RORγt+ APCs require a distinct cis-regulatory element to instruct tolerance to dietary antigens

Zhao, Jie; Hao, Jiacheng; Chen, Jincheng; Lyu, Mengze; Liu, Haoyu; Li, Na; Song, Panwei; Wang, Wenyan; Chu, Coco; Sonnenberg, Gregory F.; Guo, Xiaohuan

doi:10.1038/s41467-026-69886-z

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Published: 21 February 2026

RORγt⁺ APCs require a distinct cis-regulatory element to instruct tolerance to dietary antigens

Jie Zhao ORCID: orcid.org/0009-0000-6039-5866^1,2,3,4^na1,
Jiacheng Hao ORCID: orcid.org/0000-0003-4025-7241^1,2,3,4^na1,
Jincheng Chen^1,2,3,4^na1,
Mengze Lyu ORCID: orcid.org/0000-0002-8509-7969^5,6,7,
Haoyu Liu^1,2,3,4,
Na Li^1,2,3,4,
Panwei Song^1,2,3,4,
Wenyan Wang ORCID: orcid.org/0000-0001-7663-9124^1,2,4,
Coco Chu^1,2,3,
Gregory F. Sonnenberg ORCID: orcid.org/0000-0003-0948-5908^5,6,7 &
…
Xiaohuan Guo ORCID: orcid.org/0000-0001-5152-3010^1,2,3,4,8

Nature Communications volume 17, Article number: 3019 (2026) Cite this article

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Abstract

Oral tolerance represents a hallmark of intestinal mucosal immunity to prevent inflammatory responses to harmless natural antigens, such as dietary components or commensal organisms. According to recent studies, RORγt⁺ antigen-presenting cell (APC) contributes to intestinal homeostasis, including oral tolerance, through inducing microbiota- and dietary antigen-specific Tregs. Here we identify a cis transcriptional regulatory element that distinguishes RORγt⁺ APCs from other of RORγt⁺ cell types. This sequence within Rorc gene loci, OCR369 governs RORγt expression in ILC3s and other RORγt⁺ APCs, but not T cells, through interaction with RUNX3 and formation of chromatin loops. OCR369 deletion results in a significant reduction of RORγt⁺ APCs in mLN around the weaning period and ILC3s in mLN and intestines of adult mice, accompanied by a decrease in RORγt⁺ Tregs and spontaneous inflammation in the small intestine. Mechanistically, the reduction in RORγt⁺ APCs, including both DC-like cells and MHCII⁺ ILC3s, impairs the development of both dietary antigen-specific and microbiota-specific RORγt⁺ Tregs and results in the loss of oral tolerance, thereby increasing allergy susceptibility. Thus, our findings identify a specific regulatory mechanism for RORγt expression in RORγt⁺ APCs and underscore the pivotal role of these cell types in mediating oral tolerance and maintaining intestinal health.

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Introduction

Group 3 innate lymphoid cells (ILC3), which are enriched in the gut and gut-associated lymphoid tissues, play a critical role in protecting the host from various infections and maintaining intestinal homeostasis^1,2,3,4,5. ILC3s can directly or indirectly regulate various cells within the intestinal microenvironment^6,7,8. Notably, ILC3s express major histocompatibility complex class II (MHCII) and are capable of antigen processing and presentation⁹. Unlike traditional antigen-presenting cell (APC) such as dendritic cell (DC), intestinal MHCII⁺ ILC3s lack the expression of co-stimulatory molecules like CD80 and CD86 at steady state, which tends to prevent the over-activation of CD4⁺ T cells, thereby contribute to the maintenance of immune homeostasis in the intestine, tumors, and during allergic airway inflammation^9,10,11,12. However, under inflammatory condition, ILC3 subset can upregulate these molecules and become pro-inflammatory¹³. ILC3s also promote the differentiation and survival of commensal-specific RORγt⁺ Treg cells through multiple mechanisms, including OX40L-OX40 interaction, integrin-mediated TGF-β release, and the production of IL-2 and CTLA-4 by ILC3s^14,15,16,17. Recent studies have expanded the group of RORγt⁺ MHCII⁺ APCs, identifying new groups of Thetis cells (TC I-IV) or Janus cells. These cells exhibit phenotypic overlaps with ILC3s and are involved in promoting the generation of RORγt⁺ Treg cells^18,19,20. However, further research is needed to better understand the unique characteristics of these innate RORγt⁺ APCs and to develop methods for their precise identification. In addition, intestinal Tregs mediate oral tolerance and prevent food allergy, while the detailed contribution of RORγt⁺ APCs and their regulatory mechanisms remains unclear.

ILC3s are innate-adaptive counterparts to adaptive Th17 cells, sharing similar lineage-gene profiles and functional roles. RORγt, a master lineage transcription factor (TF), regulates the development of both ILC3s and Th17 cells, and is also required for the survival of CD4⁺CD8⁺ double positive (DP) thymocytes and TCRα rearrangement²¹. Despite the similar requirement of RORγt, emerging evidence highlights distinct regulatory mechanisms between ILC3s and T cells^22,23. Th17 cell differentiation is driven by RORγt, which relies on STAT3 signaling in response to IL-6 and IL-23 stimulation^24,25. In contrast, the mechanism underlying RORγt expression initiation in ILC3s from their progenitors remains poorly understood. The development of ILC3s appears to be independent of STAT3²³, and even transient inhibition or ablation of RORγt has limited effects on the function and population of mature ILC3s²⁶, indicating distinct requirements and regulatory mechanisms of RORγt. Recent studies identified several cis-regulatory elements (CRE) at the Rorc locus, like conserved non-coding sequence (CNS) 6, CNS9, CNS11 and RORCE2, which play important roles in regulating RORγt expression in T cells^27,28,29. However, whether ILC3-specific CREs exist to regulate RORγt expression remains largely unknown.

In this study, we identify an open chromatin region (OCR369) as a specific CRE that regulates high-level RORγt expression in ILC3s and related RORγt⁺ APC subsets, but not T cells. OCR369 deletion impairs the development and function of ILC3s and other RORγt⁺ APC subsets, leading to disrupted Treg-induced oral tolerance and increased allergy susceptibility. These findings reveal that a distinct CRE can specifically regulate RORγt⁺ APCs, including ILC3s, to maintain oral tolerance by inducing dietary and microbial antigen-specific Tregs.

Results

A distinct open chromatin region in the Rorc locus is essential for ILC3s

Chromatin accessibility is closely linked to gene activity, and the landscape of open chromatin regions (OCR) reflects the regulatory networks specific to distinct cell types. To identify the specific cis-regulatory elements in ILC3s and T cells, we performed ATAC-seq analysis at the Rorc locus with intestinal ILC3s, Th17 cells and RORγt⁺ Tregs^22,30,31 (Th17 from GSM3638386 and ILC3 subsets from GSE137319 of the GEO database). While all ILC3 subsets, Th17 cells and RORγt⁺ Tregs showed high chromatin accessibility at the RORγt promoter, two regions (OCR225 and OCR369) with higher accessibility were identified in ILC3s, located downstream of the RORγt Promoter within the first intron (Fig. 1a). Interestingly, these two regions were located in the CNS9 (Fig. 1a) identified previously²⁷. As a part of CNS, OCR369 also showed higher accessibility in human ILC3s than Th17 cells (Supplementary Fig. 1a), suggesting its potential role as an ILC3-specific CRE regulating RORγt expression in both human and mouse.

**Fig. 1: OCR369 is required for high levels of RORγt and ILC3 development.**

To investigate the role of OCR225 and OCR369 in RORγt⁺ cells, we used the CRISPR-Cas9 system to generate germline-depleted mice lacking OCR225 (Rorc^Δ225) and OCR369 (Rorc^Δ369) (Supplementary Fig. 1b). Although no significant differences were observed in the proportion, absolute numbers, or RORγt expression levels of intestinal ILC3s and RORγt⁺ T cells between Rorc^Δ225 and wild type mice (Supplementary Fig. 1c, d), Rorc^Δ369 mice exhibited reduced RORγt⁺ ILC3s, increased RORγt⁺ Th17 cells, and decreased RORγt⁺ Tregs in the small intestine (Fig. 1b–d). This contrasts with findings in CNS9-deficient mice²⁷, indicating a distinct regulatory mechanism. Wild-type ILC3s showed much higher RORγt level than Th17 cells and RORγt⁺ Tregs, and OCR369 depletion in ILC3s significantly reduced RORγt expression to levels comparable to those in Th17 cells, with no effect on RORγt expression in Th17 or RORγt⁺ Tregs (Fig. 1e). In contrast to unchanged ILC1 and ILC2 cell numbers (Supplementary Fig. 1e), Rorc^Δ369 mice displayed reduced RORγt expression and cell numbers across all ILC3 subsets (CCR6⁺, NKp46⁺, and CCR6⁻NKp46⁻ double-negative (DN) ILC3s) in the small intestine, with the most pronounced reduction in CCR6⁺ ILC3s (Fig. 1f, g). Similarly, c-Kit⁺ ILC3s and NRP1⁺ ILC3s were significantly reduced in Rorc^Δ369 mice (Supplementary Fig. 1f), suggesting that OCR369 is critical for maintaining ILC3 numbers, rather than merely influencing surface marker expression. Decreased ILC3 numbers and RORγt expression were also observed in the large intestine and mesenteric lymph nodes (mLN) (Supplementary Fig. 1g–i), indicating OCR369’s broad role in regulating the ILC3 population. In addition, OCR369-deficient ILC3s exhibited reduced production of IL-22 and IL-17, particularly IL-17A (Fig. 1h). While transient RORγt deletion in ILC3s has been reported to preserve CD3⁻IL-22⁺ cells²⁶, Rorc^Δ369 mice showed decreased IL-22⁺ ILCs in the small intestine (Fig. 1i), possibly due to reduced total ILC3 numbers. Notably, RORγt expression in thymic DP thymocytes and the development of CD4⁺ and CD8⁺ T cells were unaffected in Rorc^Δ369 mice (Supplementary Fig. 1j), indicating that OCR369 is not necessary for T cell development.

