IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease

Abstract

Coeliac disease is a complex, polygenic inflammatory enteropathy caused by exposure to dietary gluten that occurs in a subset of genetically susceptible individuals who express either the HLA-DQ8 or HLA-DQ2 haplotypes1,2. The need to develop non-dietary treatments is now widely recognized3, but no pathophysiologically relevant gluten- and HLA-dependent preclinical model exists. Furthermore, although studies in humans have led to major advances in our understanding of the pathogenesis of coeliac disease4, the respective roles of disease-predisposing HLA molecules, and of adaptive and innate immunity in the development of tissue damage, have not been directly demonstrated. Here we describe a mouse model that reproduces the overexpression of interleukin-15 (IL-15) in the gut epithelium and lamina propria that is characteristic of active coeliac disease, expresses the predisposing HLA-DQ8 molecule, and develops villous atrophy after ingestion of gluten. Overexpression of IL-15 in both the epithelium and the lamina propria is required for the development of villous atrophy, which demonstrates the location-dependent central role of IL-15 in the pathogenesis of coeliac disease. In addition, CD4+ T cells and HLA-DQ8 have a crucial role in the licensing of cytotoxic T cells to mediate intestinal epithelial cell lysis. We also demonstrate a role for the cytokine interferon-γ (IFNγ) and the enzyme transglutaminase 2 (TG2) in tissue destruction. By reflecting the complex interaction between gluten, genetics and IL-15-driven tissue inflammation, this mouse model provides the opportunity to both increase our understanding of coeliac disease, and develop new therapeutic strategies.

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Fig. 1: DQ8-Dd-villin-IL-15tg mice: a gluten-dependent model of coeliac disease with villous atrophy.
Fig. 2: Cytotoxic IELs are the key effector cells that mediate tissue destruction.
Fig. 3: HLA-DQ8, CD4+ T cells and IFNγ are required for tissue destruction.
Fig. 4: DQ8-Dd-villin-IL-15tg mice represent a preclinical model for coeliac disease.

Data availability

Source Data for Figs. 14 and Extended Data Figs. 110 are provided with the paper. The RNA sequencing experiments have been deposited into the Gene Expression Omnibus (GEO) database under the accession code GSE134900. All other data are available from the corresponding author(s) upon reasonable request.

References

  1. 1.

    Abadie, V., Sollid, L. M., Barreiro, L. B. & Jabri, B. Integration of genetic and immunological insights into a model of celiac disease pathogenesis. Annu. Rev. Immunol. 29, 493–525 (2011).

  2. 2.

    Green, P. H. & Cellier, C. Celiac disease. N. Engl. J. Med. 357, 1731–1743 (2007).

  3. 3.

    Plugis, N. M. & Khosla, C. Therapeutic approaches for celiac disease. Best Pract. Res. Clin. Gastroenterol. 29, 503–521 (2015).

  4. 4.

    Jabri, B. & Sollid, L. M. Tissue-mediated control of immunopathology in coeliac disease. Nat. Rev. Immunol. 9, 858–870 (2009).

  5. 5.

    Husby, S. et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J. Pediatr. Gastroenterol. Nutr. 54, 136–160 (2012).

  6. 6.

    Dieterich, W. et al. Identification of tissue transglutaminase as the autoantigen of celiac disease. Nat. Med. 3, 797–801 (1997).

  7. 7.

    Jabri, B. & Abadie, V. IL-15 functions as a danger signal to regulate tissue-resident T cells and tissue destruction. Nat. Rev. Immunol. 15, 771–783 (2015).

  8. 8.

    Waldmann, T. A. The biology of interleukin-2 and interleukin-15: implications for cancer therapy and vaccine design. Nat. Rev. Immunol. 6, 595–601 (2006).

  9. 9.

    Setty, M. et al. Distinct and synergistic contributions of epithelial stress and adaptive immunity to functions of intraepithelial killer cells and active celiac disease. Gastroenterol. 149, 681–691 (2015).

  10. 10.

    Black, K. E., Murray, J. A. & David, C. S. HLA-DQ determines the response to exogenous wheat proteins: a model of gluten sensitivity in transgenic knockout mice. J. Immunol. 169, 5595–5600 (2002).

  11. 11.

    Jabri, B. & Sollid, L. M. Mechanisms of disease: immunopathogenesis of celiac disease. Nat. Clin. Pract. Gastroenterol. Hepatol. 3, 516–525 (2006).

