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PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Fig. 2ECs in CD.a, UMAP plot of small intestinal epithelial EPCAM cells in HCs (n = 3) and in participants with CD (n = 5).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
b, Bubble plot showing the expression of selected genes defining specific cluster identities.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Scaled gene expression indicated by color; proportion of cells expressing the gene indicated by bubble size.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
c, Local neighborhood enrichment of EPCAM cells in ACD versus HCs.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Color indicates enrichment (log fold change (FC)) of cells in ACD versus HCs in that UMAP neighborhood; size of dot indicates false discovery rate (FDR)-adjusted −log10 values.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
d, TA cells (left) and early enterocytes (right) in HCs and CD, as a proportion of total EPCAM cells.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
e, Pseudotime trajectory of gene expression of EPCAM ECs, colored by pseudotime axis (left), cluster identity (middle) and lineage (right).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Arrows indicate putative direction of cell differentiation.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
f, Density of cells along pseudotime trajectory axis split by disease state: ACD (red), TCD (blue) and HCs (gray).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
g, Smoothed heat map showing expression of selected genes related to intestinal absorption along pseudotime trajectories relating to secretory (toward left) and absorptive (toward right) lineage.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
h, Volcano plot displaying differentially expressed gene transcripts between HCs and ACD in total ECs.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
d, Unpaired two-tailed t-test.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Data are presented as mean values ± s.e.m.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
a, UMAP plot of small intestinal epithelial EPCAM cells in HCs (n = 3) and in participants with CD (n = 5).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
b, Bubble plot showing the expression of selected genes defining specific cluster identities.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Scaled gene expression indicated by color; proportion of cells expressing the gene indicated by bubble size.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
c, Local neighborhood enrichment of EPCAM cells in ACD versus HCs.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Color indicates enrichment (log fold change (FC)) of cells in ACD versus HCs in that UMAP neighborhood; size of dot indicates false discovery rate (FDR)-adjusted −log10 values.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
d, TA cells (left) and early enterocytes (right) in HCs and CD, as a proportion of total EPCAM cells.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
e, Pseudotime trajectory of gene expression of EPCAM ECs, colored by pseudotime axis (left), cluster identity (middle) and lineage (right).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Arrows indicate putative direction of cell differentiation.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
f, Density of cells along pseudotime trajectory axis split by disease state: ACD (red), TCD (blue) and HCs (gray).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
g, Smoothed heat map showing expression of selected genes related to intestinal absorption along pseudotime trajectories relating to secretory (toward left) and absorptive (toward right) lineage.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
h, Volcano plot displaying differentially expressed gene transcripts between HCs and ACD in total ECs.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
d, Unpaired two-tailed t-test.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Data are presented as mean values ± s.e.m.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
A LYZ Paneth cell-like population (MMP7REG1ASOD3PLA2G2A) was also identified (Fig. 2a,b), although defensin gene expression was not detected.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
This population expressed PGC, mucins including MUC5AC, MUC1 and MUC6 and AQP5, suggesting it also contained Brunner’s gland cells or ectopic gastric pyloric gland cells.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
This cell type was enriched in active celiac disease (ACD; Fig. 2c,d), perhaps in response to IFNγ.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Thus, this population could represent inflammation-driven gastric cell metaplasia.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Transit-amplifying (TA) cells were increased in CD, along with enrichment of uniform manifold approximation and projection (UMAP) areas corresponding to EC progenitors (stem cells, TA cells and early enterocytes; Fig. 2c,d).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
This persisted in treated celiac disease (TCD; Extended Data Fig. 1b,c).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
In parallel, more actively cycling ECs were observed in ACD and TCD (Extended Data Fig. 1d,e).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Pseudotime analyses identified epithelial developmental trajectories, from undifferentiated progenitor states toward absorptive and secretory lineages (Fig. 2e).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
In CD, ECs were shifted to earlier pseudotime states, with loss of mature ECs (Fig. 2f).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
CCL25, encoding the ligand for CCR9 (implicated in CD pathogenesis), was expressed predominantly by progenitor cells (Fig. 2b and Extended Data Fig. 1f).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