To further investigate the role of OCR369 in RORγt⁺ ILCs and T cells, we performed single-cell RNA sequencing (scRNA-seq) on innate lymphocytes and CD4⁺ T cells sorted from the small intestinal lamina propria of 6-week-old control and Rorc^Δ369 mice (Supplementary Data 1). After quality control, five distinct ILC clusters and five T cell clusters were identified based on marker gene expression (Supplementary Fig. 2a) and visualized using uniform manifold approximation and projection (UMAP) analysis (Fig. 1j and Supplementary Fig. 2b). Rorc^Δ369 mice exhibited a significant reduction in the normalized frequencies of ILC3 clusters, including both LTi-like and NCR1⁺ ILC3s by relative frequencies analysis of these clusters (Fig. 1k). Reduced expression levels of Rorc, Il17f, and Nfil3 were also observed in OCR369-deficient ILC3s (Fig. 1l). Interestingly, alongside an increased ex-ILC3/ILC1 cluster, a dysregulated ILC3 cluster emerged in Rorc^Δ369 mice, characterized by downregulation of all ILC3 marker genes, including Rorc, Ncr1, Ccr6, Il17f, Il22, and Nfil3 without upregulated typical ILC1 or ILC2 genes (Fig. 1k, l, and Supplementary Fig. 2a). Moreover, Rorc^Δ369 mice exhibited increased RORγt⁺ Th17 cell cluster and reduced RORγt⁺ Treg cluster by scRNA-seq (Supplementary Fig. 2c). These data suggest that OCR369 is required for ILC3 homeostasis and may also influence T cell homeostasis in the intestine.

OCR369 is required for RORγt⁺ APCs

Recent studies have highlighted the importance of MHCII⁺ RORγt⁺ APCs, including MHCII⁺ ILC3s¹⁶, Janus cells¹⁸, and Thetis cells¹⁹, in the development of microbiota-specific RORγt⁺ Treg cells, which are essential for maintaining colon homeostasis³². We noticed that MHCII⁺ ILC3s, along with their RORγt and MHCII expression levels, were significantly reduced in the small intestine and mLN of adult Rorc^Δ369 mice (Fig. 2a–d). ATAC-seq analysis of published datasets also showed high chromatin accessibility at the OCR369 locus in described Thetis cells (TC I-IV)¹⁸ (Fig. 2e), indicating OCR369 regulates RORγt expression in RORγt⁺ APCs. To examine the role of OCR369 in RORγt⁺ APCs, lineage-negative (CD3, B220, F4/80, TCRβ, TCRγδ, Siglec-F, Gr-1, Ter-119, F4/80) MHCII⁺ cells were sorted from mLNs of control and Rorc^Δ369 mice at postnatal day 18 (P18) and then subjected to scRNA-seq analysis (Supplementary Data 2). After quality control, the scRNA-seq data were integrated and analyzed with published datasets^18,33 (Supplementary Fig. 3a). Based on marker gene expression and recent annotations, four clusters of RORγt⁺ APCs, including MHC-I⁺ ILC3s, RORγt⁺ DC-like cells (TC II), RORγt⁺ eTAC I (TC I), and RORγt⁺ eTAC II (TC III), were identified (Fig. 2f and Supplementary Fig. 3a, b). MHCII⁺ ILC3s expressed Rora, Rorc, Cxcr6, Il22, and RORγt⁺ DC-like cells (TC II) highly expressed Rorc, Prdm16, Col17a1, Ccr6, Nrp1, while RORγt⁺ eTAC I (TC I) and RORγt⁺ eTAC II (TC III) expressed Aire and a lower level of Rorc (Supplementary Fig. 3b, c). In addition, RORγt⁺ eTAC I highly express Ncam1 and Sirpa (Supplementary Fig. 3b). Although no big difference in the normalized frequencies of MHCII⁺ ILC3s and RORγt⁺ eTAC I/II were observed between control and Rorc^Δ369 group, the normalized frequency of RORγt⁺ DC-like cells was largely reduced in the mLN of P18 Rorc^Δ369 mice (Fig. 2g). Moreover, although the decreased expression of Rorc could be detected in both MHCII⁺ ILC3s and RORγt⁺ DC-like cells, only OCR369-deficient RORγt⁺ DC-like cells showed decreased expression of MHCII genes (e.g., H2-Ab1, H2-Aa, H2-Dma, and H2-DMb1) (Supplementary Fig. 3c, d), suggesting that OCR369 regulates the antigen-presenting capacity of RORγt⁺ DC-like cells during early life.

Fig. 2: OCR369 regulates multiple RORγt+ APC subsets. — **Fig. 2: OCR369 regulates multiple RORγt⁺ APC subsets.**

To validate the impact of OCR369 on RORγt⁺ APCs, cells from the mLNs of 3-week-old control and Rorc^Δ369 mice were analyzed by flow cytometry (Fig. 2h). Consistent with scRNA-seq findings, only the number and proportion of RORγt⁺ DC-like cells were reduced in OCR369 deficiency mice, whereas other RORγt⁺ APCs including MHCII⁺ ILC3s remained comparable (Fig. 2i). And OCR369 deficiency led to significant reductions in RORγt expression by MHCII⁺ ILC3s and RORγt⁺ DC-like cells, but not by RORγt⁺ eTAC I and eTAC II (Fig. 2j), in line with scRNA-seq findings. Moreover, reduced MHCII was observed only in RORγt⁺ DC-like cells, not in other RORγt⁺ APCs, including ILC3s (Fig. 2j). These results suggest that OCR369 is required for the regulation of RORγt⁺ DC-like cells during early life. Furthermore, RORγt⁺ APCs from 6-week-old control and Rorc^Δ369 mice were also examined by flow cytometry. In 6-week-old Rorc^Δ369 mice, both MHCII⁺ ILC3s and RORγt⁺ DCs exhibited significant reductions in absolute numbers, proportions, and RORγt and MHCII expression levels (Fig. 2k). These results indicate that OCR369 is required for the regulation of both MHCII⁺ ILC3s and RORγt⁺ DC-like cells in adult mice. Notably, MHCII⁺ ILC3s were the most abundant RORγt⁺ APCs in the mLN of both 3-week-old and 6-week-old mice (Fig. 2i, k). Taken together, these data suggest that OCR369 regulates the maintenance and function of RORγt⁺ APC subsets, particularly RORγt⁺ DC-like cells during early life and both MHCII⁺ ILC3s and RORγt⁺ DC-like cells in adults.

OCR369 intrinsically controls ILC3 development

To confirm the intrinsic role of OCR369 in ILC3s, mixed bone marrow chimeric mice were generated on CD45.1 irradiated mice (Fig. 3a). Among CD4⁺ T cells and ILCs, only OCR369-deficient ILC3s and their RORγt expression were significantly decreased, while RORγt⁺ Th17 cells and Tregs remained unaffected (Fig. 3b, c, and Supplementary Fig. 4a). Further analysis of ILC3 subsets revealed heterogeneous effects of OCR369, with decreased CCR6⁺ and DN ILC3s, particularly MHCII⁺ ILC3s, but not NKp46⁺ ILC3, in Rorc^Δ369 chimeric mice (Fig. 3d). This suggests varying RORγt requirements among ILC3 subsets. In the mLN, OCR369-deficient MHCII⁺ ILC3s exhibited reduced proportion and RORγt expression, while the CD127⁻CXCR6⁻ eTAC I (Janus cells) remained unaffected in Rorc^Δ369 chimeric mice (Supplementary Fig. 4b). Furthermore, IL-17A, but not IL-22, was significantly reduced in OCR369-deficient ILC3s (Fig. 3e), consistent with a previous report of limited RORγt effect on IL-22 production on ILC3s²⁶. Overall, these results confirm the specific and intrinsic role of OCR369 in ILC3s and their RORγt expression.

Fig. 3: OCR369 intrinsically controls RORγt+ ILC3 development. — **Fig. 3: OCR369 intrinsically controls RORγt⁺ ILC3 development.**

In adult mice, ILC3s develop from bone marrow progenitors, and RORγt expression is essential for their differentiation³⁴. To determine how OCR369 influences ILC3s, ILC progenitors including CLPs, CHILPs, and ILCPs, were examined, and we found no differences in their percentages or numbers between Rorc^Δ369 and control mice (Supplementary Fig. 4c, d). When CHILPs were sorted and co-cultured with OP9-DL1 feeder cells to generate ILCs (Fig. 3f), OCR369-deficient CHILPs could develop ILC1 and ILC2 normally, but failed to generate ILC3s in contrast to control CHILPs (Fig. 3g, h). In vitro co-culture of CD45.2⁺ OCR369-deficient CHILPs with CD45.1⁺ control CHILPs confirmed that OCR369 deficiency significantly impaired ILC3 development (Fig. 3i, j), indicating that high RORγt expression, controlled by OCR369, is essential for in vitro ILC3 development. Meanwhile, naïve CD4⁺ T cells were sorted and cultured under Th17 cell- or Treg-polarizing conditions. Both Th17 and Treg cells were normally differentiated after OCR369 deletion (Supplementary Fig. 4e–g). No cell number differences in IL-17⁺ Th17 cells or RORγt⁺ Tregs were observed, and there was a slight reduction in RORγt⁺ Th17 cells (Supplementary Fig. 4e–g). Considering the reduced RORγt⁺ Tregs and increased Th17 cells in Rorc^Δ369 mice (Fig. 1b–d), these data suggest that OCR369 extrinsically regulates Th17 cells and RORγt⁺ Tregs through its effects on RORγt⁺ APCs. Taken together, these results demonstrate that OCR369 intrinsically controls high RORγt expression and development of ILC3s but does not affect CD4⁺ T cells.