  12. 12.

    Korneychuk, N. et al. Interleukin 15 and CD4+ T cells cooperate to promote small intestinal enteropathy in response to dietary antigen. Gastroenterology 146, 1017–1027 (2014).

  13. 13.

    DePaolo, R. W. et al. Co-adjuvant effects of retinoic acid and IL-15 induce inflammatory immunity to dietary antigens. Nature 471, 220–224 (2011).

  14. 14.

    Di Niro, R. et al. High abundance of plasma cells secreting transglutaminase 2-specific IgA autoantibodies with limited somatic hypermutation in celiac disease intestinal lesions. Nat. Med. 18, 441–445 (2012).

  15. 15.

    Nilsen, E. M. et al. Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology 115, 551–563 (1998).

  16. 16.

    Meresse, B. et al. Reprogramming of CTLs into natural killer-like cells in celiac disease. J. Exp. Med. 203, 1343–1355 (2006).

  17. 17.

    Meresse, B. et al. Coordinated induction by IL15 of a TCR-independent NKG2D signaling pathway converts CTL into lymphokine-activated killer cells in celiac disease. Immunity 21, 357–366 (2004).

  18. 18.

    Hüe, S. et al. A direct role for NKG2D/MICA interaction in villous atrophy during celiac disease. Immunity 21, 367–377 (2004).

  19. 19.

    Bodd, M. et al. HLA-DQ2-restricted gluten-reactive T cells produce IL-21 but not IL-17 or IL-22. Mucosal Immunol. 3, 594–601 (2010).

  20. 20.

    van de Wal, Y. et al. Selective deamidation by tissue transglutaminase strongly enhances gliadin-specific T cell reactivity. J. Immunol. 161, 1585–1588 (1998).

  21. 21.

    Molberg, O. et al. Tissue transglutaminase selectively modifies gliadin peptides that are recognized by gut-derived T cells in celiac disease. Nat. Med. 4, 713–717 (1998).

  22. 22.

    Klöck, C., Herrera, Z., Albertelli, M. & Khosla, C. Discovery of potent and specific dihydroisoxazole inhibitors of human transglutaminase 2. J. Med. Chem. 57, 9042–9064 (2014).

  23. 23.

    Freitag, T. L. et al. Gliadin-primed CD4+CD45RBlowCD25 T cells drive gluten-dependent small intestinal damage after adoptive transfer into lymphopenic mice. Gut 58, 1597–1605 (2009).

  24. 24.

    Meisel, M. et al. Interleukin-15 promotes intestinal dysbiosis with butyrate deficiency associated with increased susceptibility to colitis. ISME J. 11, 15–30 (2017).

  25. 25.

    Pinto, D., Robine, S., Jaisser, F., El Marjou, F. E. & Louvard, D. Regulatory sequences of the mouse villin gene that efficiently drive transgenic expression in immature and differentiated epithelial cells of small and large intestines. J. Biol. Chem. 274, 6476–6482 (1999).

  26. 26.

    Lefrancois, L. & Lycke, N. Isolation of mouse small intestinal intraepithelial lymphocytes, Peyer's patch, and lamina propria cells. Curr. Protoc. Immunol. Chapter 3, Unit 3.19 (2001).

  27. 27.

    Bouziat, R. et al. Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease. Science 356, 44–50 (2017).

  28. 28.

    DiRaimondo, T. R. et al. Elevated transglutaminase 2 activity is associated with hypoxia-induced experimental pulmonary hypertension in mice. ACS Chem. Biol. 9, 266–275 (2014).

  29. 29.

    Du, P., Kibbe, W. A. & Lin, S. M. lumi: a pipeline for processing Illumina microarray. Bioinformatics 24, 1547–1548 (2008).

  30. 30.

    Lin, S. M., Du, P., Huber, W. & Kibbe, W. A. Model-based variance-stabilizing transformation for Illumina microarray data. Nucleic Acids Res. 36, e11 (2008).

  31. 31.

    Smyth, G. K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3, Article3 (2004).

  32. 32.

    Benjamini, Y. H. Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. A Stat. Soc. 57, 289–300 (1995).

  33. 33.

    Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L. Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol. 34, 525–527 (2016).

  34. 34.

    Robinson, M. D., McCarthy, D. J. & Smyth, G. K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

  35. 35.

