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A human autoimmune organoid model reveals IL-7 function in coeliac disease

Nature volume 632, pages 401–410 (2024)Cite this article

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Abstract

In vitro models of autoimmunity are constrained by an inability to culture affected epithelium alongside the complex tissue-resident immune microenvironment. Coeliac disease (CeD) is an autoimmune disease in which dietary gluten-derived peptides bind to the major histocompatibility complex (MHC) class II human leukocyte antigen molecules (HLA)-DQ2 or HLA-DQ8 to initiate immune-mediated duodenal mucosal injury1,2,3,4. Here, we generated air–liquid interface (ALI) duodenal organoids from intact fragments of endoscopic biopsies that preserve epithelium alongside native mesenchyme and tissue-resident immune cells as a unit without requiring reconstitution. The immune diversity of ALI organoids spanned T cells, B and plasma cells, natural killer (NK) cells and myeloid cells, with extensive T-cell and B-cell receptor repertoires. HLA-DQ2.5-restricted gluten peptides selectively instigated epithelial destruction in HLA-DQ2.5-expressing organoids derived from CeD patients, and this was antagonized by blocking MHC-II or NKG2C/D. Gluten epitopes stimulated a CeD organoid immune network response in lymphoid and myeloid subsets alongside anti-transglutaminase 2 (TG2) autoantibody production. Functional studies in CeD organoids revealed that interleukin-7 (IL-7) is a gluten-inducible pathogenic modulator that regulates CD8+ T-cell NKG2C/D expression and is necessary and sufficient for epithelial destruction. Furthermore, endogenous IL-7 was markedly upregulated in patient biopsies from active CeD compared with remission disease from gluten-free diets, predominantly in lamina propria mesenchyme. By preserving the epithelium alongside diverse immune populations, this human in vitro CeD model recapitulates gluten-dependent pathology, enables mechanistic investigation and establishes a proof of principle for the organoid modelling of autoimmunity.

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Fig. 1: Human ALI small-intestine organoids preserve epithelium, mesenchyme and diverse immune populations without exogenous reconstitution.
Fig. 2: Gliadin induces epithelial IL-15 production and apoptosis in coeliac organoids.
Fig. 3: Gliadin induces T-cell expansion in CeD organoids.
Fig. 4: Gliadin induces network adaptive and innate immune responses in CeD organoids.
Fig. 5: IL-7 necessity and sufficiency during gluten-induced CeD organoid cytotoxicity.
Fig. 6: IL-7 production is increased by gliadin in CeD organoids and upregulated in active CeD biopsies.

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Data availability

Data sets for scRNA-seq have been deposited in Gene Expression Omnibus with the accession code GSE200075. Source data are provided with this paper.

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Acknowledgements

We thank members of the Kuo, Davis, Mellins and Sollid groups for discussions; the Stanford FACS, Functional Genomics, Human Histology (P. Chu), Cell Sciences Imaging and Human Immune Monitoring cores for technical expertise; the Stanford Tissue Bank for providing surgical samples; E. Sanjines, A. Adiao, D. Souki, G. Tan and G. Masarweh for collection and delivery of endoscopy samples; B. Simonsen for the HLA-DQ molecules used for tetramer assembly; and J. and R. Triebsch for support from the Stanford Celiac Translational Research Program. This work was also supported by funding from the Stanford Medicine Children’s Health Center for IBD and Celiac Disease. V.v.U. was supported by a Netherlands Organization for Scientific Research Rubicon grant (452181214). We acknowledge funding from the South-Eastern Norway Regional Health Authority (projects 2016113 and 2020027 to L.M.S.), a Stanford Maternal Child Health Research Institute seed grant (C.J.K.), NIH RM1-HG007735 (H.Y.C.), NIH U19AI057229 (M.M.D.), NIH U01DK085527, U19AI116484, R01CA251514, R01DK130414, R01DK115728 (C.J.K.), the NIDDK Intestinal Stem Cell Consortium and the NIAID Biomimetic U19 Consortium. We dedicate this work to the memory of Elizabeth D. Mellins, whose experimental design and guidance were crucial for this study.