OCR369 interacts with RUNX3 and facilitates chromatin loop formation at the Rorc locus

To determine how OCR369 specifically regulates high RORγt expression in ILC3s, the mRNA level of RORγt was first detected. RT-qPCR analysis showed significantly decreased RORγt mRNA in OCR369-deficient ILC3s compared to controls (Fig. 4a), indicating that OCR369 regulates RORγt expression at the transcription level. The binding of specific TFs with cis-regulatory elements is one of the key mechanisms for CREs regulating gene expression. To determine TFs binding to OCR369, the biotin-labeled OCR369 DNA or control Rorc(γt) promoter DNA was used to pull-down the specific binding proteins from ILC3 nuclei, followed by mass-spectrometry (MS) analysis (Fig. 4b, Supplementary Fig. 5a and Supplementary Data 3). Combining MS results with TF binding site (TFBS) prediction from JASPAR database (Supplementary Data 4), four candidate TFs were identified, two of which belonged to the RUNX family: RUNX3 and RUNX1 (Fig. 4c). RUNX1 and RUNX3 share similar binding motifs, which are conserved between mouse and human. RUNX3 has been previously reported to be the most abundant RUNX family member in ILC3s, playing an essential role during ILC3 development through directly binding the Rorc promoter and inducing RORγt expression³⁵. We confirmed that Runx3 expression was significantly higher than that of Runx1, Runx2, and Cbfb in ILC3s, and that RUNX3 expression was higher in ILC3s compared to Th17 cells, iTregs, and other ILCs (Fig. 4d and Supplementary Fig. 5b, c). In the small intestinal lamina propria, ILC3s expressed higher RUNX3 levels than Th17 cells and Tregs (Fig. 4e). Consistent with predicted RUNX3 binding sites at OCR369 and the RORγt promoter (RORγt-P) (Fig. 4f), CUT&RUN assay revealed stronger RUNX3 binding to both OCR369 and the RORγt promoter in ILC3s compared to Th17 cells (Fig. 4g). OCR369 deficiency significantly influenced RUNX3 binding at OCR369 sites but not at the promoter (Fig. 4g). To test the effect of RUNX3 binding to OCR369 on RORγt expression, a Dual-Luciferase Reporter system with RORγt promoter and OCR369 in HEK293T cells was utilized (Supplementary Fig. 5d). As previously reported³⁵, RUNX3 significantly enhanced RORγt promoter activity (Supplementary Fig. 5d). However, the addition of OCR369 did not further enhance transcription (Supplementary Fig. 5d), likely due to the limitations of in vitro reporter systems for studying distal cis-regulatory elements. In addition, RUNX3 is expressed by other RORγt⁺ APCs, particularly RORγt⁺ DC-like cells (TC II) (Supplementary Fig. 5e), suggesting its potential role in regulating the RORγt⁺ APC family. Together, these results indicate that OCR369 interacts with RUNX3 in ILC3s, contributing to their higher RORγt expression compared to Th17 cells.

Chromatin looping has been well recognized as a way for enhancer-promoter interaction to promote gene expression. Since OCR369 is in the first intron, ~ 7 kb downstream of the Rorc transcription start site (TSS), it may form a chromatin loop with the Rorc promoter to enhance RORγt expression in ILC3s. To test this hypothesis, a chromosome conformation capture-qPCR (3C-qPCR) assay was performed in MNK3 cells³⁶, an ILC3-like cell line with high chromatin accessibility at OCR369 (Supplementary Fig. 5f). Using the restriction enzyme NlaIII, which was previously used to study chromatin interactions at the Rorc locus²⁹, two NlaIII restriction recognition sites (OCR369-1, 2) within OCR369 and one at the Rorc promoter were analyzed as the anchor site (Fig. 4h). Another upstream NlaIII site without interaction with the Rorc promoter was set as a negative control site (NCS)²⁹. Compared to the negative control, chromatin crosslinking between OCR369 and the Rorc promoter was detected in MNK3 cells but not B16 melanoma cells (Fig. 4i), indicating the formation of the chromatin loop between OCR369 and the Rorc promoter in MNK3 cells. This interaction was further confirmed in ILC3s isolated from the mouse small intestine (Fig. 4j). Together, these data suggest that OCR369 is involved in the formation of chromatin higher-order structures, which contribute to enhanced expression of RORγt in ILC3s.

OCR369-deficient mice develop spontaneous small bowel inflammation

ILC3s play an important role in maintaining intestinal homeostasis and anti-infection immunity. To dissect the in vivo role of OCR369 in the intestine, Rorc^Δ369 mice were orally challenged with Citrobacter rodentium. However, no significant differences were observed between OCR369-deficient and control mice in terms of body weight change or fecal pathogen load (Supplementary Fig. 6a–c). Although Rorc^Δ369 mice exhibited defects in total ILC3s, IL-17-producing, and IL-22-producing ILC3s in the colon after infection, IL-17-producing CD4⁺ T cells were significantly increased in Rorc^Δ369 mice (Supplementary Fig. 6d–f), which may compensate for host defense against C. rodentium in Rorc^Δ369 mice. Interestingly, compared to their littermate controls, Rorc^Δ369 mice developed spontaneous small intestinal remodeling as they aged, characterized by increased small intestine length (Fig. 5a and Supplementary Fig. 7a), villus length (Supplementary Fig. 7b), and goblet cell and DCLK1⁺ tuft cell numbers (Fig. 5b and Supplementary Fig. 7c). These changes of small intestine resemble type 2 inflammation, such as that seen during helminth infection. Indeed, bulk RNA-seq data from 20-week-old littermates showed increased expression of type 2 immune response genes (e.g., Pla2g4c, Dclk1, Mcpt1) and Gsdmc genes in the small intestine of Rorc^Δ369 mice (Supplementary Fig. 7d and Supplementary Data 5), which are also upregulated during helminth infection^37,38. Mcpt1, a marker of the intraepithelial mast cells (IEMCs), was significantly increased in the small intestines of Rorc^Δ369 mice (Fig. 5c), indicating mast cell-driven type 2 inflammation. Gene Ontology (GO) enrichment and Gene Set Enrichment Analysis (GSEA) revealed up-regulation of extracellular matrix (ECM) genes in the small intestine of Rorc^Δ369 mice (Supplementary Fig. 7e–g, and Supplementary Data 6), which was further confirmed by Sirius Red staining showing ECM deposition (Fig. 5d), suggesting progressive fibrosis.

**Fig. 5: OCR369-deficient mice developed spontaneous inflammation in the small intestine.**

To further understand the intestinal immune changes in Rorc^Δ369 mice, scRNA-seq was performed on small intestine samples from 28-week-old Rorc^Δ369 and control mice (Supplementary Fig. 8a and Supplementary Data 7). Consistent with findings in 6-week-old mice, 28-week-old Rorc^Δ369 mice exhibited more significant reductions in ILC3 clusters, including both LTi-like and NCR1⁺ ILC3s, along with increased ex-ILC3/ILC1 and dysregulated ILC3 cluster (Fig. 5e, f, and Supplementary Fig. 8b). For CD4⁺ T cells, RORγt⁺ Treg proportion was significantly decreased, while Th2 cell and effector Th17 cell proportions were increased in OCR369-deficient mice (Fig. 5g). Accordingly, the inflammatory effector cytokines, such as Il4, Il5, Il13, Il17a, produced by these T cells were increased, while inhibitory cytokine Il10 was slightly decreased in Rorc^Δ369 mice (Fig. 5h). These data suggest that OCR369 deficiency-induced small intestinal inflammation results from altered T cell balance.

Further analysis of Th cell and ILC3 dynamics at different ages revealed that ILC3 proportions in the small intestine remained relatively stable after weaning, with OCR369 deficiency causing persistent reductions (Supplementary Fig. 9a, b). In control mice, RORγt⁺ Tregs developed post-weaning and increased with age in the small intestine, while Th2, Th17 and Th1 cells remained low (Fig. 5i, j and Supplementary Fig. 9c). However, Rorc^Δ369 mice exhibited a significant reduction of RORγt⁺ Tregs and age-dependent increases in Th2, Th17 and Th1 cells (Fig. 5i, j and Supplementary Fig. 9c). Significantly increased inflammatory cytokine production by T cells and innate cells was also observed in the small intestine of Rorc^Δ369 mice (Supplementary Fig. 9d–f). Given the intrinsic role of OCR369 in supporting the RORγt⁺ APC family but not T cells, these findings indicate that OCR369-dependent RORγt⁺ APCs regulate Th/Treg balance in response to antigens from the small intestine, probably dietary or microbiota-derived antigens, thereby maintaining small intestinal homeostasis. Indeed, when Rorc^Δ369 mice were crossed with Rag1^-/- mice, no difference of the small intestinal or villus length was observed between Rorc^Δ369Rag1^-/- and control Rag1^-/- mice at 20 weeks old (Supplementary Fig. 9g), though Rorc^Δ369Rag1^-/- mice still showed reduced ILC3s, particularly CCR6⁺ and DN subsets (Supplementary Fig. 9h, i), suggesting that adaptive immune cells are involved in the small intestinal remodeling in Rorc^Δ369 mice. Collectively, these data indicate that OCR369 deficiency leads to progressive inflammation in the small intestine, likely due to Th2/Th17-mediated immune dysregulation.