    Ritchie, M. E. et al. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 43, e47 (2015).

  36. 36.

    Eden, E., Navon, R., Steinfeld, I., Lipson, D. & Yakhini, Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists. BMC Bioinformatics 10, 48 (2009).

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Acknowledgements

We thank patients with coeliac disease and their family members, as well as the University of Chicago Celiac Disease Center, for supporting our research. We thank BZ-Histo Services for the histological processing of intestinal samples. We also thank the Human Tissue Resource Center and the Integrated Light Microscopy Core Facility at the University of Chicago. This work was supported by grants from NIH: R01 DK67180 and R01 DK63158, and Digestive Diseases Research Core Center P30 DK42086 at the University of Chicago as well as by funding from F. Oliver Nicklin associated with the First Analysis Institute of Integrative Studies and the Regenstein Foundation to B.J., from the CIHR (catalyst grant in environments, genes, and chronic disease) to V.A. and L.B.B., the SickKids Foundation (NI15-040) and the Canadian Celiac Association to V.A., from NIH: R01 DK063158 and R01 DK100619 to C.K., award from the Wallonie-Bruxelles International-World Excellence and from FRQNT to T.L. The Carlino Fellowship for Celiac Disease Research from the University of Chicago Celiac Disease Center supported V.D.

Author information

V.A. and B.J. designed the research and supervised all investigations. S.M.K., T.L., J.D.E., B.A.P., C.C., J.V., R.B., K.P., M.A.Z., A.D., V.Y. and V.A. performed experiments and analysed the data. V.D. and I.L. performed the human RNA sequencing experiments. E.V.M. and I.H. undertook serology experiments. O.T., J.-C.G., M.B.F.H. and L.B.B. performed the computational analysis. C.K., J.A.M. and L.B.B. provided intellectual input and technical support. V.A. and B.J. wrote the manuscript with the contribution of S.M.K., T.L., B.A.P. and C.K. All authors provided critical review of the manuscript.

Correspondence to Valérie Abadie or Bana Jabri.

Ethics declarations

Competing interests

B.J. and J.A.M. are consultants to Celimmune and Bionix. C.K. is a member of the Board of Directors of Protagonist Therapeutics. All other authors declare no competing interests.

Additional information

Peer review information Nature thanks Nadine Cerf-Bensussan, Arthur Kaser, Daniel Mucida and Emil Unanue for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Development of villous atrophy requires IL-15 expression in both the lamina propria and epithelium.

ag, DQ8-Dd-IL-15tg, DQ8-villin-IL-15tg and DQ8-Dd-villin-IL-15tg mice that were raised on a GFD were maintained on a GFD (sham) or fed gluten for 30 days. a, Expression of Ifng in the lamina propria was measured by qPCR (DQ8-Dd-IL-15tg sham, n = 7, gluten n = 16; DQ8-villin-IL-15tg sham, n = 13, gluten n = 12). b, Serum anti-gliadin IgG2c levels were measured by ELISA 30 days after gluten feeding (DQ8-Dd-IL-15tg sham, n = 16, gluten n = 16; DQ8-villin-IL-15tg sham, n = 15, gluten n = 15). c, Serum levels of anti-DGP IgG were measured by ELISA 30 days after gluten feeding (DQ8-Dd-IL-15tg sham, n = 10, gluten n = 10; DQ8-villin-IL-15tg sham, n = 9, gluten n = 9). d, Quantification of IELs among IECs was performed on H&E-stained ileum sections (DQ8-Dd-IL-15tg sham, n = 11, gluten n = 12; DQ8-villin-IL-15tg sham, n = 17, gluten n = 16; DQ8-Dd-villin-IL-15tg sham, n = 14, gluten n = 17). e, Left, H&E staining of paraffin-embedded ileum sections. Right, the ratio of the morphometric assessment of villus height to crypt depth (DQ8-Dd-IL-15tg sham, n = 10, gluten n = 10; DQ8-villin-IL-15tg sham, n = 17, gluten n = 19; DQ8-Dd-villin-IL-15tg sham, n = 21, gluten n = 28). f, g, Expression of Rae1 (f) and Qa-1 (g) in the intestinal epithelium was measured by qPCR. Relative expression levels in sham and gluten-fed mice for each strain are shown (DQ8-Dd-IL-15tg sham, n = 16, gluten n = 16; DQ8-villin-IL-15tg sham, n = 15, gluten n = 15; DQ8-Dd-villin-IL-15tg sham, n = 20, gluten n = 26). Data are mean ± s.e.m. (a, f, g) or mean values (be) from three (ac, f, g), four (d) or six (e) independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparison test (ae) or unpaired, two-tailed, t-test (f, g). Source data

