−, B6. Rag^−/−Il2rg^−/− and B6.I-A^b−/− mice. Quantification shown right (n = 4 per group from 3 experiments). (E) Fluorescence images of ileum from 24h post-TBI of B6.WT, B6.Rag^−/− and B6.Rag^−/−Il2rg^−/− mice. Representative images (left) and quantification from 2 replicate experiments are shown (n = 3 – 5 per group). (F) Representative IFNγ-YFP expression (left) and quantified data (right) in hematopoietic cell subsets within the ileum from naïve mice are shown (n = 3 – 6 per group from 3 replicate experiments). (G) IFNγ-YFP expression shown in the indicated populations from the ileum and mLN of naïve mice and 18h after TBI. (H) Representative confocal images of small intestinal organoids generated from B6. I-A^bβ-GFP or B6.WT mice in the presence or absence of IFNγ. Representative images from 2 replicate experiments. (I) Flow cytometric quantification of HLA class-II MFI in 7AAD^negEpCAM⁺CD45^neg cells from human duodenum organoids before and after IFNγ stimulation (healthy donors, n = 3, paired t-test). Multiple comparisons by ANOVA (B, E) or Kruskal-Wallis test (D) (mean ± SEM). *P < 0.05, ** P < 0.01, **** P < 0.0001. See also Figure S5.

Figure 5.. IEC induce MHC-II-dependent GVHD.

(A) The distribution (top: confocal images) and lineage (bottom: flow cytometry) of YFP⁺ cells in the ileum from naïve VillinCre^posRosa26-YFP, NestinCre^posRosa26-YFP or Tie2Cre^posRosa26-YFP mice. Representative of two replicate experiments. (B) B6 male Nestin, Villin or Tie2 Cre^posI-A^b-fl/fl mice and relevant Cre^negI-A^b-fl/fl mice were transplanted with BM from female B6.I-A^b−/− mice. 3 months later, these chimeric mice were transplanted with BM from female B6.I-A^b−/− mice and 25 × 10³ sorted Marilyn T cells. Survival by Kaplan-Meier analysis, combined from 2 replicate experiments (n = 9 – 11 per group). P = 0.0025 (Villin), 0.0066 (Nestin): Cre ^posI-A^b-fl/fl vs. Cre ^negI-A^b-fl/fl chimeric recipients. (C) BM chimeric recipients were transplanted as in (B), but with 0.2 × 10⁶ sorted Marilyn^luc+ T cells. Representative BLI images and quantitative data on d7 combined from 3 – 4 replicate experiments are shown (n = 12 – 21 per group). Two-tailed unpaired t test (mean ± SEM), **P = 0.0090, N.S. = not significant. See also Figure S6.

Figure 6.. MHC-II-expressing IEC elicit alloantigen reactive T cell expansion and Th1 differentiation in the GI tract.

Male (A – G) or female (H – K) Villin Cre-ER^T2-negI-A^b-fl/fl and Cre-ER^T2-posI-A^b-fl/fl or female B6.WT mice (A – G) were treated with Tamoxifen 2 weeks before transplant. (A) Tamoxifen-treated mice were transplanted with B6.WT BM with 0.5 × 10⁶ CD4 MACS-purified Marilyn T cells. Female recipients are negative controls. Survival by Kaplan-Meier analysis, combined from 2 replicate experiments (n = 11 – 12 per T cell replete group). (B – G) Recipients were transplanted as in (A) but with 0.2 × 10⁶ sorted Marilyn ^luc+ T cells. Serum TNF, BLI data, enumeration of Marilyn T cells with MFI of T-bet expression and intestinal histology on d7 are shown, combined from 3 replicate experiments (n = 12 per T cell replete group). (H – K) Lethally irradiated Tamoxifen-treated female mice were transplanted with BM and T_reg-depleted CD4⁺ T cells from PC61-treated BALB/c mice. (H) Survival by Kaplan-Meier analysis, combined from 2 replicate experiments (n = 11 per T cell replete group); (I, J) Intestinal histopathology scores on d10 (n = 6 per T cell replete group); and (K) late skin histology (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001: Villin Cre-ER^T2-negI-A^b-fl/fl vs. Cre-ER^T2-posI-A^b-fl/fl. Statistical analysis by two-tailed Mann-Whitney U test (B - F) or t-test (I) (mean ± SEM), except for survival data. See also Figure S6 - 7.

Figure 7.. Neutralizing IL-12 prior to irradiation prevents the induction of MHC-II expression on IEC pre-transplant and subsequent GVHD.

B6.WT mice (A – D) and B6.Il12/23p40-YFP mice (G, H) received either antibiotic water or normal water for 2 weeks. (A - D) Lethally irradiated male B6.WT gut decontaminated mice and control male and female mice were transplanted with 0.2 × 10⁶ Marilyn T cells. (A - C) Ileum was analysed on d3. (A) MHC-II expression quantified on IEC (EpCAM⁺CD45^neg), (B) T-bet MFI in host ILC1 and (C) serum IFNγ (n = 8 – 12 per group from 2 replicate experiments, Mann Whitney U test). (D) Quantification of MHC-II expression on IEC (left), frequency of Marilyn T cells infiltrating the epithelial fraction (middle) and ileum GVHD histopathology scores (right) with representative H&E stained images on d7 are shown (n = 8 – 12 per group from 2 replicate experiments). (E) Representative flow plots of IL-12/23p40-YFP expression on macrophages (Mac), DC, monocytes (Mono) and neutrophil (Neu) from naïve B6.Il12/23p40-YFP mice (from 3 replicate experiments). B6.WT mice were gated as negative controls. (F) Quantification of Il12/23p40-YYP⁺ Mac and DC from naïve mice or 6h post-TBI (n = 6 – 7 per group from 3 replicate experiments, unpaired t-test). (G, H) Representative flow plots (G) and frequency (left) and numbers (right) of IL-12/23p40-YFP⁺ cells (H) in the ileum of mice 24h after TBI (n = 9 – 10 per group from 2 replicate experiments, unpaired t-test). (I) B6.WT mice were treated with IL-12/23p40 mAb or isotype control at 48h and 24h prior to TBI. Representative ileal image are shown with quantification (n = 5 – 6 per group from 2 replicate experiments). (J) Survival by Kaplan-Meier estimates. Male B6.WT mice were treated with IL-12/23p40 mAb or isotype control at 48h (d-3), 24h (d −2) prior to TBI and d 0 prior to the infusion of B6.WT BM and Marilyn T cells (n = 12, combined from 2 replicate experiments). Female recipients (n = 4) were used as negative controls. Multiple comparisons by ANOVA, except D (ileal pathology), Kruskal-Wallis test, (mean ± SEM). *P < 0.05, ** P < 0.01, **** P < 0.0001. See also Figure S7.