We examined putative EC functions through functional gene-set analysis (Extended Data Fig. 1a), identifying functions of secretory Paneth-like/Brunner’s gland cells (secreted protein and vesicle pathways), BEST4 enterocytes (chloride/anion channel activity), tuft cells (taste perception) and enteroendocrine cells (EECs; peptide hormone processing/secretion).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Mature enterocytes expressed key metabolic and macronutrient catabolic pathways, and active transport and absorption mechanisms.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Early ECs and TA cells did not express these pathways.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Absorptive function genes were limited to cell states at the end of absorptive epithelium pseudotime trajectories, consistent with EC development along the crypt–villus axis (Fig. 2g).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Notably, gene sets related to lipid, carbohydrate, cholesterol, vitamin and iron processing and absorption were all downregulated in mature enterocytes in ACD (Extended Data Fig. 1g–i).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
These transcriptional changes normalized in TCD, although some pathways, including fructose metabolism and lipid catabolism, remained reduced (Extended Data Fig. 1h).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Overall, absorptive capacity is reduced in ACD not simply by reduction in villus surface area, but through a relative increase of EC progenitors lacking absorptive machinery, and pathway downregulation in mature enterocytes.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
ECs in ACD upregulated multiple antigen-presentation molecules, including classical HLA class I and class II genes (except HLA-DQ) and nonclassical genes including HLA-E and HLA-F (Fig. 2h).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Interferon-stimulated genes (types I and II) dominated the epithelial response, including STAT1 (Fig. 2h and Supplementary Table 5).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
The major disease-associated responses were observed in all EC lineages (Extended Data Fig. 1j–l), including antigen-presentation pathways, type I/II interferon responses, lymphocyte-mediated immunity and cytotoxicity and cell adhesion regulation (Extended Data Fig. 1m,n).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Some transcriptional changes were cell-type specific.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
IL32 was highly expressed in ACD by mature enterocytes (Extended Data Fig. 1k), perhaps regulated by interferons.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
The reduction of fatty acid catabolism/transport (APOA1, FABP2), metal ion transport (iron: FTH1, FTL; zinc: SLC39A4) and carbohydrate metabolism (ALDOB, PCK1) was restricted to absorptive lineages, mainly mature enterocytes (Extended Data Fig. 1k,n).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Progenitor cells upregulated genes associated with cell division and differentiation, and downregulated those associated with tissue repair and homeostasis (Extended Data Fig. 1m,n).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Secretory lineages showed increased expression of gut hormone genes, LYZ, and chemokines (CXCL17, CXCL2; Extended Data Fig. 1l).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
The duodenum, where CD inflammation predominates, has sensory and neurohormonal functions.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
We extended EEC clustering, revealing multiple transcriptional states, including NEUROG3 progenitors and EEC subtypes, which showed similar CD-related transcriptional changes to other ECs (Extended Data Fig. 2).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
EEC proportions altered in CD, with increases in NEUROG3 progenitor cells and somatostatin-producing D cells (Extended Data Fig. 2i–k).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
In adults (dataset 1), CD4 T cells formed subsets dominated by TH1-polarized and IL-17-producing helper T (TH17)-polarized effectors, as well as small naive and FOXP3 regulatory populations (Fig. 3a–c and Supplementary Table 6).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
There was a cluster of TFH-like CD4 T cells expressing PDCD1, BTLA, CD28, ICOS and intermediate CXCR5.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Dataset 2 (pediatric) contained analogous subsets (Extended Data Fig. 3a), including CD31CR2 recent thymic emigrants, a CCR7 TFH-like subset and the TFH-like subset expressing PD1, ICOS, CTLA4, BTLA and CD161 at the protein level (Fig. 3d,e).Fig.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
3CD4 T cells in CD.a–c, Intestinal CD4 T cells in health and CD in dataset 1 (adult—10x Genomics).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
a, UMAP plot of intestinal CD4 T cells in health and CD (n = 8).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
b, Bubble plot showing the expression of selected genes defining specific cluster identities.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Scaled gene expression indicated by color; proportion of cells expressing the gene indicated by bubble size.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
c, CD4 T cell UMAP plots overlaid with expression of TNFSF8, PDCD1, TOX2, CXCR3, CXCL13, CD200, CXCR5 and TRBV7-2.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Intestinal CD4 T cells in health and CD in dataset 2 (pediatric—BD Rhapsody; d–f).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
d, UMAP plot of intestinal CD4 T cells in health and CD (n = 15).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
e, Bubble plot showing the expression of selected genes and proteins defining specific cluster identities.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Scaled gene/protein expression indicated by color; proportion of cells expressing the gene/protein indicated by bubble size.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
f, Local neighborhood enrichment of CD4 cells in ACD versus HCs (dataset 1).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Color indicates enrichment (log fold change) of cells in ACD versus HCs in that UMAP neighborhood; size of dot indicates −log10FDR.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
g, Scatterplot of mean proportion (± s.e.)