Extended Data Fig. 2 Acquisition of cytotoxic properties by IELs requires IL-15 expression in both the lamina propria and epithelium.

aj, DQ8-Dd-IL-15tg, DQ8-villin-IL-15tg and DQ8-Dd-villin-IL-15tg mice were maintained on a GFD or fed gluten for 30 days (gluten). ai, The intestinal epithelium was isolated and analysed by flow cytometry. A subset of IELs was identified as TCRβ+CD4CD8αβ+ cells. In parallel, IELs were identified as TCRβ+CD4CD8+ cells by flow cytometry and quantified among IECs on H&E-stained ileum sections. a, Percentage of NKG2D+NKG2CD8αβ+ IELs are indicated (DQ8-Dd-IL-15tg sham, n = 11, gluten n = 14; DQ8-villin-IL-15tg sham, n = 20, gluten n = 20; DQ8-Dd-villin-IL-15tg sham, n = 17, gluten n = 22). b, Numbers of NKG2D+NKG2CD8+ IELs per 100 IECs (DQ8-Dd-IL-15tg sham, n = 11, gluten n = 13; DQ8-villin-IL-15tg sham, n = 20, gluten n = 20; DQ8-Dd-villin-IL-15tg sham, n = 16, gluten n = 19). c, Percentage of CD94+NKG2ACD8αβ+ IELs are indicated (DQ8-Dd-IL-15tg sham, n = 10, gluten n = 10; DQ8-villin-IL-15tg sham, n = 11, gluten n = 11; DQ8-Dd-villin-IL-15tg sham, n = 9, gluten n = 10). d, Numbers of CD94+NKG2ACD8+ IELs per 100 IECs (DQ8-Dd-IL-15tg sham, n = 10, gluten n = 10; DQ8-villin-IL-15tg sham, n = 11, gluten n = 11; DQ8-Dd-villin-IL-15tg sham, n = 9, gluten n = 10). e, Percentage of intracellular granzyme B+ IELs (DQ8-Dd-IL-15tg sham, n = 9, gluten n = 10; DQ8-villin-IL-15tg sham, n = 14, gluten n = 13; DQ8-Dd-villin-IL-15tg sham, n = 12, gluten n = 18). f, Numbers of granzyme B+ IELs per 100 IECs (DQ8-Dd-IL-15tg sham, n = 11, gluten n = 13; DQ8-villin-IL-15tg sham, n = 20, gluten n = 20; DQ8-Dd-villin-IL-15tg sham, n = 16, gluten n = 19). g, Mean fluorescent intensity (MFI) of intracellular granzyme B was measured. (DQ8-Dd-IL-15tg sham, n = 9, gluten n = 9; DQ8-villin-IL-15tg sham, n = 14, gluten n = 13; DQ8-Dd-villin-IL-15tg sham, n = 10, gluten n = 15). h, Percentage of CD8αβ+CD107a+ IELs. i, Numbers of CD8+CD107a+ IELs per 100 IECs (DQ8-Dd-IL-15tg sham, n = 7, gluten n = 6; DQ8-Dd-villin-IL-15tg sham, n = 8, gluten n = 5; DQ8-Dd-villin-IL-15tg sham, n = 5, gluten n = 4). j, Expression of Prf1 in the intestinal epithelium was measured by qPCR. Relative expression levels in sham and gluten-fed mice for each strain are shown (DQ8-Dd-IL-15tg sham, n = 16, gluten n = 16; DQ8-villin-IL-15tg sham, n = 15, gluten n = 15; DQ8-Dd-villin-IL-15tg sham, n = 20, gluten n = 26). Data are mean ± s.e.m. (j) or mean values (ag, i) from six (a, b), three (c, d, j), five (e, f), four (g) or two (h, i) independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparison test (ag, i) or unpaired, two-tailed, t-test (j). Source data

Extended Data Fig. 3 Overexpression of IL-15 in HLA-humanized DQ8 mice confers susceptibility to development of coeliac disease-like features in a gluten-dependent manner.

