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M cells are rare epithelial cells that are involved in intestinal mucosal immunity1,2,3. They have irregular microvilli on their apical surface4,5 and transcytose luminal antigens to the immune cells that reside in their basally located ‘pocket’2,3,4. Mouse M cells are derived from LGR5+ intestinal stem cells6, require RANKL7 and the transcription factors SPIB6,8 and SOX8 (ref. 9), and express Gp2 (ref. 10). The differentiation trajectory of human M cells has largely remained unknown. Mouse M cell markers are often not expressed in humans—for example, Ulex europaeus agglutinin-1 (UEA-1) uniquely recognizes mouse M cells11, whereas human M cells display sialyl Lewis A antigen12. Of note, the M cell pocket contains both dendritic cells and T cells13. M cells may directly interact with T cells through a dendritic cell-independent mechanism.

Human M cell organoids

We recently developed a culture protocol for human intestinal organoids14, which we further optimized as ‘M cell medium’ by adding RANKL, tumour necrosis factor (TNF) and retinoic acid, leading to the appearance of GP2+ cells (Fig. 1a–c). Consistent with observations in mice7, RANKL appeared to be essential for the development of M cell organoids (Fig. 1b,c). M cell organoids exhibited reduced numbers of proliferative buds (Extended Data Fig. 1a–d), as noted previously for mouse M cell organoids15. As previously reported16, lymphotoxin-β (LTB) enhanced the frequency of GP2+ cells but led to cell death in our culture (Extended Data Fig. 1e,f). LTB had little effect on absolute numbers of M cells and was thus excluded from the differentiation cocktail.

Fig. 1: A human intestinal organoid model containing M cells.

a, Schematic of M cell differentiation from human intestinal organoids. Representative cell types in the cultured organoids are indicated. Images were created in BioRender. van Es, J. (2025) https://BioRender.com/xh7haut. EEC, enteroendocrine cell; TA cell, transit-amplifying cell. b,c, Representative flow cytometry analysis (b) and quantification of GP2+ M cell percentage (c) in cultured M cell organoids. The effect of each M cell differentiation factor was evaluated by removing it from the M cell medium. c, For each tested factor, three different donors were tested. Each dot represents the mean value derived from three independent wells. Data are mean ± s.e.m. P values from one-way ANOVA with Dunnett’s test. Ctrl, control; RA, retinoic acid. d,e, Representative confocal image (d) and flow cytometry analysis (e) of SPIB-P2A-tdTomato reporter organoids cultured in M cell medium. Mature M cells are marked by tdTomato fluorescence and GP2 antibody staining (green, arrows in d). Three independent experiments were performed on cells from one donor with similar results. Scale bar, 50 µm. f, scRNA-seq analysis of 375 cells derived from human M cell organoids. Cell clusters are visualized in uniform manifold approximation and projection (UMAP) plots and coloured by cell type (left) or by duplicate plates (right, representing two biological replicates). Cell numbers are shown in brackets below each indicated cell type. g, Violin plots showing the expression levels of cell-type-specific markers for M cells and enterocytes in the M cell organoid-derived scRNA-seq dataset containing 375 cells. h, Representative TEM images of M cell organoids. M cells are identified by having fewer apical microvilli compared with neighbouring enterocytes. M cell organoids from two donors were tested with similar results (see also Extended Data Fig. 1p). Scale bars: 10 µm (left), 2 µm (middle and right).

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We next created *SPIB *reporter organoids (SPIB-P2A-tdTomato; Extended Data Fig. 1g). M cell medium efficiently generated SPIB-expressing (tdTomato+) cells (Extended Data Fig. 1h). Immunofluorescent staining and flow cytometry analysis revealed that SPIB expression was induced in more than 75% of cells, with approximately 3% co-expressing GP2 on their apical surface (Fig. 1d,e). These SPIB+GP2+ cells expressed other M cell markers such as SOX8 and CCL23 (ref. 16), as demonstrated by quantitative PCR (qPCR) analysis following fluorescence-activated cell sorting (FACS) (Extended Data Fig. 1i). Cells derived from M cell organoids were sorted by FACS for single-cell RNA-sequencing (scRNA-seq) analysis. We thereby identified three cell clusters corresponding to enterocytes, intermediate cells and M cells (Fig. 1f,g and Extended Data Fig. 1j,k).