RORγt⁺ APCs instruct T cell responses to dietary and microbiota antigens

Since RORγt⁺ APCs are crucial for the development of microbiota-specific RORγt⁺ Tregs, and OCR369 deficiency reduces multiple RORγt⁺ APC subsets, next, we investigated whether Rorc^Δ369 mice exhibited reduced tolerance to gut microbiota. After orally inoculation with Helicobacter hepaticus (H. h.), a gut pathobiont, and transfer of naïve H. h.-specific CD4⁺ T cells from Hh7-2 TCR transgenic (Hh7-2tg) mice, the differentiation of H. h.-specific CD4⁺ T cells in both adult Rorc^Δ369 and control mice was examined by flow cytometry (Fig. 6a). In control mice, Hh7-2 T cells mainly differentiated to RORγt⁺ Tregs in both the colon and colon-draining mLN (Fig. 6b–d). However, in Rorc^Δ369 mice, the development of H. h.-specific RORγt⁺ Tregs was almost completely abrogated, and Hh7-2 T cells mainly differentiated to inflammatory Th17 and Th1 cells (Fig. 6c), highlighting that OCR369 is essential for RORγt⁺ APCs to instruct microbiota-specific RORγt⁺ Tregs.

Fig. 6: OCR369-dependent RORγt+ APC instruct Treg responses to dietary and microbiota-derived antigens. — **Fig. 6: OCR369-dependent RORγt⁺ APC instruct Treg responses to dietary and microbiota-derived antigens.**

Given that OCR369-deficient mice developed spontaneous small intestinal inflammation with reduced RORγt⁺ Tregs and increased Th2 cells (Fig. 5), we explored whether OCR369-dependent RORγt⁺ APCs, particularly RORγt⁺ DC-like cells and MHCII⁺ ILC3s, are also required for the development of dietary antigen-specific Tregs. CFSE-labeled or CD45.1⁺ naïve ovalbumin (OVA)-specific CD4⁺ T cells from OT-II TCR transgenic mice were transferred into Rorc^Δ369 and control adult mice, followed by daily oral OVA administration (Fig. 6e and Supplementary Fig. 10a). At day 4 post OVA challenge, a significant reduction in CFSE⁺ RORγt⁺ Treg cells and an increase in CFSE⁺ Th17 cells in the mLN and Peyer’s patches (PP) were observed in Rorc^Δ369 mice (Supplementary Fig. 10b–d). By day 8, Rorc^Δ369 mice showed fewer CD45.1⁺RORγt⁺ OT-II Tregs and more CD45.1⁺RORγt⁺ OT-II Th17 and CD45.1⁺GATA3⁺ OT-II Th2 cells (Fig. 6f, g). Similarly, endogenous OVA-specific CD45.2⁺ T cells in Rorc^Δ369 mice exhibited reduced RORγt⁺ Tregs and increased inflammatory RORγt⁺ Th17 and GATA3⁺ Th2 cells (Supplementary Fig. 10e–g). The dependence of RORγt⁺ APCs for dietary antigen-specific Treg development was confirmed in mice with specific MHCII deletion in RORγt⁺ APCs (Rorc^creH2-Ab1^fl/fl), where OVA-specific RORγt⁺ Tregs were nearly absent, and Th2 cells dominated (Fig. 6h–j). Together, these data demonstrate that OCR369-dependent RORγt⁺ APCs are crucial for the development of dietary antigen-specific RORγt⁺ Tregs. Both MHCII⁺ ILC3s and CXCR6^-RORγt⁺ APCs (including RORγt⁺ DC-like cells) isolated from the mLN of 3-week-old RORγt-reporter mice were capable of promoting in vitro OVA-specific iTreg development (Fig. 6k). However, OCR369-deficient MHCII⁺ ILC3s from adult mLN exhibited reduced ability of promoting Treg differentiation (Fig. 6l), emphasizing the importance of OCR369 in regulating the function of RORγt⁺ APCs.

To confirm the role of Tregs in maintaining intestinal morphology and homeostasis, Rorc^Δ369 mice were adoptively transferred with Tregs every 4 weeks starting at 4 weeks of age (Supplementary Fig. 11a). Treg transfer restored small intestine length, villus length, and reduced goblet cell and tuft cell numbers (Supplementary Fig. 11b–e), while significantly reducing Th2 cells in Rorc^Δ369 mice (Supplementary Fig. 11f). Since OCR369 is required for both dietary- and microbiota-specific Treg development, we investigated whether the immunopathology in Rorc^Δ369 mice arises from intolerance to dietary or microbiota antigens. Control and Rorc^Δ369 mice were treated with antibiotics in the drinking water alone, elementary-diet alone or both since born (Supplementary Fig. 12a–d). Elementary-diet treatment, but not antibiotics alone treatment, eliminated the differences of RORγt⁺ Tregs in the small intestine between control and Rorc^Δ369 mice, indicating that OCR369-dependent RORγt⁺ Tregs in the small intestine are induced by dietary antigens. Moreover, only combined treatment with antibiotics and an elementary diet controlled Th2 and Th17 cell increases in Rorc^Δ369 mice (Supplementary Fig. 12a–d), indicating that both dietary and microbiota antigens contribute to the intestinal inflammation in OCR369-deficient mice. Together, considering the dynamic influence of OCR369 on RORγt⁺ APCs (Fig. 2), these results indicate that RORγt⁺ APC subsets critically instruct tolerance to dietary- and microbiota-derived antigens in a temporal manner across the lifespan.

OCR369-dependent RORγt⁺ APCs restrain food allergy

Given the OCR369-dependence of RORγt⁺ APC and the associated role in regulating Th/Treg balance in response to dietary antigens, we hypothesized that these APCs contribute to oral tolerance beyond microbiota homeostasis. A delayed-type hypersensitivity (DTH) model using OVA (OVA-DTH) was employed (Fig. 7a). OVA feeding before immunization induced tolerance to OVA challenge in control mice, but failed to do so in Rorc^Δ369 mice, as indicated by severe foot pad swelling and immune cell infiltration (Fig. 7b, c). Interestingly, “tolerized” Rorc^Δ369 mice not only displayed elevated Th17 and Th1 cells in the foot pad-draining lymph nodes, but also showed significant systemic allergic responses post challenge, as evidenced by decreased body temperature, increased serum OVA-specific IgE levels, as well as increased Th2 cells and IL-13-producing T cells in the spleen and peripheral blood (Fig. 7d–i and Supplementary Fig. 13a–f). These findings demonstrate that OVA pre-feeding induces a robust allergic response rather than tolerance in OCR369-deficient mice. In addition, transferring OT-II T cells into the OVA-DTH mouse model exacerbated the allergic response in Rorc^Δ369 mice, causing rapid body temperature drops and mortality within 30 minutes of OVA challenge (Supplementary Fig. 13g, h). Moreover, Rorc^Δ369 mice exhibited heightened susceptibility to food allergy, with significant body temperature drops, increased serum OVA-specific IgE and IgG1 levels (Fig. 7j–l). Together, these data demonstrate that OCR369-dependent RORγt⁺ APCs are essential for maintaining oral tolerance and preventing food allergy.

**Fig. 7: OCR369-deficient mice exhibit impaired oral tolerance.**

Discussion

Recent advances have highlighted the critical role of the MHCII-expressing RORγt⁺ APC family in shaping peripheral immunity and tolerance through direct interaction with T cells. The characterization of these cells is still evolving, and currently recognized major populations include MHCII⁺ ILC3s, extra-thymic Aire-expressing cells (eTAC), which resemble the medullary thymic epithelial cells (mTEC), and other potential CXCR6⁻IL-7R⁻ DC-like populations³⁹. The newly reclassified RORγt⁺ APC family, previously called Thetis cells (TC I-IV), is thought to encompass eTACs and DC-like cells^18,40, aligning with the resent identifications of RORγt⁺ DCs^33,41. Through integrated analysis of published and our own scRNA-seq data, we identified four distinct clusters of RORγt⁺ APCs: MHCII⁺ ILC3s, RORγt⁺ DC-like cells (TC II), RORγt⁺ eTAC I (TC I, Janus cell), and RORγt⁺ eTAC II (TC III). Here we identify OCR369 as a specific cis-regulatory element governing RORγt expression in multiple RORγt⁺ APC populations, particularly in ILC3s and RORγt⁺ DC-like cells. During the weaning period, OCR369 is essential for maintaining the population and function of RORγt⁺ DC-like cells, but not other RORγt⁺ APCs, in the mLNs. In adult mice, OCR369 deficiency causes a pronounced reduction in both the number and function of RORγt⁺ DC-like cells and MHCII⁺ ILC3s. Both cell types can promote Treg development in vitro, and OCR369 deficiency specifically impairs the capacity of MHCII⁺ ILC3s to drive this process. Consequently, OCR369 deficiency leads to impaired development of food- and microbiota-specific pTreg cells and exacerbates intestinal inflammation. These findings collectively indicate that OCR369-dependent RORγt⁺ APCs are required for establishing oral tolerance (Supplementary Fig. 14).