Author information

Authors and Affiliations

  1. Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    António J. M. Santos, Vincent van Unen, Zhongqi Lin, Steven M. Chirieleison, Nhi Ha, Arpit Batish, Joshua E. Chan, Jose Cedano, Elisa T. Zhang, Qinghui Mu, Alexander Guh-Siesel, Madeline Tomaske, Shannon S. Choi, Kasper Karlsson, Andrew Ha & Calvin J. Kuo

  2. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA

    Vincent van Unen & Mark M. Davis

  3. Institute for Immunity, Transplantation and Infection, Stanford University School of Medicine, Stanford, CA, USA

    Vincent van Unen, Jing Li & Mark M. Davis

  4. Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA

    Steven M. Chirieleison, Deana Colburg, Sushama Varma & Bing M. Zhang

  5. K. G. Jebsen Coeliac Disease Research Centre, Institute of Clinical Medicine, University of Oslo, Oslo, Norway

    Asbjørn Christophersen & Ludvig M. Sollid

  6. Department of Immunology, Oslo University Hospital, Oslo, Norway

    Asbjørn Christophersen & Ludvig M. Sollid

  7. Department of Rheumatology, Dermatology and Infectious Diseases, Oslo University Hospital, Oslo, Norway

    Asbjørn Christophersen

  8. Department of Chemistry, Stanford University School of Medicine, Stanford, CA, USA

    Ani Baghdasaryan & Hongjie Dai

  9. Center for Personal Dynamic Regulomes, Stanford University School of Medicine, Stanford, CA, USA

    Kathryn E. Yost & Howard Y. Chang

  10. Department of Dermatology, Stanford University School of Medicine, Stanford, CA, USA

    Kathryn E. Yost & Howard Y. Chang

  11. Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Kasper Karlsson

  12. Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA

    Kasper Karlsson & Howard Y. Chang

  13. Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

    Zachary M. Sellers & Elizabeth D. Mellins

  14. Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, CA, USA

    Howard Y. Chang & Mark M. Davis

  15. Department of Pediatric Surgery, Stanford University School of Medicine, Stanford, CA, USA

    James C. Y. Dunn

  16. Division of Gastroenterology and Hepatology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA

    Nielsen Q. Fernandez-Becker

Authors
  1. António J. M. Santos
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  2. Vincent van Unen
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  3. Zhongqi Lin
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  10. Qinghui Mu
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  11. Alexander Guh-Siesel
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  14. Sushama Varma
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  15. Shannon S. Choi
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  16. Asbjørn Christophersen
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  17. Ani Baghdasaryan
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  18. Kathryn E. Yost
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  19. Kasper Karlsson
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  20. Andrew Ha
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  21. Jing Li
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  22. Hongjie Dai
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  24. Howard Y. Chang
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  25. James C. Y. Dunn
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  26. Bing M. Zhang
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  27. Elizabeth D. Mellins
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  28. Ludvig M. Sollid
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  29. Nielsen Q. Fernandez-Becker
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  30. Mark M. Davis
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  31. Calvin J. Kuo
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Contributions

A.J.M.S. conceived, designed and performed experiments, analysed data and wrote the manuscript. V.v.U. conceived experiments and analysed scRNA-seq data. Z.L. designed experiments, did organoid cultures, confocal imaging and analysis, analysed scRNA-seq data and did RT–qPCR. S.M.C. collected tissue FFPE blocks and coordinated sectioning for haplotyping and IL7 in situ hybridization and analysed data. N.H. did organoid cultures and RT–qPCR. A. Batish did RT–qPCR. J.E.C. measured and analysed organoid sizes. J.C. did organoid cultures, cryopreservation and recovery, imaging and analysis. E.T.Z. sectioned frozen tissue blocks and helped with their staining and imaging. Q.M. did organoid cultures. A.G.-S. did confocal imaging and analysis. M.T. did RT–qPCR and data analysis. D.C. sectioned FFPE tissue blocks and did IL7 in situ hybridization. S.V. did IL7 in situ hybridization. S.S.C. did confocal imaging and schematic design. A.C. produced HLA-DQ monomers for tetramer assembly. A. Baghdasaryan provided resources. K.E.Y. prepared libraries for scRNA-seq. K.K. used Cell Ranger for scRNA-seq. A.H. assisted with 10x Genomics cell capture. J.L. did HLA genotyping. H.D. provided resources and supervision. Z.M.S. provided samples and guidance. H.Y.C. provided resources and supervision. J.C.Y.D. provided surgical samples. B.M.Z. did HLA genotyping and analysis. E.D.M. conceived and designed experiments and provided resources and supervision. L.M.S. provided resources, supervision and guidance. N.Q.F.-B. identified CeD patients and controls, coordinated and collected endoscopy samples and provided guidance. M.M.D. provided resources, supervision and guidance. C.J.K. conceived and designed experiments, analysed data and wrote the manuscript.