To identify marker genes for primary M cells, we re-analysed the published scRNA-seq resource17, which reportedly contained around 400 M cells among more than 420,000 total cells. This dataset was integrated with three other scRNA-seq datasets that covered all other intestinal cell types (Extended Data Fig. 1l). We noted that more than half of the previously annotated M cells expressed GP2 but no other M cell markers (Extended Data Fig. 1l,m). These cells were marked by TFF2 and PGC and derived from the duodenum (Extended Data Fig. 1m,n). GP2 is known to mark human Brunner’s gland cells18. Immunohistochemistry (IHC) staining for TFF2 on human duodenum sections stained Brunner’s gland (Extended Data Fig. 1o). Exclusion of these cells defined a core set of primary human M cell markers, including GP2, SPIB, SOX8, SLC2A6, PTAFR and GABRP (Extended Data Fig. 1m), which were also highly expressed in organoid M cells (Fig. 1g). M cells exhibit far fewer and shorter apical microvilli compared with enterocytes4,5. Transmission electron microscopy (TEM) imaging of organoid M cells visualized this difference (Fig. 1h and Extended Data Fig. 1p). We further established a two-dimensional (2D) air–liquid interface (ALI) culture based on M cell medium (Extended Data Fig. 1q–s). Apical microvillus structures were clearly visible with actin staining, and co-staining for GP2 confirmed that cells with fewer microvilli were M cells (Extended Data Fig. 1t). We conclude that our organoid model generates genuine human M cells.

ICAM2 marks M-lineage cells

GP2 is the only known surface marker for rare, fully mature M cells10. The primary M cell dataset suggested ICAM2 as a broader M cell marker (Fig. 2a). Indeed, ICAM2 was expressed in more than 90% of GP2+ organoid M cells (Fig. 2b), yet about 50% of GP2− cells also expressed ICAM2 (Fig. 2b). SPIB+ M-lineage organoid cells could be subdivided into ICAM2− and ICAM2+ cells (Fig. 2c,d). Thus, we could distinguish three stages within the M-lineage cells—SPIB+ICAM2−GP2− early M cells, SPIB+ICAM2+GP2− immature M cells and SPIB+ICAM2+GP2+ mature M cells—in addition to SPIB−ICAM2−GP2− non-M cells (Fig. 2d,e). These four populations were sorted for transcriptome profiling (Fig. 2d,f). SPIB− non-M cells expressed enterocyte markers that decreased in abundance during M cell differentiation (Fig. 2g and Extended Data Fig. 2a). Immature M cell markers (TNFAIP2 (ref. 19), SOX8 and CCL23) were enriched in ICAM2+ cells (Fig. 2g). M cell maturation coincided with appearance of previously reported human M cell markers and RANKL-responsive genes16 (Extended Data Fig. 2b). In total, 1,639 differentially expressed genes (DEGs) were identified (Fig. 2h). Gene ontology (GO) analysis identified enterocytes and M cells as the most relevant cell types on the basis of the downregulated and upregulated genes in M cells, respectively (Fig. 2i). Additionally, gene set enrichment analysis (GSEA) revealed nutrient metabolic pathways and brush border components in enterocytes, whereas M cells expressed genes related to pathogen infection and immune cell interaction (Extended Data Fig. 2c–e). Markers for other intestinal cell types were undetectable in M cell organoids (Extended Data Fig. 2f). In human Peyer’s patch tissues, immunofluorescent staining confirmed the presence of ICAM2+ M cells (Extended Data Fig. 3a). Some ICAM2+ cells exhibited features of mature M cells, yet most represented immature M cells with enterocyte-like brush borders. scRNA-seq analysis of primary M cells confirmed that early, immature and mature stages were distinguishable by SPIB, ICAM2 and GP2 expression (Extended Data Fig. 3b). GP2− and GP2+ ICAM2-enriched subpopulations equally expressed immature M cell markers, such as TNFAIP2 and CCL23 (Extended Data Fig. 3b). Thus, organoid M cells faithfully recapitulated human M cell differentiation. Although GP2 uniquely marks mature M cells (Fig. 2g and Extended Data Fig. 3c,d), the transcriptome of ICAM2+GP2− M cells strongly resembled that of GP2+ M cells (Extended Data Fig. 3c,d). Only nine genes were upregulated more than twofold in GP2+ cells (Extended Data Fig. 3c,d). Indeed, CLU is known to mark GP2+ M cells10. The minimal transcriptomic difference was also observed in primary M cells (Extended Data Fig. 3e,f). Of note, some GP2− M cells also displayed the typical M cell microvillus morphology (Extended Data Fig. 1t, arrows).