The field of RORγt⁺ APCs continues to develop rapidly, underscoring their essential role in regulating the Th/Treg balance, particularly in generating microbiota- and dietary antigen-specific RORγt⁺ Treg cells in the gut^{15,16,18,19,40}. Consistent with our observation that OCR369 regulates RORγt⁺ DC-like cells during weaning, prior studies have shown that RORγt⁺ APCs are most active in early life, when Thetis cells peak in abundance before declining in adulthood, thereby promoting tolerance to dietary antigens^18,40. Thus, OCR369-dependent RORγt⁺ DC-like cells appear to be the dominant APCs controlling food-specific pTreg development and oral tolerance in early life. In adult mice, however, recent evidence indicates that only MHCII⁺ ILC3s, not RORγt⁺ DC-like cells, interact with antigen-specific OT-II T cells after OVA challenge⁴². Given the exceptionally low abundance of RORγt⁺ DC-like cells and the more than 20-fold greater number of MHCII⁺ ILC3s in adults, along with data demonstrating that RORγt⁺ APCs remain necessary for Treg generation in adulthood using Rorc^{Venus-creERT2}H2-Ab1^fl/fl mice^18,40, we propose that in adult mice, these two subsets cooperate in pTreg differentiation and the maintenance of oral tolerance. Further experimental evidence will be required to validate this model and clarify the precise contributions of each subset.

Furthermore, the mechanisms by which RORγt regulates the function of RORγt⁺ APCs remain incompletely understood. Our data demonstrate that OCR369 modulates the expression levels of both RORγt and MHCII in RORγt⁺ DC-like cells and MHCII⁺ ILC3s, suggesting that RORγt influences antigen-presenting capacity via MHCII regulation. Indeed, OCR369-deficient MHCII⁺ ILC3s exhibit a reduced ability to drive Treg differentiation. Whether OCR369-deficient RORγt⁺ DC-like cells are similarly impaired in promoting pTreg development requires further investigation. In addition, other pathways, such as IL-2 production¹⁵, active TGF-β release via integrin^18,19,43, have been shown to be important for RORγt⁺ APCs in regulating Tregs. Whether these pathways are also involved in Treg-dependent food tolerance regulation by RORγt⁺ APCs requires further investigation. A notable question is why OCR369 selectively regulates different RORγt⁺ APC subsets, such as ILC3s and RORγt⁺ DC-like cells, and T cells. One plausible explanation is that MHCII⁺ ILC3s and RORγt⁺ DC-like cells express higher levels of RORγt relative to other cells. Notably, RORγt⁺ DC-like cells exhibit the highest RORγt expression, even surpassing ILC3s around weaning. This suggests that OCR369 is required for high RORγt expression, which in turn is critical for the maintenance and function of RORγt⁺ DC-like cells and MHCII⁺ ILC3s. These observations indicate that, in addition to lineage-specific determinants, RORγt expression levels play a crucial role in regulating distinct immune cells, a hypothesis that warrants further validation.

Despite their similar transcription profiles and cytokine production, ILC3s differ from their Th17 cell counterparts in their regulatory mechanism. The differences can be traced to their distinct chromatin landscapes at lineage-determining genes. For RORγt regulation, previous studies have identified multiple CREs, including RORCE2, essential for RORγt induction by STAT3 and SOX-5²⁹, CNS9 and CNS6 were found to be indispensable for RORγt expression through IL-6-STAT3 and TGF-β signaling²⁷. More recently, a + 7 kb CRE has been reported to regulate RORγt in ILC3s and RORγt⁺ DCs⁴¹. Nevertheless, the precise mechanisms that govern the development and function of distinct RORγt⁺ immune populations remain incompletely understood. Herein, we highlighted the OCR369, a region closely overlapping with the previously reported + 7 kb locus, as a CRE amplifying high-level of RORγt expression in ILC3s and RORγt⁺ DC-like cells but not T cells, thereby refining our understanding of distinct CREs regulating type 3 immune cells. The opening of a unique cis-regulatory element provides docking sites for TFs that regulate transcription, particularly those conserved in non-coding regions. Here, we find that RUNX3, highly expressed in ILC3s, binds OCR369 in ILC3s but not T cells. Although RUNX3 directly induces RORγt expression and is essential for ILC3 development³⁵, the functional implications of its direct binding to OCR369 in ILC3s remain to be investigated. In addition, whether other TFs interact with OCR369 to promote high RORγt expression is an area requiring further exploration. Moreover, the distinct distribution of multiple CREs is linked to chromatin loop formation anchored by lineage-determining TF complexes in higher-order chromatin structures^29,44. Interestingly, RUNX3 has been identified as a core TF in genome-wide chromatin loop formation in CD8⁺ T cells⁴⁵. In this study, we identified a chromatin loop connecting the Rorc promoter and OCR369, suggesting that RUNX3 may collaborate with other key TFs to link multiple CREs and regulate RORγt expression. The involvement of distinct CREs likely fine-tunes RORγt expression kinetics across different cell types, such as RORγt initiation (which requires further investigation) and amplification via elements like OCR369. Furthermore, given that RUNX3 is also expressed by RORγt⁺ DC-like cells, we hypothesize that RUNX3-OCR369 interaction could also sustain their high RORγt expression and development, thereby regulating the differentiation of RORγt⁺ Tregs and the maintenance of intestinal tolerance.

Overall, we have identified OCR369 as a specific CRE regulating RORγt expression in ILC3s and RORγt⁺ DC-like cells and highlighted the importance of OCR369-dependent RORγt⁺ APCs in oral tolerance induction. These findings provide new insights into potential therapeutic strategies for oral tolerance-related diseases.

Methods

Mice

Rorc^Δ369 and Rorc^Δ225 mice were generated on the C57BL/6 J background using the CRISPR-Cas9 system. Guide RNAs (gRNA) were designed with the CRISPOR web tool (CRISPOR.org). The efficiency of candidate gRNAs were tested using a commercial kit (TransGen), and gRNAs with high efficiency were in vitro transcribed with the MEGAshortscript kit. Micro-injection of gRNA and Cas9 mRNA was performed by the Laboratory Resources Animal Center of Tsinghua University. The edited mice were confirmed by genotyping, and were then selected and backcrossed for multiple generations to establish a stable germline-deficient strain. Rag1^–/– mice were purchased from Jackson Laboratory. OT-II TCR transgenic mice were generously provided by Chen Dong (Westlake University, Hangzhou). CD45.1 mice were bought from the Laboratory Resources Animal Center of Tsinghua University. Foxp3-RFP mice were generously provided by Yan Shi (Tsinghua University, Beijing). Rorc^GFP mice were provided by Coco Chu (Tsinghua University, Beijing). Rorc^creH2-Ab1^fl/fl mice were generously provided by Gregory F. Sonnenberg (Weill Cornell Medicine, New York). All mice were bred and maintained under specific pathogen-free (SPF) conditions on a 12/12-hour light–dark cycle. Unless otherwise noted, 6–12-week-old and sex matched mice were used for experiments. Sex was not considered in the study design or analysis because the primary objective was to investigate the regulating mechanism of ILC3 and RORγt⁺ APCs, which has not been reported to be sex-dependent in either humans or mice. To minimize biological variability, all experiments were performed with age-matched adult animals of the same sex. Consequently, sex-based differences were not analyzed. All animal experiments in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of Tsinghua University.

Isolation of immune cells

The isolation of intestinal lamina propria lymphocytes (LPLs) were performed as previously reported⁴⁶. Mice were firstly euthanized with CO₂ followed by cervical dislocation. Briefly, the intestines were longitudinally opened, Peyer’s patches (PPs) and luminal contents were removed, and the tissue was cut into around 2 cm pieces. The tissue was washed twice in PBS, and incubated in HBSS containing 1 mM DTT (Sigma, D0632), 5 mM EDTA, 15 mM HEPES, and 3% FBS for 15 min twice. Then the tissue was washed in HBSS with 15 mM HEPES and transferred to digestion buffer (3% FBS RPMI 1640 medium containing 0.05% DNase I (Sigma, DN25) and 0.1 mg/mL Liberase (Roche, 05401020001)) in C tubes (Miltenyi Biotech) for 20 (small intestine) or 30 min (large intestine) at 37 °C. Then the tissues were homogenized by gentleMACS Dissociator (Miltenyi Biotech) and passed through a 70 μm cell strainer. Single-cell suspensions were collected from 40–80% Percoll Density Gradient Media (GE Healthcare) after centrifuge at 400 × g for 20 min without brake. For other tissues, including the thymus, spleen, PPs, and lymph nodes, cells were mechanically dissociated, passed through a 70 μm strainer, and used for further analysis.

Flow cytometry analysis, ex vivo stimulation and FACS sorting

Isolated single-cell suspensions were washed twice with 3% FBS RPMI 1640 medium, resuspend in FACS buffer (PBS buffer containing 2% FBS and 0.02% NaN₃), and blocked with anti-CD16/CD32 antibody (eBioscience). Cells were stained with antibody cocktails for surface markers and Fixable Viability Dye (eBioscience) at 4 °C for 40 min. For transcription factor staining, cells were fixed in Foxp3/Transcription Factor Staining Buffer Set (eBioscience) at 4 °C overnight, then permeabilized and stained with antibody cocktails for 30 min at room temperature. Antibodies used for surface markers were used at 1:500, for the intracellular cytokines and TFs staining, antibodies were used at 1:250. For ex vivo stimulation and intracellular cytokine staining, cells were resuspended in 10% FBS RPMI 1640 medium containing Glutamax (Gibco), 10 mM HEPES, sodium pyruvate (Gibco), and 2-Mercaptoethanol (Gibco). Cells were stimulated with PMA and Ionomycin (P.I.) or IL-23 and IL-1β (10 ng/mL) for 2 h, followed by the addition of Brefeldin A for the next 2 h. Cells were collected, stained for surface markers, fixed overnight in IC fixation buffer (eBioscience), permeabilized, and stained with anti-IL-22, anti-IL-17A, anti-IL-5, anti-IL-13, and anti-IFN-γ antibodies (Biolegend). Flow cytometry was performed on Fortessa instruments (BD Biosciences) and analyzed with FlowJo software. Gating strategies of Th/Treg cells and ILC1-3 were shown in Supplementary Fig. 15a. For FACS sorting, cells were resuspended in PBS buffer containing 2% FBS and 5 mM EDTA, and sorted by BD FACSAria™ flow cytometer. ILC3s were gated as live lineage^–KLRG1^–CD90^highCD45^low cells from intestinal LPLs.