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
of CD4 T cell clusters in HCs (n = 3) versus ACD (n = 5) in dataset 1.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Clusters above the line of unity are enriched in ACD.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
h,i, Treg (h) and TFH (i) CD4 T cell populations in HCs and CD, as a proportion of total CD4 T cells in dataset 1 (HCs n = 3, ACD n = 5) and dataset 2 (HCs n = 5, ACD n = 10).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
j, UMAP plot of CD4 T cells in dataset 2, overlaid with IL21 and IFNG expression.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
k, UMAP plot of CD4 T cells in dataset 1, overlaid with CXCL13, IL21, IFNG and TNFSF8 expression.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
h,i, Two-sided Mann–Whitney test.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Data are presented as mean values ± s.e.m.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Ab, antibody; Tc17, IL17CD8 T cells; DP, CD4CD8 double positive cells.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
a–c, Intestinal CD4 T cells in health and CD in dataset 1 (adult—10x Genomics).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
a, UMAP plot of intestinal CD4 T cells in health and CD (n = 8).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
b, Bubble plot showing the expression of selected genes defining specific cluster identities.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Scaled gene expression indicated by color; proportion of cells expressing the gene indicated by bubble size.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
c, CD4 T cell UMAP plots overlaid with expression of TNFSF8, PDCD1, TOX2, CXCR3, CXCL13, CD200, CXCR5 and TRBV7-2.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Intestinal CD4 T cells in health and CD in dataset 2 (pediatric—BD Rhapsody; d–f).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
d, UMAP plot of intestinal CD4 T cells in health and CD (n = 15).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
e, Bubble plot showing the expression of selected genes and proteins defining specific cluster identities.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Scaled gene/protein expression indicated by color; proportion of cells expressing the gene/protein indicated by bubble size.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
f, Local neighborhood enrichment of CD4 cells in ACD versus HCs (dataset 1).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Color indicates enrichment (log fold change) of cells in ACD versus HCs in that UMAP neighborhood; size of dot indicates −log10FDR.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
g, Scatterplot of mean proportion (± s.e.)
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
of CD4 T cell clusters in HCs (n = 3) versus ACD (n = 5) in dataset 1.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Clusters above the line of unity are enriched in ACD.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
h,i, Treg (h) and TFH (i) CD4 T cell populations in HCs and CD, as a proportion of total CD4 T cells in dataset 1 (HCs n = 3, ACD n = 5) and dataset 2 (HCs n = 5, ACD n = 10).
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
j, UMAP plot of CD4 T cells in dataset 2, overlaid with IL21 and IFNG expression.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
k, UMAP plot of CD4 T cells in dataset 1, overlaid with CXCL13, IL21, IFNG and TNFSF8 expression.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
h,i, Two-sided Mann–Whitney test.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Data are presented as mean values ± s.e.m.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Ab, antibody; Tc17, IL17CD8 T cells; DP, CD4CD8 double positive cells.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
This TFH-like population in adults and children showed similar phenotypic profiles to those of gut-resident gluten-specific CD4 T cells in CD (Extended Data Fig. 3b), and expressed TOX2, CD200, IL21 and CXCL13.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
The cluster showed enrichment of TRBV7-2, a V-gene enriched in gluten-specific CD4 T cell HLA-DQ2.5 TCR repertoires.
PMC12133578
Immune-epithelial-stromal networks define the cellular ecosystem of the small intestine in celiac disease.
Treg and TFH-like CD4 T cells were increased in ACD in adults and children (Fig. 3f–i).