27 okt. 2025
Zhao et al. (ResearchSquare) DOI: 10.21203/rs.3.rs-4865841/v1
Keywords
Mucosal immunity
Oral tolerance
ILC3
Main Findings
How does the mucosal immune system distinguish innocuous antigens from real threats? This central question in mucosal immunology is still being actively investigated. Recently, several antigen-presenting cell (APC) subsets have been identified that regulate homeostatic responses to commensal microbiota and dietary antigens. These include APCs expressing RORγt and sometimes AIRE, such as type 3 innate lymphoid cells (ILC3s) and Thetis cells (TC). These APCs can instruct downstream regulatory T cell (Treg) responses and ultimately establish a memory of harmless antigens. Despite these advances, it remains unclear how these specialized APCs are themselves regulated. In the discussed preprint, Zhao et al. identified a regulatory element, OCR369 (Open Chromatin Region 369), required for the proper development of RORγt+ ILC3s. OCR369 was discovered through analysis of publicly available ATAC-seq datasets and was found to be selectively accessible only in RORγt+ APCs. To investigate the impact of OCR369 deficiency on cell development in vivo, the authors use OCR369-whole body knockout mice (RorcΔ369), which exhibit reduced frequencies of gut ILC3s (especially of RORγt+ CCR6+ subtypes) and (DC)-like RORγt+ APCs. On a more physiological level, these mice develop age-associated spontaneous gut inflammation and show impaired antigen-specific Treg induction upon exposure to microbial (Helicobacter hepaticus) or dietary antigens (ovalbumin, OVA). To establish the molecular mechanism of OCR369 regulation of RORγt, the authors used CUT&RUN and 3C-qPCR assays to demonstrate that OCR369 regulates RORγt expression in cis, acting on the same DNA molecule. Taking this further, the authors identified RUNX3 as a transcription factor that binds to OCR369 and regulates RORγt expression more strongly in ILC3s compared to TH17 cells. Finally, using a food allergy model, the authors show that OCR369-deficient mice failed to develop oral tolerance to OVA, reinforcing the functional importance of this regulatory element. In summary, this preprint advances our understanding of how (mostly) ILC3 development is regulated and how this regulation impacts oral tolerance. It is a carefully executed study that highlights OCR369 as a novel cis-regulatory element and opens new avenues for exploring the molecular mechanisms underlying oral tolerance in other RORγt+ APCs.
Limitations
The authors should put the proposed RORγt⁺ APC types in the up-to-date and consistent nomenclature, e.g. RORγt⁺ eTAC II are not TC III. These cells could be named as RORγt⁺ TC I-IV.
The regulation of dietary Tregs by Thetis cells (TC) was recently shown by Cabric et al. (2025), and this could be mentioned in the introduction section.
Would it be possible to add in the methods more details on the bioinformatic analysis of the ATAC-seq data (published) that led to the OCR369 locus finding? Ideally, the scripts for the analyses should be deposited in a public repository.
Does the RorcΔ369 model have reduced lymph node formation, if the deletion affects mostly the CCR6+ ILC3 subset (Fig. 1g)? Another function of ILC3s is to promote IgA class switching. Is the IgA response impaired in RorcΔ369?
In the bone marrow chimera experiments, it is not fully clear how many cells were used to repopulate the recipient mice, and how many cells could be found after reconstitution: only normalized proportions were shown in the results part. Maybe this could be elaborated further in the results and methods.
In the spontaneous gut inflammation model (Fig. 5), is it possible to revert fibrosis (5d) with anti-IL-4/IL-5/IL-13 cytokine treatment? Is this inflammation CD4 T cell dependent?
Fig. 5e-f represents scRNA-seq experiment on ILCs in 28-week old RorcΔ369 mice; could there be similar effects in RUNX3-deficient ILC3s? For example, on a RorcCreRunx3fl/fl model.
Since definitive phenotypic evidence for the role of OCR369 in RORγ+ APC development is mainly presented for ILC3 in this preprint, statements claiming the importance for OCR369 in all RORγt+ APCs should be made more carefully.
In the food allergy model (Fig. 7a), the authors should clarify why non-tolerized mice (WT and RorcΔ369) did not develop an IgE response and were protected from anaphylaxis. At the same time, only the WT tolerized group did not develop foot pad inflammation after s.c. OVA injection. What would be the positive control then?
The oral challenge with C. rodentium showing no difference in response is a very interesting experiment and would also fit into the main figures, as it highlights the fact that the described regulation of OCR369 on ILC3s is more subtle and might be preferentially required for oral tolerance to more harmless antigens, while in the context of acute infection there could be different mechanisms.
Significance/Novelty
This preprint describes a new mechanism on how RORγt+ ILC3s are regulated by a distinct cis-regulatory element OCR369 and how a deficiency in this element can change effector responses to microbial and dietary antigens. The study opens up many interesting questions to investigate next, e.g. how regulation happens in the rest of RORγt+APCs, and what is the role of RUNX3-OCR369-RORγt axis in the human disease context. Knowing this new line of regulation can also help to study ILC3s more specifically.
Credit
Reviewed by Alisa Iakupova as part of a cross-institutional journal club between the Max-Delbrück Center Berlin, the Ragon Institute Boston (Mass General, MIT, Harvard), the University of Virginia, the Medical University of Vienna and other life science institutes in Vienna.
The author declares no conflict of interests in relation to their involvement in the review.