Fig. 2: Differentiation trajectory of human M cells.

a, Violin plot showing the expression levels of ICAM2 across multiple human intestinal cell types from tissue-derived scRNA-seq datasets containing 15,543 cells. b, Representative flow cytometry analysis of ICAM2 and GP2 expression in M cell organoids. Three independent experiments were performed on two donors with similar results. c, Representative confocal images of ICAM2 antibody staining in SPIB-P2A-tdTomato reporter organoids cultured in M cell medium. M cells are marked by tdTomato fluorescence (red) and show positive staining of ICAM2 antibody (green, primarily detected on the basolateral cell surface of M cells). Three independent experiments were performed with cells from one donor with similar results. Scale bar, 50 µm. d, Representative flow cytometry analysis of ICAM2 and GP2 expression in SPIB-P2A-tdTomato reporter organoids cultured in M cell medium. Three independent experiments were performed on one donor with similar results. e, Schematic of spatial-temporal differentiation of human M cells, showing markers associated with M cells at different stages. Images were created in BioRender. van Es, J. (2025) https://BioRender.com/xh7haut. f, Principal components analysis plot showing transcriptomal changes during M cell differentiation. n = 2 biological replicates from one donor. Each dot represents one biological replicate for the FACS-sorted cell population. g, Expression levels (normalized counts) of cell-type-specific markers across the indicated cell populations. n = 2 biological replicates. Data are mean values. h, Heat map showing expression patterns of DEGs during M cell differentiation. n = 2 biological replicates. i, Volcano plot showing GO analysis based on downregulated and upregulated DEGs in M cells, which identifies the most relevant cell types as enterocytes and M cells, respectively. P values from one-sided Fisher’s exact test.

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ICAM1 and ICAM2 are co-expressed by primary (Fig. 2a and Extended Data Fig. 3g) and organoid (Extended Data Figs. 2b and 3h) M cells. ICAM2 is located basolaterally (Fig. 2c and Extended Data Fig. 3a). ICAM molecules can facilitate adhesion between T cells and antigen-presenting cells20. We next analysed the role of ICAM2 in CD4+ T cell binding to M cell organoids. We genetically deleted ICAM2 (Extended Data Fig. 3i) and blocked ICAM1 with a neutralizing antibody, which resulted in decreased numbers of T cells attached to the M cell organoids (Extended Data Fig. 3j,k). Notably, ICAM2 knockout did not affect M cell differentiation (Extended Data Fig. 3l,m).

Human M cell development requires RUNX2

Expression of 63 transcription factors increased during M cell differentiation (Extended Data Fig. 4a), including the known M cell transcription factors SPIB, SOX8, EHF21, RELB, NFKB1 (nuclear factor (NF)-κB p105 subunit) and NFKB2 (NF-κB p100 subunit)22 (Extended Data Fig. 4a), whereas expression of 59 transcription factors decreased. ONECUT2 drives enterocyte differentiation in mice15. Its expression was markedly decreased during human M cell maturation (Extended Data Fig. 4b). Treatment with the ONECUT2 inhibitor CSRM617 resulted in more than twofold increase in the number of GP2+ M cells in organoids (Extended Data Fig. 4c,d). SPIB is described as a master regulator for MHC-II-expressing B cells23 and dendritic cells24. Spib deficiency depletes GP2+ M cells in mouse Peyer’s patch epithelium8. SPIB-knockout organoids revealed the same essential role of SPIB in human M cell development (Extended Data Fig. 4e,f).

RANKL signalling is an essential niche factor for dendritic cell activation25. Several RANKL-induced M cell transcription factors, besides SPIB, are known from dendritic cell biology (that is, EHF, RELB and NFKB1/2)26. We hypothesized that dendritic cells and M cells might share a RANKL-induced transcription regulatory network. One transcription factor expressed by M cells, RUNX2, attracted our interest (Extended Data Fig. 4a,b). Like SPIB, RUNX2 is a master regulator for dendritic cells27 and its expression is induced by RANKL28. Addition of CADD522, a RUNX2-specific inhibitor, resulted in dose-dependent loss of GP2+ M cells (Extended Data Fig. 4g–i). Similarly, RUNX2 knockout led to significant loss of GP2+ organoid M cells (Extended Data Fig. 4g–j).