Corresponding author

Correspondence to Calvin J. Kuo.

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Competing interests

C.J.K. and A.J.M.S. are inventors on patent WO 2020/247528 describing methods and uses of patient-derived celiac intestinal organoids. C.J.K. and M.M.D. are founders of Mozart Therapeutics and NextVivo, Inc. L.M.S has been a consultant during the last 3 years for BMS, GSK, Mozart Therapeutics, Ono Pharmaceutical, Precigen ActoBio, Sanofi-Aventis, SQZ Biotech, Takeda and Topas Therapeutics. All other authors declare no competing interests.

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Nature thanks Nadine Cerf-Bensussan, Toshiro Sato and Detlef Schuppan for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Small intestine ALI organoids possess different mesenchymal and epithelial cell types.

a, IF whole-mount staining of small intestine organoids at day 14 showing SMA+ or PDGFRA+ fibroblasts, CD31+ endothelial cells and PGP9.5+ neurons (green), ECAD+ epithelium (white) and DAPI (blue) (representative images from n = 3 biological replicates). b, IF whole-mount staining of small intestine organoids at day 14 showing MUC2+ goblet cells, CHGA+ enteroendocrine cells, LYZ1+ Paneth cells (green), ECAD+ epithelium (white) and DAPI (blue) (representative images from n = 3 biological replicates). c, Enlargement of organoid cup-shaped MUC2+ goblet cells (representative image from n = 3 biological replicates). d, Violin plots of CD14 and CD68 mRNA expression from CeD organoid scRNA-seq and a scatter plot of CD14 and CD68 mRNA co-expression in the myeloid compartment. e, Whole-mount IF staining of small intestine ALI organoid CD4+ (green) and CD8+ (red) T cells, showing enrichment of CD8+ T cells within the EPCAM+ epithelial compartment (white). DAPI (blue). In contrast, CD4+ T cells localize to non-epithelial lamina propria-like areas (representative image from n = 3 biological replicates). All scale bars are 100 µm, except (c) in which the scale bar is 50 µm.

Extended Data Fig. 2 Duodenal ALI organoids contain diverse immune populations, related to Fig. 1.

a, Integrated UMAP plot of CD45+-sorted cells from scRNA-seq, revealing diverse immune populations in small intestine ALI organoids at day 14, n = 6 CeD patients. b, Violin plots showing expression of genes used to identify the immune populations shown in (a). c, UMAP plots of overlap between tissue and organoid CD45+ immune populations as in (a, b). d, scRNA-seq Jaccard index of TCR overlap between fresh small intestine tissue (n = 1 CeD patient) and ALI organoids (n = 4 CeD patients). e, Integrated UMAP from scRNA-seq of active CeD organoid T cells (n = 6 patients). Cells expressing KIR3DL1 or KIR2DL3 are rendered in red. f, Plot of CD8+ T cells from (e). g, Integrated UMAP from scRNA-seq of active CeD organoid T cells (top left) (n = 6 patients). Cells in red exhibit expression of KIR3DL1 or KIR2DL3 (top right), NKG2C (bottom left) and NKG2D (bottom right). h, Pie bar graph showing organoid-derived TCR counts in which each segment represents a unique clonotype, n = 5 patients. Expanded clonotypes (TCR counts ≥ 2) are indicated in red.

Extended Data Fig. 3 Cytokine supplementation and cryopreservation of intestinal ALI organoids.

a, FACS-based tSNE plots depicting time course abundance of EPCAM+ and CD45+ cells (top) and CD4+ and CD8+ T cells (bottom) as a percentage of total live single ileal organoid cells with or without addition of IL-2 and IL-7, representative experiment of n = 3 biological replicates. b, Organoids grown for 14 days (control) have similar percentages of epithelium and immune components as organoids grown for 5 days, frozen in-gel at −80 °C, cryorecovered, and replated for the indicated durations. c-d, ALI organoids demonstrate persistent growth after being frozen in-gel at −80 °C, cryorecovered and replated (c, arrows), with maintenance of epithelial protrusions by H&E (d). Numerous air bubbles in the collagen are present on initial plating post-cryorecovery and progressively disappear with culture. (b-d) depict representative experiments from n = 4 biological replicates. Scale bar is 5 mm for (c) and 100 µm for (d).