ak, DQ8 and DQ8-Dd-villin-IL-15tg mice were maintained on a GFD or fed gluten for 30 days. a, Left, H&E staining of paraffin-embedded ileum sections. Scale bars, 100 µm. Right, the ratio of the morphometric assessment of villus height to crypt depth (DQ8 sham, n = 12, gluten n = 13; DQ8-Dd-villin-IL-15tg sham, n = 12, gluten n = 15). b, Villus/crypt ratio from sham-fed DQ8-Dd-villin-IL-15tg mice and DQ8-Dd-villin-IL-15tg mice fed a standard rodent chow without supplementary gluten (sham, n = 4, gluten n = 8). c, IgA (red) and CD138+ (green) plasma cells were distinguished by immunohistochemical staining of frozen ileum sections. Scale bars, 50 μm. d, Serum levels of anti-DGP IgG as measured by ELISA. Serum was collected 30 days after gluten feeding (DQ8 sham, n = 6, gluten n = 7; DQ8-Dd-villin-IL-15tg sham, n = 6, gluten n = 7). e, Quantification of IELs among IECs performed on H&E-stained ileum sections (DQ8 sham, n = 10, gluten n = 16; DQ8-Dd-villin-IL-15tg sham, n = 12, gluten n = 14). fj, The intestinal epithelium was isolated and analysed by flow cytometry. IELs were identified as TCRβ+CD4CD8+ and TCRβ+CD4CD8αβ+ cells. f, Percentage of granzyme B+ IELs (DQ8 sham, n = 13, gluten n = 15; DQ8-Dd-villin-IL-15tg sham, n = 11, gluten n = 15). g, Numbers of CD8+ granzyme B+ IELs per 100 IECs (DQ8 sham, n = 11, gluten n = 14; DQ8-Dd-villin-IL-15tg sham, n = 11, gluten n = 13). h, MFI of granzyme B (DQ8 sham, n = 11, gluten n = 12; DQ8-Dd-villin-IL-15tg sham, n = 9, gluten n = 12). i, Percentage of NKG2D+NKG2 IELs (DQ8 sham, n = 13, gluten n = 17; DQ8-Dd-villin-IL-15tg sham, n = 11, gluten n = 15). j, Numbers of NKG2D+NKG2 IELs per 100 IECs (DQ8 sham, n = 12, gluten n = 16; DQ8-Dd-villin-IL-15tg sham, n = 11, gluten n = 13). k, The intestinal epithelium was isolated and analysed by flow cytometry. IECs were identified as EpCAM+CD45 cells. Percentage of Rae-1+ IECs (n = 5 mice per group). Data are mean values (a, b, dk) from four (a, eg, i, j), two (b, d, k) or three (c, h) independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparison test (a, dk) or unpaired, two-tailed, t-test (b). Source data

Extended Data Fig. 4 A GFD decreases the anti-gluten antibody response and the number of cytotoxic IELs.