CSF2 promotes human M cell maturation

We next interrogated the dataset for M cell-expressed receptors (Extended Data Fig. 4k). Consistent with the functional effects of RANKL, TNF and retinoic acid, their receptors (TNFRSF11A, TNFRSF1B and RARG, respectively) were expressed in M cells (Extended Data Fig. 4k). We performed FACS-based screening to identify potential niche factors that could promote M cell maturation (Extended Data Fig. 4l,m). Among 12 tested ligands, CSF2 resulted in a more than fourfold increase of GP2+ organoid M cells (Extended Data Fig. 4l–n). CSF2 is widely used for dendritic cell differentiation in vitro. The CSF2 receptor was expressed in primary human M cells, indicating its potential function in vivo (Extended Data Fig. 4o). Notably, expression of CSF2 receptors was undetectable by qPCR analysis in organoid-derived GP2+ mouse M cells (Extended Data Fig. 5a,b) and was minimal in a published mouse scRNA-seq dataset containing RANKL-induced M cells29 (Extended Data Fig. 5c), demonstrating an interspecies difference.

Human M cells express dendritic cell-related genes

The shared transcription factors (SPIB, RUNX2, RELB and NF-κB) and signalling regulators (RANKL and CSF2) between M cells and dendritic cells suggested a shared gene regulatory network. This prompted us to search for additional dendritic cell genes in our RNA-seq dataset. A series of lymphoid dendritic cell marker genes was induced during M cell maturation (Fig. 3a). Intestinal lymphoid dendritic cells have been defined by signature genes including CD83, LAMP3, IL7R and FSCN1 (refs. 17,30) and resemble conventional dendritic cells (cDCs) more than plasmacytoid dendritic cells (pDCs)30. We integrated scRNA-seq datasets of primary human intestinal epithelial cells with various immune cell types (Extended Data Fig. 5d,e). Unsupervised, transcriptomic similarity-based clustering of individual epithelial and immune cells identified a unique cell cluster (cluster 9), in which both primary M cells and lymphoid dendritic cells resided (Extended Data Fig. 5d,e, with cell types annotated by the previous study17). Signature genes defining cluster 9 included the lymphoid dendritic cell markers (Extended Data Fig. 5f) and were expressed by both lymphoid dendritic cells and M cells (Fig. 3b and Extended Data Fig. 5f). Within cluster 9, M cells, but not lymphoid dendritic cells, specifically expressed conventional M cell markers including ICAM2, CCL23 and SOX8 (Extended Data Fig. 5f). Notably, although M cells clustered with lymphoid dendritic cells, a number of cDC genes (CD74, DC-SIGN and CD11B) and several pDC genes (CXCR3 and RUNX2) also appeared upon M cell differentiation (Extended Data Fig. 5g). Thus, M cells did not resemble a specific dendritic cell subtype, but rather exhibited broader expression of dendritic cell-related genes. Finally, GO analysis using all DEGs induced during M cell maturation identified activated dendritic cells and antigen-presenting cells as being most closely related (Fig. 3c).

Fig. 3: Human M cells express dendritic cell markers including MHC-II.