Extended Data Fig. 4 Gliadin induces loss of villus-like structures in CeD organoids.

a, Duodenal ALI organoids from celiac (CeD) or non-celiac control donors were established for 9–12 days followed by gliadin or CLIP treatment for 2 days before analysis, unless stated otherwise. The gliadin peptides were a 1:1 mixture of deamidated immunodominant, HLA-DQ2.5-restricted, glia-α1 (LQPFPQPELPYPGS) and glia-α2 (APQPELPYPQPGS) gluten epitopes. b-d, Confirmatory IF staining of sections of human duodenum tissue showing IL-15 (red) in (a), SI (red) in (b) and APOA4 (red) in (c); DAPI (blue) (representative images from n = 3 biological replicates). e, Quantification of SI mRNA in FACS-sorted organoid EPCAM+ cells from 2-day gliadin-treated control or active CeD organoids. RT-qPCR, expressed as a ratio of gliadin:CLIP treatment, from control (n = 4) or CeD (n = 5) biological replicates. Box plots show the median as the center line, the interquartile range as the box limits and the whiskers represent the min and max. *, P = 0.0381; two-tailed Mann-Whitney test. f, Representative H&E staining of different sections of control or active CeD organoids after 2-day gliadin or CLIP treatment. Arrows denote regions where epithelial protrusions are absent. g, Quantification of epithelial protrusions per organoid circumference from (f); control (N = 6 biological replicates), CeD (N = 7 biological replicates), each data point is from an individual organoid. Scatter plots show the median as the center line and the whiskers represent the min and max. ***=P < 0.0001; two-tailed Mann-Whitney test. All scale bars are 100 µm. All CeD organoids were DQ2.5+.

Source Data

Extended Data Fig. 5 Gliadin induces epithelial proliferation in CeD organoids.

a, Representative IF staining of sections of active CeD organoids after 2-day gliadin or CLIP treatment in EN media showing proliferative KI67+ cells (green), ECAD (red) and DAPI (blue). Scale bar is 50 µm. b, Quantification of KI67 fluorescence from (a), control (n = 3 biological replicates), CeD (n = 5 biological replicates); each data point is from an individual organoid. ***, P < 0.0001; two-tailed Mann-Whitney test. c, Representative brightfield images of active CeD organoids before and after 2-day treatment with gliadin or CLIP peptides. Scale bar is 5 mm. d, Automated quantification of fold change in CeD organoid area from (c), 2 days after treatment with gliadin or CLIP. n = 10 CeD patients. **, P = 0.002; two-tailed Wilcoxon test. e, LGR5 RT-qPCR from FACS-sorted organoid EPCAM+ cells as ratio of gliadin:CLIP treatment for 2 days in organoids from control (n = 7 biological replicates) or active CeD (n = 8 biological replicates). **, P = 0.0012; two-tailed Mann-Whitney test. f, PCNA RT-qPCR from FACS-sorted organoid EPCAM+ cells as ratio of gliadin:CLIP treatment for 2 days in control or active CeD organoids, (n = 7 biological replicates each). **, P = 0.007; two-tailed Mann-Whitney test. g, CCND1 RT-qPCR from FACS-sorted organoid EPCAM+ cells as ratio of gliadin:CLIP treatment for 2 days in organoids from control (n = 8 biological replicates) or active CeD (n = 7 biological replicates). ***, P = 0.0003; two-tailed Mann-Whitney test. All box plots show the median as the center line, the interquartile range as the box limits and the whiskers represent the min and max. All CeD organoids were DQ2.5+.

Source Data

Extended Data Fig. 6 TCR sequencing from CeD organoid scRNA-seq reveals known and suspected gliadin-specific TCR motifs.

a, scRNA-seq integrated UMAP plot highlighting TCR-expressing T cells in active CeD organoids at day 14, n = 5 CeD patients. b, GLIPH homology analysis showing conserved CDR3 motifs (red) found between active CeD organoids and gliadin-specific published sequences, n = 5 CeD patients. All CeD organoids in this figure were HLA-DQ2.5+.