ah, k, l, DQ8-Dd-villin-IL-15tg mice raised on a GFD were maintained on a GFD, fed gluten for 30 days, or fed gluten for 30 days and then reverted to a GFD for 30 days. a, Left, CD3ε+ T cells (red) and CD138+ plasma cells (green) were distinguished by immunohistochemistry staining of frozen ileum sections. Scale bars, 50 μm. Right, the number of CD138+ cells per section, normalized to lamina propria area (sham, n = 8 mice; gluten, n = 8 mice; gluten→GFD, n = 9 mice). b, c, Serum levels of anti-gliadin IgG (b) and anti-gliadin IgA (c) were measured by ELISA. Serum was collected sequentially in the same mice before gluten feeding (untreated), 30 days after gluten feeding (gluten d30), and 30 days after reversion to a GFD (GFD d60) (n = 12 or 13 mice per group for anti-gliadin IgG or IgA, respectively). d, Mucosal IgA deposits (red) and TG2 (green) were identified by immunohistochemistry staining of frozen ileum sections. Scale bars, 100 μm. e, Serum levels of anti-TG2 IgG antibody measured by ELISA 30 days after gluten feeding (n = 13 mice per group). f, Quantification of IELs among IECs was performed on H&E-stained ileum sections (sham, n = 8 mice; gluten, n = 12 mice; gluten→GFD, n = 9 mice). g, Left, granzyme B staining by immunohistochemistry on paraffin-embedded ileum sections. Scale bars, 20 μm. Right, number of granzyme B+ IELs per 100 IECs per mouse (sham, n = 6 mice; gluten, n = 10 mice; gluten→GFD, n = 9 mice). h, Expression of Gzmb in the intestinal epithelium was measured by qPCR. Relative expression levels in gluten and gluten→GFD groups were normalized against the expression levels observed in sham-fed DQ8-Dd-villin-IL-15tg mice (n = 6 mice per group). i, j, DQ8-Dd-villin-IL-15tg mice raised on a GFD were maintained on a GFD, fed gluten for 30 days, or fed gluten for 30 days and then reverted to a GFD for 60 or 90 days. The intestinal epithelium was isolated and analysed by flow cytometry, and IELs were identified as TCRβ+CD4CD8αβ+ cells. Granzyme B+ IELs are indicated by percentage (i) and MFI (j) (sham, n = 6 mice; gluten, n = 8 mice; gluten→GFD, n = 8 mice). k, Expression of Prf1 in the intestinal epithelium was measured by qPCR. Analysis was performed as in h (gluten, n = 6 mice; gluten→GFD, n = 7 mice). l, The intestinal epithelium was isolated and analysed by flow cytometry. IELs were identified as TCRβ+CD4CD8+ cells. In parallel, IELs were quantified among IECs on H&E-stained ileum sections. NKG2D+NKG2 IELs are indicated by absolute number per 100 IECs (sham, n = 8 mice; gluten, n = 11 mice; gluten→GFD, n = 9 mice). Data are mean ± s.e.m. (b, c, gl) or mean values (a, e, f) from three (a, d, g), four (b, c, e, f) or two (hl) independent experiments. P values were determined by one-way ANOVA with Tukey’s multiple comparison test (ac, f, g, i, j, l) or unpaired, two-tailed, t-test (e, h, k). Source data

Extended Data Fig. 5 Effect of CD8+ T cell depletion on antibody production and epithelial stress markers.

af, Ten-week-old DQ8-Dd-villin-IL-15tg mice were treated with 200 μg anti-CD8α antibody (clone 2.43) or its isotype control (rat IgG2b) twice before and during the course of gluten feeding. a, Experimental scheme. b, c, Representative dot plots showing depletion efficiency in the blood (b) and epithelium (c) of DQ8-Dd-villin-IL-15tg mice after 30 days of anti-CD8α treatment. d, e, Expression of Rae1 (d) and Qa-1 (e) genes in the intestinal epithelium was determined by qPCR. Relative expression levels were normalized against the expression levels observed in sham-fed DQ8-Dd-villin-IL-15tg mice (gluten + isotype, n = 16; gluten + anti-CD8, n = 14). f, Serum levels of anti-DGP IgG 30 days after gluten feeding (n = 11 mice per group). Data are mean ± s.e.m. (d, e) or mean values (f) from four (be) or three (f) independent experiments. P values determined by unpaired, two-tailed, t-test (df). Source data

Extended Data Fig. 6 HLA-DQ8 is required for the expansion of cytotoxic IELs.

a, Cells were isolated from the mesenteric lymph nodes of DQ8-Dd-villin-IL-15tg and Dd-villin-IL-15tg mice. CD11c+CD103+ dendritic cells were analysed by flow cytometry for their expression of MHC class II and HLA-DQ8 molecules. bf, DQ8-Dd-villin-IL-15tg and Dd-villin-IL-15tg mice raised on a GFD were maintained on a GFD or fed gluten for 30 days. b, Serum levels of anti-DGP IgG antibodies were measured by ELISA. Serum was collected 30 days after gluten feeding (DQ8-Dd-villin-IL-15tg, sham n = 17, gluten n = 28; Dd-villin-IL-15tg, sham n = 17, gluten n = 28). c, Expression of Ifng in the lamina propria was measured by qPCR. Relative expression levels in gluten groups were normalized against the expression levels observed in sham-fed DQ8-Dd-villin-IL-15tg mice and sham-fed Dd-villin-IL-15tg (DQ8-Dd-villin-IL-15tg, n = 8; Dd-villin-IL-15tg, n = 9). df, The intestinal epithelium was isolated and analysed by flow cytometry. IELs were identified as TCRβ+CD4CD8+ cells. d, NKG2D+NKG2 IELs are indicated by absolute number per 100 IECs (DQ8-Dd-villin-IL-15tg, sham n = 8, gluten n = 16; Dd-villin-IL-15tg, sham n = 9, gluten n = 22). e, Granzyme B+ IELs are indicated by absolute number per 100 IECs (DQ8-Dd-villin-IL-15tg, sham n = 8, gluten n = 8; Dd-villin-IL-15tg, sham n = 9, gluten n = 10). f, MFI of intracellular granzyme B (DQ8-Dd-villin-IL-15tg, sham n = 5, gluten n = 6; Dd-villin-IL-15tg, sham n = 5, gluten n = 5). Data are mean values (b, df) or mean ± s.e.m. (c) from six (b, d), three (c), four (e) or two (f) independent experiments. P values were determined by ANOVA with Tukey’s multiple comparison test (b, df) or unpaired, two-tailed, t-test (c). Source data