a, Heat map showing the expression patterns of a set of lymphoid dendritic cell markers during M cell differentiation. n = 2 biological replicates. b, Dot plot showing the expression levels of a set of lymphoid dendritic cell markers (cluster 9 markers) across multiple intestinal epithelial and immune cell types from tissue-derived scRNA-seq datasets. n = 20,210 single cells. Dot colour relates to normalized mean expression values and dot size indicates the fraction of expressing cells. c, Volcano plot showing GO analysis based on upregulated DEGs in M cells, which identifies the most relevant cell types as activated dendritic cells (top) or antigen-presenting cells (bottom). P values from one-sided Fisher’s exact test. d, Expression levels (normalized counts) of MHC-II-related genes across the indicated cell populations. n = 2 biological replicates. Data are mean values. e, Representative flow cytometry analysis of MHC-II and GP2 expression in cultured M cell organoids (wild type or CIITA-knockout (KO)) versus normal intestinal organoids (with or without IFNγ). Three independent experiments were performed with similar results in wild-type organoids derived from three donors and in two CIITA-knockout clonal organoid lines derived from one donor. Abs, antibodies; WT, wildtype. f, Representative confocal images of MHC-II antibody staining in SPIB-P2A-tdTomato reporter organoids cultured in M cell medium. M cells are marked by tdTomato fluorescence (red) and show positive staining of MHC-II antibody (green, detected in both cytoplasm and primarily on basolateral surface of M cells). Three independent experiments were performed on cells from one donor with similar results. Scale bar, 50 µm. g, Representative immuno-electron microscopy images showing MIIC structures in endosomes, identified by immunogold staining of MHC-II antibody in organoid M cells. Arrows indicate MHC-II vesicles in the endosome. M cell organoids from two donors were tested with similar results (see also Extended Data Fig. 8a). Scale bars, 200 nm.

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Human M cells express MHC-II

Dendritic cells take up exogenous antigens and process these into peptides, which are then presented by MHC-II complexes. FACS-sorted M cells exhibited distinct morphology from SPIB− cells when cultured in 2D, with multiple protrusions resembling dendritic cells (Extended Data Fig. 5h). These protrusions facilitated phagocytosis of bacteria particles (Supplementary Video), which could be blocked by the phagocytosis inhibitor cytochalasin B (Extended Data Fig. 5i,j). Notably, expression of MHC-II genes and the invariant chain (CD74) increased during M cell maturation (Fig. 3d and Extended Data Fig. 5k). Flow cytometry analysis for MHC-II and GP2 confirmed expression of MHC-II proteins on GP2+ as well as GP2− M cells (Fig. 3e). Notably, MHC-II proteins were expressed by SPIB+ M-lineage cells, with the staining signals primarily detected on the basolateral cell surface (Fig. 3f), whereas SPIB− cells did not express MHC-II genes (Fig. 3d). Flow cytometry analysis confirmed that MHC-II+ M cells were more mature, with more than 98% of MHC-II+ cells co-expressing ICAM2 (Extended Data Fig. 5l). Consistently, FACS-sorted MHC-II+ cells expressed various markers of immature and mature M cells (Extended Data Fig. 5m).

Notably, intestinal organoids cultured without M cell-inducing factors did not express MHC-II in the absence of IFNγ (Fig. 3e), as also shown elsewhere16. By contrast, human organoid M cells spontaneously expressed MHC-II without IFNγ under homeostatic conditions (Fig. 3e). To test whether the IFNγ-independent expression of MHC-II in M cells required the transcription factor CIITA, we generated *CIITA-*knockout organoids (Extended Data Fig. 5n). These organoids contained similar numbers of M cells, yet MHC-II expression was absent (Fig. 3e). IRF1 is the classical upstream transcription factor that induces CIITA expression in the presence of IFNγ. Expression of IRF1 was not induced during M cell differentiation (Fig. 3d and Extended Data Fig. 3b), indicating that CIITA expression in M cells does not depend on IRF1, which further confirms that MHC-II expression in M cell organoids is independent of IFNγ signalling. Beyond IRF1, several IFNγ-independent transcription factors have been reported as potential upstream regulators of CIITA (for example, RFX5 (ref. 31) and NF-κB32). These genes were induced by threefold to fivefold during M cell differentiation (Fig. 3d).

In primary intestine, MHC-II can be expressed by enterocytes33. The expression of MHC-II in enterocytes crucially depends on IFNγ stimulation during inflammation33,34. MHC-II expression is almost completely abolished in Ifngr1-knockout mouse enterocytes, even with pathogenic bacterial infection34. In human intestinal tissues, MHC-II+ enterocytes were not uniformly detectable but showed different distributions between donors and tissue areas (Extended Data Fig. 6a), indicating that expression of MHC-II is not a spontaneous, intrinsic property, but is induced by exogenous factors such as IFNγ. Notably, unlike M cells, the transcriptome of MHC-II+ enterocytes did not resemble that of lymphoid dendritic cells (Extended Data Fig. 6b). In the scRNA-seq datasets, MHC-II+ enterocytes did not cluster with any dendritic cell subtype (Extended Data Fig. 6c). Transcriptomic comparison between MHC-II-expressing M cells and enterocytes identified 414 DEGs that were expressed at higher levels in M cells (Extended Data Fig. 6d,e). These DEGs were again highly co-related with blood dendritic cells, as revealed by GO analysis (Extended Data Fig. 6f).