Construction of bone marrow chimera mice

Bone marrow (BM) cells from WT CD45.1 mice were 1:1 mixed with BM cells from the Rorc^Δ369 or Rorc^WT mice and intravenously transferred to irradiated (5.5 Gy × 2) CD45.1 mice. Mice were analyzed 8 weeks later with flow cytometry.

Analysis of bone marrow progenitors

Bone marrow cells were isolated from Rorc^WT or Rorc^Δ369 mice and treated with red blood cells (RBCs) lysis buffer to remove RBCs. Cells were stained with a lineage antibodies cocktail (CD3, NK1.1, Ter-119, CD11b, Gr-1, B220) and other antibodies. CLPs (lineage^–CD127⁺Flt3⁺α4β7^–), CHILPs (lineage^–CD127⁺Flt3^–α4β7⁺CD25^–PD-1^–), and CHILPs (lineage^–CD127⁺Flt3^–α4β7⁺CD25^–PD-1⁺) were analyzed by flow cytometry.

CHILP in vitro differentiation

OP9-DL1 stromal cells were cultured and irradiated 1 day before use. CHILPs were isolated and purified from bone marrow of age- and sex-matched donor mice, negatively selected by magnetic Dynabeads (Invitrogen) with biotin-labeled lineage antibodies (CD3, NK1.1, Ter-119, CD11b, Gr-1, B220), and then sorted as lineage^–CD127⁺Flt3^–α4β7⁺CD25^–PD-1^–cells using a BD FACSAria™ flow cytometry. CHILPs were cultured on OP9-DL1 cells in α-MEM medium including 20% FBS, 10 mM HEPES, 100 U/mL Penicillin-Streptomycin (Thermo Fisher), 1 mM sodium pyruvate, 8 mg/mL Glutamine, 2-Mercaptoethanol (Gibco), and mIL-7 (Novoprotein, CC73, 20 ng/mL) and mSCF (Novoprotein, C775, 10 ng/mL). The medium was refreshed at day 5, and cells were harvested at day 10 for flow cytometry analysis.

Th17 and Treg in vitro differentiation

Naïve T cells were sorted as live CD45⁺CD3⁺CD8α^–CD4⁺CD62L^highCD44^low cells from the spleen and lymph nodes and cultured in anti-CD3/CD28 antibody (10 μg/mL)-coated cell culture plates in 10% FBS complete 1640 medium with different cytokine cocktails: Th17 condition (IL-6 (Peprotech, 216-16, 20 ng/mL), TGF-β (R&D, 240-B-010, 1 ng/mL), IL-1β (Peprotech, 211-11B, 10 ng/mL), IL-23 (R&D, 1887-ML-010, 25 ng/mL)); Treg condition (TGF-β (R&D, 7666-MB, 2 ng/mL) and IL-2 (Peprotech, 212-12, 10 ng/mL)). Cells were analyzed at day 4 after 2 h stimulation with P.I. and Golgi Stop (Thermo Fisher, 00-4980-93) for 4 h for intracellular staining and flow cytometry analysis.

DNA-pulldown and Mass spectrometry

Biotin-labeled DNA-pulldown assay was performed according to the previous report^47,48 with modifications. In brief, 1 × 10⁶ FACS sorted ILC3s were washed twice in cold PBS and nuclear proteins were extracted with a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0027). Protein concentration was determined by BCA assay (Thermo Fisher, 23225), and adjusted with DNA-protein binding buffer (120 mM NaCl, 0.44 mM EDTA, 0.8 mM MgCl₂ and 6% glycerol). The biotin-labeled DNA probes were prepared by PCR using Q5^® High-Fidelity DNA Polymerase (NEB, M0491S) with 5’-biotin labeled primers and purified from 2% agarose gel. Pulldown assays were performed with BeyoMag™ Streptavidin Magnetic Beads (Beyotime, P2151). DNA probes were incubated with Streptavidin Magnetic Beads in Binding & Washing Buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl, 0.01%-0.1% Tween-20) at 4 °C for 3 h, washed twice, and incubated with nuclear extracts in DNA-protein binding buffer (containing 100 μg/mL salmon sperm DNA (Thermo Fisher, 15632011) and 0.125 mg/mL poly (dIdC) to nonspecific binding reduction) at 4°C overnight. Beads were washed twice with 0.05% PBST and eluted with 1 × SDS-PAGE loading buffer at 95 °C for 5 min. Eluted proteins were analyzed by 15% SDS-PAGE followed by Silver staining (Thermo Fisher, 24612), and bands were excised for mass spectrometry identification using a Q Exactive LC-MS/MS (Thermo Fisher) by Technology Center for Protein Science, Tsinghua UniversityThermo Fisher.

CUT&RUN assay

5 × 10⁵ sorted ILC3 and in vitro differentiated Th17 cells were collected for CUT&RUN library preparation (YEASEN 12598) following the manufacturer’s instructions. Anti-RUNX3 (CST, #9647) and Isotype Rabbit IgG (Abcam, ab171870) were used for immunoprecipitation (1:50). RUNX3-bound DNA was amplified via 15 cycles of PCR and then purified with magnetic beads. DNA fragments were detected by SYBR Green qPCR analysis (YEASEN, 11203ES), normalized by Ct value relative to E. coli spike-in controls. Primers are listed in Supplementary Data 8.

Dual-Luciferase reporter assay

The OCR369 and RORγt-promoter elements were cloned into the firefly luciferase pGL3 vector. RUNX3-CDS was inserted into the pcDNA3.4 vector (provided by Chen Dong, Westlake University, Hangzhou). pGL3 vectors, pcDNA3.4 vectors and renilla luciferase control vector pRL-TK were 5:5:1 co-transfected into HEK293T cells (provided by Yang-Xin Fu, Tsinghua University, Beijing) by Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher, L3000001). The luciferase activity was measured using the Dual-Luciferase^® Reporter Assay System (Promega, E1910) on a Glomax Multi detection system (Promega). Relative Luciferase activity was calculated as firefly luciferase activity divided by renilla luciferase activity.

Quantitative analysis of chromosome conformation capture assays (3C-qPCR)

B16 and MNK-3 cell line were provided by Yun-Cai Liu (Tsinghua University, Beijing) and James R. Carlyle (University of Toronto, Toronto)/David S.J. Allan (NHLBI, NIH, Bethesda), respectively. 3C-qPCR was performed as previously reported^29,49 with modifications. In brief, 2 ~ 5 × 10⁶ cells were harvested and resuspended in cold PBS with 10% FBS, fixed with 1% formaldehyde (Thermo Fisher, 28908) for 10 min at room temperature, then quenched by 1 M cold glycine. Nuclei were extracted by cold lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 0.2% NP-40, pH 7.5) with protease inhibitor (Roche, 11836145001) on ice for 20 min. The pellets of nuclei were incubated in rCutsmart buffer (NEB) with 0.3% SDS for 1 h at 37 °C, followed by the addition of 1% Triton X-100 for 1 h at 37 °C. Nuclei were digested by NlaIII restriction enzyme at 37 °C overnight. The reaction was inactivated by the addition of 1.6% SDS and incubate at 65 °C for 10 min. Samples were diluted with T4 DNA ligase buffer (NEB), added with 1% Triton X-100 and incubate for 1 h. 400U T4 DNA ligase was added and incubated at 16 °C overnight. Ligation was stopped by adding protease K (Beyotime), and then the crosslinks were reversed at 65 °C overnight. The samples were digested with 10 µg/mL of RNase I (Beyotime) at 37 °C for 45 min, and DNA was purified by Universal DNA Purification Kit (TIANGEN, DP214). The 3 C products were analyzed with TaqMan qPCR master mix (YEASEN, 11208ES) on a StepOnePlus Real-Time PCR System (Thermo Fisher). Relative crosslinking was calculated by normalizing Ct values to Gapdh. Primers for the 3C-qPCR are listed in Supplementary Data 8.

Bulk RNA sequencing

RNA was extracted from ~ 0.5 cm of small intestine using TRIzol reagent (Invitrogen, 15596026CN). RNA quality was confirmed by 1% agarose gel electrophoresis, and samples were sent to The Beijing Genomics Institute (BGI) for library preparation and sequencing. Clean reads were aligned to the mouse reference genome (mm10). Differential gene expression analysis was conducted using DESeq2 (DEG identified by q-value < 0.05 and fold change > 1). Gene Ontology (GO) enrichment, KEGG pathway enrichment were performed by Dr. Tom tool (biosys.bgi.com).

Gene set enrichment analysis (GSEA)

The gene-level count matrix was generated by the BGI data analysis server, and then the genes were pre-ranked by signal2noise. GSEA was performed on a custom set of MSigDB gene sets, including Reactome pathways and a gene set of the top 300 up-regulated genes in the intestinal epithelium of mice infected with N. brasiliensis compared to non-infected controls, using clusterProfiler (v.4.13.3) package⁵⁰. The enrichplot (v.1.24.4) package was used for visualization.