Extended Data Fig. 7 CD4+ T cells are required for the pathogenesis of coeliac disease.

al, DQ8-Dd-villin-IL-15tg mice were treated with 200 or 400 μg of depleting anti-CD4 antibody (clone GK1.5) or isotype control (rat IgG2b) twice before and during the course of gluten feeding. a, Experimental scheme. b, c, Representative dot plots showing depletion efficiency in the blood (b) and mesenteric lymph nodes (MLN) (c) of DQ8-Dd-villin-IL-15tg mice. d, The intestinal epithelium was isolated and analysed by flow cytometry. IELs were identified as TCRβ+CD4CD8+ cells. NKG2D+NKG2 IELs are indicated by absolute number per 100 IECs. (sham, n = 8; gluten + isotype, n = 20, gluten + anti-CD4, n = 12). eh, Expression of Gzmb (e), Prf1 (f), Rae1 (g) and Qa-1 (h) in the intestinal epithelium as measured by qPCR. Relative expression levels were normalized against the expression levels observed in sham-fed DQ8-Dd-villin-IL-15tg mice (gluten + isotype, n = 12 to 17, gluten + anti-CD4, n = 8). il, Serum levels of anti-DGP IgG (i), anti-gliadin IgG2c (j), anti-gliadin IgG (k) and anti-gliadin IgA (l) antibodies were measured by ELISA from serum collected 30 days after gluten feeding (sham, n = 11; gluten + isotype, n = 25-26, gluten + anti-CD4, n = 11). Data are mean values (d, il) or mean ± s.e.m. (eh) from four (bl) independent experiments. P values determined by ANOVA with Tukey’s multiple comparison test (d, il) or unpaired, two-tailed, t-test (eh). Source data

Extended Data Fig. 8 Transcriptional programs promoted by HLA-DQ8, IL-15 and gluten.

a, b, We contrasted the enrichment scores in the epithelium and the lamina propria for DQ8-Dd-villin-IL-15tg (x axis) and DQ8 (y axis) mice for all pathways enriched at an FDR < 5% in at least one of the strains. Positive or negative scores represent enrichments among genes that are more highly or lowly expressed in gluten-fed mice, respectively. The bottom right quadrant refers to pathways that in response to gluten are upregulated in DQ8-Dd-villin-IL-15tg but downregulated in DQ8 mice. c, Gene Ontology terms significantly enriched among genes differently expressed in response to gluten challenge in DQ8, Dd-villin-IL-15tg and DQ8-Dd-villin-IL-15tg mice. d, Heat map of genes showing a stronger response to gluten in DQ8-Dd-villin-IL-15tg mice as compared to DQ8 and Dd-villin-IL-15tg mice. The colours reflect the magnitude of the response to gluten (log2-transformed scale), and the stars highlight Gene Ontology terms associated with each of the genes plotted.

Extended Data Fig. 9 Effect of IFNγ and IL-21 neutralization on the development of coeliac disease.