Extended Data Fig. 7 Two additional biological replicates of scRNA-seq-derived dot plots.

Dot plots from organoid scRNA-seq from two active CeD patients (a, b); a third patient is shown in Fig. 4a. Depiction of mean expression levels and corresponding percent population expression amongst active CeD organoid CD4+ and CD8+ T cells, Treg, plasma B cells and myeloid cells after 2-day gliadin or CLIP treatment. The patient in (a) is HLA-DQ2.5, as is the patient in Fig. 4a. The patient in (b) is HLA-DQ2.2, which manifests low-affinity binding to the HLA-DQ2.5 gliadin peptides used in the study (Bodd et al, Gastroenterology, 2012 Mar;142(3):552-61).

Extended Data Fig. 8 ScRNA-seq-based interactome analysis of novel gliadin-induced immune interactions in CeD organoids.

a, Overview of unique CellPhoneDB immune interactions found in 2-day CLIP- or gliadin-treated active CeD organoids, stratified by immune cell type (CD4+ T, CD8+ T, myeloid, NK and Treg cells). Columns indicate sending:receiving cell type and rows indicate ligand-receptor pairs. P values are indicated by circle size. The mean (log2) average expression levels of interacting molecule 1 and interacting molecule 2 are indicated by the color gradient. b, Corresponding schematic showing potential interactions between immune cells in CeD. Integrated data from n = 4 CeD patients, 3 DQ2.5+ and 1 DQ2.2+.

Extended Data Fig. 9 Sc-RNAseq-based interactome of B cells and plasma cells, and BCR sequence consensus.

a, B cell- and plasma B cell-specific immune interactomes derived from scRNA-seq CellPhoneDB analysis showing 117 unique B and plasma-cell driven interactions in gliadin-treated organoids and absence of unique interactions in CLIP, integrated data from n = 4 CeD patients. b, BCR sequence consensus analysis from scRNA-seq of matched CDR3 sequences from active CeD organoids and published anti-TG2 CDR3 sequences categorized by length; n = 3 CeD patients.

Extended Data Fig. 10 BCR sequencing from CeD organoid scRNA-seq reveals extensive overlap with public anti-TG2 CeD-specific motifs.

a, scRNA-seq integrated UMAP plot highlighting BCR-expressing B and plasma cells in active CeD organoids, n = 3 CeD patients. b, Homology analysis showing conserved CDR3 sequences (red) found between active CeD organoids and anti-TG2 CeD-specific published sequences, n = 3 CeD patients.

Source Data

Extended Data Fig. 11 IL-7 is upregulated in active celiac duodenal biopsy tissue.

a, Luminex protein analysis of organoid conditioned media from active CeD (N = 7 biological replicates) or control (N = 4 biological replicates) showing fold-increases of IL-7 as ratio of gliadin:CLIP treatment after 2 days. Box plots show the median as the center line, the interquartile range as the box limits and the whiskers represent the min and max. ns, P = 0.072; two-tailed Mann-Whitney test. b-c, Representative IF staining using a rabbit (Rb) anti-IL-7 antibody (red) in fresh duodenal biopsies from (b) 14 remission CeD patients (previously diagnosed with CeD but on gluten-free diet) versus (c) 14 CeD patients with active disease, showing increased IL-7 levels in the latter. Epithelium (CK19, green); DAPI (blue). Figure 6c shows staining for a 15th patient in remission and a 15th patient with active CeD, and quantitation is presented in Fig. 6d. (GFD, n = 15 donors) or active CeD (n = 15 donors). d, Representative IF staining using a mouse (Ms) anti-IL-7 antibody (red) in fresh duodenal biopsies from (b), 4 remission CeD patients versus 4 patients with active CeD. This confirmed elevated IL-7 expression in active CeD seen with a different antibody than in (b) and (c). Scale bars are 100 µm.

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This file contains sequential FACS gating strategy, donor DQ-typing and demographics, and primer sequences used for RT-qPCR.

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Santos, A.J.M., van Unen, V., Lin, Z. et al. A human autoimmune organoid model reveals IL-7 function in coeliac disease. Nature 632, 401–410 (2024). https://doi.org/10.1038/s41586-024-07716-2

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