Immunofluorescence staining and histology analysis

Intestinal tissue was coiled into a “Swiss roll” and fixed in the 4% paraformaldehyde (PFA) and paraffin-embedded. After dewaxing, rehydration and antigen retrieval, 10 μm tissue sections were blocked in PBS with 3% BSA and 0.3% Triton X-100 for 1 h at room temperature. Sections were stained with the indicated primary antibody (sheep anti-MCPT1, R&D AF5146; rabbit anti-DCAMKL1, abcam ab31704, 1:500) overnight at 4 °C, followed by secondary antibody (Goat anti-rabbit IgG, eBioscience a21244; Donkey Anti-Sheep, abcam ab150179, 1:1000) and DAPI. Images were acquired using an FV3000 Olympus confocal microscope (20 × or 40 × magnification), and 5 random images per tissue were selected for statistical analysis. For histology, Hematoxylin and eosin (H&E) staining was performed after dewaxing and rehydration. Intestinal goblet cells were stained with AB-PAS staining kit (Solarbio, G1285). Deposition of extracellular matrix was stained by Sirius Red Stain Kit (Solarbio, G1572). Tissue sections were scanned by a PANNORAMIC SCAN II scanner (3DHISTECH). The average length of villi and crypt, the numbers of tuft cell, mast cell and goblet cell were calculated from at least 10 villi/crypt units for each section of mice.

RNA extraction and real-time PCR

RNA was extracted from ~0.5 cm of small intestine using TRIzol reagent (Invitrogen, 15596026CN). cDNA synthesis was conducted with RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, K1622). qPCR was performed with SYBR Green qPCR mix (YEASEN, 11203ES) on a StepOnePlus Real-Time PCR System (Thermo Fisher). Relative gene expression was normalized by Gapdh or Actb. Primers are listed in Supplementary Data 8.

OT-II T cell adoptive transfer & Treg rescue

For the OT-II T cell adoptive transfer experiment in Rorc^Δ369 mice, naïve OT-II CD4⁺ T cells were sorted as live CD45⁺CD3⁺CD8α^–CD4⁺CD62L^highCD44^lowcell from the spleen and lymph nodes (Supplementary Fig. 15b). 1 × 10⁶ cells were intravenously transferred to each mouse, which were then orally treated with 50 mg ovalbumin (OVA) dissolved in water (YEASEN, 36121ES) daily. Mice were euthanized at day 8 for immune cell analysis in SI-LPL, PPs, and mLN. For the CFSE labeling assay, the sorted OT-II T cells were labeled with 1 μM CFSE for 15 min at 37 °C, washed twice and then transferred. For the OT-II T cell adoptive transfer experiment in Rorc^creH2-Ab1^fl/fl mice, after OT-II T cell transfer, mice received drinking water containing 1% OVA (v/w) throughout the experiment and also received OVA by oral gavage (20 mg/mouse; A5503; Sigma) every other day. Cells of SI-LP and mLN were analyzed 13 days post-transfer. For the Treg rescue experiment, Treg cells sorted as live CD45⁺CD3⁺CD4⁺FOXP3-RFP⁺ cells from the spleen and lymph nodes. 5 × 10⁵ Treg cells were intravenously transferred to Rorc^Δ369 mice every 4 weeks starting at 4 weeks of age. Mice were euthanized and analyzed at around 20 weeks old.

Citrobacter rodentium infection model

Mice were orally gavaged with C. rodentium strain DBS100 (ATCC 51459). C. rodentium was cultured in LB broth overnight, and the bacterial concentration was determined by measuring the optical density at 600 nm (OD600). Mice were infected with 2 × 10⁹ CFU in 200 μL PBS. Bodyweight was measured every 2 days, and fecal pellets were collected, homogenized, and plated on MacConkey agar to determine CFU counts.

OVA tolerance generation in DTH model

Mice were tolerized by oral gavage with 50 mg OVA (YEASEN, 36121ES) for 2 consecutive days. Two weeks later, mice were immunized subcutaneously with 200 μg OVA (Sigma, A5503) in PBS mixed 1:1 with Complete Freund’s Adjuvant (Sigma, F5881). One week later, mice were challenged subcutaneously with 50 μg OVA in the left footpad, along with 20 μL PBS in the right footpad as a control. Rectal temperatures were measured every 5 min after challenge for at least 40 min. Footpad thickness was measured in a blinded fashion before and 48 h after injection with a digital precision caliper. Footpad swelling was calculated as [(left footpad thickness − right footpad thickness)] at 48 h – [(left footpad thickness − right footpad thickness)] at 0 h. Serum was collected at 0 h and 72 h for OVA-specific Ig detection. Mice were euthanized at 72 h for flow cytometry analysis, and footpads were fixed in 4% FPA for histology analysis.

Enzyme-linked immunosorbent assay (ELISA)

To detect OVA-specific antibodies in the serum, 96-well plates were coated with 75 μL OVA/PBS (10 μg/mL) at 4 °C overnight. Plates were blocked with 3% FBS/PBS for 1 h, washed with 0.05% PBST, and incubated with 5-fold serially diluted serum. Secondary HRP-conjugated antibodies were added (Goat Anti-Mouse IgE-HRP, SouthernBiotech, 1110-05 1:5000; Goat Anti-Mouse IgG1-HRP, SouthernBiotech, 1071-05 1:6000), and plates were developed with TMB solution (Thermo Fisher, 00-4201-56). Absorbance was measured at 450–570 nm using an ELX808IU microplate readerThermo Fisher (BioTek).

Helicobacter hepaticus colonization and HH7-tg T cell transfer

H. hepaticus and HH7-tg mice were provided by Mo Xu (National Institute of Biological Sciences, Beijing), and the experiment was conducted as previous reported⁵¹. In brief, H. hepaticus was cultured on 5% sheep blood agar plates in a micro-aerobic atmosphere (1% O₂) at 37 °C. For infection, H. hepaticus was resuspended in Brucella broth at the OD600 readings of 1-1.5, then each mouse was orally administrated in two doses (0.2 mL each). For adoptive transfer, Naïve CD4⁺ T cells were sorted from the spleen and LNs of HH7-tg mice, and 2 × 10⁵ cells were intravenously transferred to each mouse. Mice were analyzed 14 days later for LI-LPLs and colon-draining mLN (c-mLN) cells by flow cytometry.

Antibiotics cocktail treatment and protein-free elemental diet administration

To deplete gut microbiota, 3-week-old mice received an antibiotics cocktail in drinking water (vancomycin (0.5 g/L), ampicillin(1 g/L), neomycin (1 g/L), metronidazole (1 g/L)) for 5 weeks. For protein-free elemental diet studies, mice were fed a modified XT-93G diet (protein replaced with amino acids) (Xietong Pharmaceutical Bio-Engineering) from birth. Mice were analyzed at 8 weeks old.

Analysis of RORγt⁺ APCs

Mice were euthanized, and the mLNs were digested in RPMI1640 medium containing 10% FBS (Gibco), 1% L-glutamine, 1% penicillin–streptomycin, 10 mM HEPES, 1 mg/mL collagenase A (Sigma, 11088793001), and 1 U/mL DNase I (Sigma, 10104159001) for 45 min at 37 °C, 250 rpm. Digested samples were filtered through 70 μm strainers and centrifuged (300 × g). Cells were resuspended in RPMI 1640 medium with 10% FBS for further analysis. For flow cytometry analysis, cells were firstly stained with biotin-labeled lineage antibodies (CD3, B220, TCRβ, TCRγδ, NK1.1, Ter-119, F4/80, Gr-1, SIGLEC-F), and then cells were stained with other antibodies for analysis. MHCII⁺ ILC3s were gated in lineage^–MHCII⁺RORγt⁺CXCR6⁺; CXCR6^–RORγt⁺ APCs (non-ILC3 RORγt⁺ APCs) were gated in lineage^–MHCII⁺RORγt⁺CXCR6^–; RORγt⁺ DC-like cells were gated in lineage^–MHCII⁺RORγt⁺CXCR6^–NCAM-1^–EpCAM-1⁺CCR6⁺NRP1⁺; RORγt⁺ eTAC I cells were gated in lineage^–MHCII⁺RORγt⁺CXCR6^–NCAM-1⁺; RORγt⁺ eTAC II cells were gated in lineage^–MHCII⁺RORγt⁺CXCR6^–NCAM-1^–EpCAM-1⁺CCR6^–NRP1^–CD11b^–; migratory DCs were gated in lineage^–MHCII⁺RORγt⁺CXCR6^–NCAM-1^–EpCAM-1⁺CCR6^–NRP1^–CD11b⁺. Besides, Janus cells were gated as Dump^– (TCRβ, TCRγδ, CD3, B220, EpCAM) MHCII⁺RORγt ⁺CD127^–CXCR6^–.