ai, DQ8-Dd-villin-IL-15tg mice were treated with 500 μg of anti-IFNγ (clone XMG1.2) and/or anti-IL21R (clone 4A9) antibodies or corresponding isotype controls (rat IgG1 and rat IgG2a, respectively) once before, and every 3 days during the course of gluten feeding as indicated. a, Ratio of the morphometric assessment of villus height to crypt depth (sham, n = 8, gluten + isotype, n = 7, gluten + anti-IL-21R, n = 6). b, Quantification of IELs among IECs was performed on H&E-stained ileum sections (sham, n = 8, gluten + isotype, n = 7, gluten + anti-IL-21R, n = 6). c, The intestinal epithelium was isolated and analysed by flow cytometry. IELs were identified as TCRβ+CD4CD8+ cells. Granzyme B+ IELs are indicated by absolute number per 100 IECs (sham, n = 8, gluten + isotype, n = 7, gluten + anti-IL-21R, n = 6). d, Granzyme B+ IELs are indicated as in c (sham, n = 12, gluten + isotype, n = 16, gluten + anti-IL-21R + anti- IFNγ, n = 11). e, Quantification of IELs among IECs was performed on H&E-stained ileum sections (sham, n = 13, gluten + isotype, n = 17, gluten + anti-IL-21R+ anti-IFNγ, n = 11). f, g, Serum levels of anti-DGP IgG (f) and anti-gliadin IgG2c (g) antibodies were measured by ELISA. Serum was collected 30 days after gluten feeding (sham, n = 5 and 6, gluten + isotype, n = 8 and 7, gluten + anti-IL-21R, n = 6). h, i, Serum levels of anti-DGP IgG (h) and anti-gliadin IgG2c (i) antibodies were measured as in f and g (sham, n = 6 and 6, gluten + isotype, n = 8, gluten + anti-IFNγ, n = 6). Data are mean values (ai) from two (ac, fi) or four (d, e) independent experiments. P values determined by ANOVA with Tukey’s multiple comparison test (ai). Source data

Extended Data Fig. 10 Validation of DQ8-Dd-villin-IL-15tg mice as a preclinical mouse model of coeliac disease.

a, Serum anti-native gliadin peptides and anti-DGP IgG levels in gluten-fed DQ8-Dd-villin-IL-15tg mice treated with TG2 inhibitors or vehicle (gluten) were compared by ELISA (gluten n = 17; gluten + TG2 inhibitors n = 13). In all cases, serum samples were obtained on day 30 after initiating the gluten challenge. b, Similar gene regulatory mechanisms underlie the development of coeliac disease in humans and DQ8-Dd-villin-IL-15tg mice. Contrast between the Gene Ontology terms enriched among genes induced by gluten challenge in DQ8-Dd-villin-IL-15tg mice (depicted in the form of −log10(P values) on the x axis) and the Gene Ontology terms enriched among genes differently expressed between patients with coeliac disease and healthy controls (depicted in the form of −log10(P values) on the x axis) in the epithelial compartment (a) or the lamina propria (b). The transcriptional comparison was made between the intestinal epithelium and lamina propria of gluten-fed DQ8-Dd-villin-IL-15tg mice and whole duodenal biopsies of patients with active coeliac disease. Data in a are mean values from four independent experiments. P value determined by paired, two-tailed, t-test). Source data

Extended Data Fig. 11 Interplay between IL-15, TG2 and HLA-DQ8 promote the development of villous atrophy.

a, Representation of the respective roles of HLA-DQ8, IL-15, IFNγ, TG2, CD4+ and CD8+ T cells in promoting villous atrophy. IL-15 upregulation in the lamina propria is required to induce the adaptive anti-gluten TH1 response, and HLA-DQ8 facilitates and enhances the IFNγ response that is required for the development of villous atrophy. However, the adaptive TH1 immune response promoted by HLA-DQ8 and IL-15 in the lamina propria is insufficient to cause tissue destruction. It needs to synergize with IL-15 in the epithelium to further promote the expansion of cytolytic IELs and their degranulation, leading to CD8+ T cell-dependent killing of epithelial cells and villous atrophy. The value of this mouse model as a gluten- and HLA-DQ8-dependent pre-clinical model for coeliac disease is further emphasized by the finding that TG2 inhibition prevents villous atrophy. b, Coeliac disease can be represented as a jigsaw puzzle in which each piece representing one component of the anti-gluten immune response must interlock to lead to the development of villous atrophy—the diagnostic hallmark of active coeliac disease.

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Reporting Summary

Supplementary Data

Data Set 1: GSEA hallmark pathways.

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Data Set 2: Differentially expressed genes between sham and gluten-fed mice.

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Abadie, V., Kim, S.M., Lejeune, T. et al. IL-15, gluten and HLA-DQ8 drive tissue destruction in coeliac disease. Nature 578, 600–604 (2020). https://doi.org/10.1038/s41586-020-2003-8

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