It has remained controversial whether human M cells can directly present antigens to CD4+ T cells5,35,36. We performed immunofluorescent staining of MHC-II on primary human Peyer’s patch tissues. MHC-II signals could be detected intracellularly and on the basolateral cell surface of M cells (Extended Data Fig. 6g,h). Of note, scRNA-seq analysis based on reliable M cell marker genes revealed that both ICAM2+GP2− and ICAM2+GP2+ primary M cells expressed MHC-II genes and CD74 (Extended Data Fig. 6i). This analysis avoids limitations of IHC staining regarding the uncertainty of MHC-II signals between neighbouring cells35,36. Furthermore, flow cytometry analysis of MHC-II in ICAM2+ or GP2+ primary M cells confirmed MHC-II protein expression in more than 97% of ICAM2+ and GP2+ M cells (Extended Data Fig. 6j,k). Although two previous M cell studies reached contradictory conclusions regarding MHC-II expression in M cells, both detected MHC-II signals in human follicle-associated epithelium (FAE) tissues35,36. It should be noted that besides GP2+ M cells, FAE also contains many M-lineage cells that do not express GP2 (refs. 9,19) and morphologically resemble non-M enterocytes. In M cell organoids, many GP2− immature M cells were found to express MHC-II in the absence of IFNγ, in addition to GP2+ M cells (Fig. 3e). Therefore, we hypothesized that in primary tissue, MHC-II+ FAE cells (GP2− normal microvilli) are likely to represent immature M cells that constitutively express MHC-II, rather than representing IFNγ-induced MHC-II+ enterocytes. Indeed, many ICAM2+ immature M cells carried normal microvillus structures in human FAE (Extended Data Fig. 3a), and a majority of these primary ICAM2+ M cells expressed MHC-II (Extended Data Fig. 6k). Of note, the presence of occasional MHC-II+ enterocytes (induced by IFNγ) in human FAE could not be formally excluded.

Notably, MHC-II expression was not observed in mouse M cells derived either from organoids (Extended Data Fig. 5b,c) or from primary FAE tissues (Extended Data Fig. 7a–c). Most dendritic cell markers identified in human M cells were not expressed in mouse (Extended Data Fig. 5c). GO analysis of mouse M cell markers identified terms related to conventional M cell functions, but no terms related to antigen presentation (Extended Data Fig. 7d). These differences may explain why an antigen-presenting function of M cells has not been observed in mouse experiments despite clear similarities between mouse and human M cells (Extended Data Fig. 7e).

A hallmark of antigen presentation via MHC-II molecules is the MHC-II compartment (MIIC), where antigen processing and loading occur37. We assessed the existence of MIIC in M cells by immuno-electron microscopy imaging of MHC-II and CD63, two marker proteins for MIIC structures37, and identified structures that represent early, multivesicular, intermediate and multilaminar MIICs in M cells and resemble the corresponding MIIC structures in dendritic cells37,38 (Fig. 3g and Extended Data Fig. 8a). We also analysed MIIC in normal intestinal organoids. Consistent with undetectable levels of MHC-II in unstimulated normal intestinal organoids (Fig. 3e), no MHC-II+ vesicles were observed in the endosomes (Extended Data Fig. 8b). Upon IFNγ stimulation, normal intestinal organoids predominantly exhibited multivesicular MIIC structures, whereas tubular extensions and the multilaminar structures were rarely observed (Extended Data Fig. 8b). Given the proposed role of multilaminar MIICs in high-fidelity peptide exchange39, these structural differences may underlie the limited capacity for complete antigen processing and presentation by IFNγ-induced enterocytes, despite the presence of surface MHC-II. Additionally, like dendritic cells, various pattern recognition receptors and key components of the antigen processing and presentation machinery were expressed in M cells, further supporting their potential antigen-presenting function (Extended Data Fig. 8c–e).

Human M cells present gluten antigen

Next, we established an organoid/T cell co-culture assay focused on T cell