In vitro OT-II CD4⁺ T cell culture

Naive CD3⁺CD4⁺CD25^–CD62L^highCD44^low OT-II T cells were isolated from OT-II mice. Different APCs in mLNs, including cDC2s (lineage^–RORγt-GFP^–CD11c⁺MHCII⁺), CXCR6^–RORγt⁺ APCs (lineage^–MHCII⁺RORγt-GFP⁺CXCR6^–) and MHCII⁺ ILC3s (lineage^–MHCII⁺RORγt-GFP⁺CXCR6⁺) were isolated from Rorc^GFP mice, with the biotin labeled lineage antibodies cocktail (CD3, B220, TCRβ, TCRγδ, NK1.1, Ter-119, F4/80, Gr-1, SIGLEC-F) (Supplementary Fig. 15c). MHCII⁺ ILC3s from mLNs of Rorc^WT and Rorc^Δ369 mice were gated in lineage^–MHCII⁺CXCR6⁺. T cells were co-cultured at a ratio of 300 APCs to 1.5 × 10³T cells in the presence of OVA_323-339 peptide (1 µg/mL, MCE, HY-P0286) with 2 ng/mL TGF-β1 (R&D Systems, 240-B-010) and 10 ng/mL IL-2 (Peprotech, 212-12). Cell number, proportion and FOXP3 expression was assessed after 4 days of culture.

scRNA-seq sample preparation

For analysis of APCs in P18 Rorc^WT and Rorc^Δ369 mice, live CD45⁺lineage^–MHCII⁺ cells in mLNs were sorted and prepared for scRNA-seq. For analysis of immune cells in 6-week-old Rorc^WT and Rorc^Δ369 mice, live CD45⁺lineage^–CD127⁺ ILCs and CD45⁺CD3⁺CD4⁺ T cells from the small intestines were sorted and mixed at a 1:1 ratio. For analysis of immune cells in 28-week-old Rorc^WT and Rorc^Δ369 mice, live CD45⁺B220^– cells from the small intestines were sorted and prepared for scRNA-seq.

scRNA-seq data analysis

Celescope (v.2.1.0) developed by Singleron Biotechnologies was employed for barcode processing, alignment to the mm10 reference genome, and single-cell counting. Seurat (v.5.2.1) was used for downstream analysis. For quality control, scDblFinder (v.1.18.0) was used to remove doublets. According to the data quality of each sample, barcodes were filtered based on the percentage of mitochondrial transcripts ( < 5% to 10%), the number of detected genes (> 500 and < 5000), and the number of detected transcripts (> 1000 and < 25000 to 50000). As described above, we performed scRNA-seq for control and Rorc^Δ369 mice for each condition. After quality control and clustering, data were merged and normalized using SCTransform function. For dimensional reduction, we performed principal component analysis (total number of principal components = 50) and used the top 30 principal components for FindNeighbor, FindCluster, and RunUMAP functions. For FindCluster, leiden algorithm was used with resolution = 0.4 to 0.5. After manual annotation, for scRNA-seq data performed with cells from the small intestine lamina propria of 6-week-old and 28-week-old mice, we divided the ILC compartment and CD4⁺ T cell compartment into various subsets for downstream analysis. For scRNA-seq data performed with cells from mLNs of P18 mice, residual B cells and Plasma cells were removed for downstream analysis. FindMarkers was used for identifying differentially expressed genes. And the scRNA-seq data of P18 mice was also integrated with datasets from Akagbosu et al. (GSE174405)¹⁸ and Narasimhan et al. (S-BSST1322)³³. Batch correction and integration was achieved with the IntegrateLayers function using anchor-based RPCA integration.

ATAC-seq

RORγt⁺ Treg cells were isolated from the intestine of 8-week-old Rorc^GFP mice as live CD45⁺CD3⁺CD4⁺RORγt-GFP⁺CD25⁺ cells. ATAC-seq was performed using the Hieff NGS^® ATAC-Seq Library Prep Kit for Illumina^® (YEASEN) according to the manufacturer’s instructions. DNA fragments were extracted and purified by the magnetic beads, then a DNA library was constructed and used for sequencing. Sequenced ATAC-seq datasets were mapped to the mouse mm10 genome with Bowtie2. Mapped reads were deduplicated and then converted to a bigwig file using bamcoverage with RPKM normalization. The results were presented by the Integrative Genomics Viewer software.

Public multiome scATAC/scRNA-seq data processing

Public multiome scRNA/scATAC-sequencing data of RORγt⁺ APCs were used (GSE174405) for evaluating the accessibility of the OCR369 locus¹⁸. The scRNA-seq data were processed using scanpy (v.1.10.1), and the scATAC-seq data were processed using Snapatac2 (v.2.7.1). Data preprocessing and cell annotation were performed as previously described¹⁸. The coverage tracks of each RORγt⁺ APC were exported by the snapatac2.ex.export_coverage function with RPKM normalization and saved as bigwig format. The results were presented by the Integrative Genomics Viewer software.

Statistical analyses

P-values of mouse datasets were determined by a parametric unpaired two-tailed Student’s t test with a 95% confidence interval. Variance was analyzed using the F-test. Welch’s correction was performed in case of unequal variance. Where appropriate, ordinary two-way ANOVA followed by Bonferroni post-tests, or ordinary one-way ANOVA with Tukey’s multiple comparisons test were performed. All statistical tests were performed with GraphPad Prism v.10 software. P-values or adjusted P-values less than 0.05 were considered significant. The exact P-values are labeled within the Figures. Results are presented as mean ± SEM unless otherwise indicated.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The scRNA-seq data generated in this study have been deposited in the Genome Sequence Archive under accession code CRA037466 (https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA056298). The scRNA-seq data used and analyzed in this study are available in the NCBI GEO database under accession code GSE174405 and in BioStudies under accession code S-BSST1322. Bulk RNA-seq data generated in this study have been deposited in the NCBI GEO database under accession code GSE274080 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE274080). ATAC-seq data used in this study are available in the NCBI GEO database under accession codes GSM3638386, GSE137319, and GSE191312. The scATAC-seq data analyzed in this study are available in the NCBI GEO database under accession code GSE174405. Other data supporting the findings of this study are available within the article and its supplementary materials. Source data are provided in this paper.

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Acknowledgements

We thank all members from Xiaohuan Guo lab for helpful discussion. We also thank the Core Facility of the Institute for Immunology and the Laboratory Animal Resources Center at Tsinghua University for their support. We are grateful to Yan Shi (Tsinghua University, Beijing) for Foxp3-RFP mice; Mo Xu (National Institute of Biological Sciences, Beijing) for H. hepaticus and HH7-tg mice; James R. Carlyle (University of Toronto, Toronto) and David S.J. Allan (NHLBI, NIH, Bethesda) for MNK-3 cells. This work was supported by the National Natural Science Foundation of China (32530036, 32170872, 82122030 and 31821003), the National Key R&D Program of China (2023YFC2306202), Beijing Natural Science Foundation (Z210015). The Guo laboratory was also supported by the SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, the Institute for Immunology, Tsinghua University, the Open Funding Project of the State Key Laboratory of Biopharmaceutical Preparation and Delivery (No. 2024KF-07), and the Bayer Investigator Fellowship. Research in the Sonnenberg Laboratory is supported by the National Institutes of Health (R01AI143842, R01AI123368, R01AI145989, U01AI095608, R01AI162936, R01CA274534, and R37AI174468), M.L. is supported by NCI K99 (K99CA290052). G.F.S. is a CRI Lloyd J. Old STAR. Figures 1a, 2e, 3a, f, i and Supplementary Figs. 1b, 12a–d, 14, and the mouse artwork (Figs. 6a, e, h, 7a, j and Supplementary Figs. 6a, 10a, 11a, 13g) have been created with BioRender (https://BioRender.com).

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These authors contributed equally: Jie Zhao, Jiacheng Hao, Jincheng Chen.

Authors and Affiliations

Institute for Immunology, Tsinghua University, Beijing, China
Jie Zhao, Jiacheng Hao, Jincheng Chen, Haoyu Liu, Na Li, Panwei Song, Wenyan Wang, Coco Chu & Xiaohuan Guo
School of Basic Medical Sciences, Tsinghua Medicine, Tsinghua University, Beijing, China
Jie Zhao, Jiacheng Hao, Jincheng Chen, Haoyu Liu, Na Li, Panwei Song, Wenyan Wang, Coco Chu & Xiaohuan Guo
Beijing Key Laboratory of Immunological Research of Allergy (LIRA); Tsinghua University, Beijing, China
Jie Zhao, Jiacheng Hao, Jincheng Chen, Haoyu Liu, Na Li, Panwei Song, Coco Chu & Xiaohuan Guo
State Key Laboratory of Molecular Oncology, School of Basic Medical Sciences; Tsinghua University, Beijing, China
Jie Zhao, Jiacheng Hao, Jincheng Chen, Haoyu Liu, Na Li, Panwei Song, Wenyan Wang & Xiaohuan Guo
Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, Cornell University, New York, NY, USA
Mengze Lyu & Gregory F. Sonnenberg
Joan and Sanford I. Weill Department of Medicine, Division of Gastroenterology & Hepatology, Weill Cornell Medicine, Cornell University, New York, NY, USA
Mengze Lyu & Gregory F. Sonnenberg
Department of Microbiology & Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA
Mengze Lyu & Gregory F. Sonnenberg
SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine, Shanxi Medical University, Taiyuan, Shanxi, 030001, China
Xiaohuan Guo

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Contributions

X.G. is the senior and corresponding author. X.G. conceived and designed the study. X.G., J.Z., J.H., and J.C. prepared the manuscript. J.Z., J.H., and J.C. designed and performed most of the experiments and analyzed the data. M.L., H.L., N.L., P.S., and W.W. participated in some experiments. C.C. and G. F.S. provided critical materials and suggestions.

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Correspondence to Xiaohuan Guo.

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Zhao, J., Hao, J., Chen, J. et al. RORγt⁺ APCs require a distinct cis-regulatory element to instruct tolerance to dietary antigens. Nat Commun 17, 3019 (2026). https://doi.org/10.1038/s41467-026-69886-z

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Received: 29 July 2025
Accepted: 09 February 2026
Published: 21 February 2026
Version of record: 30 March 2026
DOI: https://doi.org/10.1038/s41467-026-69886-z