Although intestinal epithelial cells (IECs) can express major histocompatibility complex class II (MHC II), especially during intestinal inflammation, it remains unclear if antigen presentation by IECs favours pro- or anti-inflammatory CD4 + T cell responses. Using selective gene ablation of MHC II in IECs and IEC organoid cultures, we assessed the impact of MHC II expression by IECs on CD4 + T cell responses and disease outcomes in response to enteric bacterial pathogens. We found that intestinal bacterial infections elicit inflammatory cues that greatly increase expression of MHC II processing and presentation molecules in colonic IECs. Whilst IEC MHC II expression had little impact on disease severity following Citrobacter rodentium or Helicobacter hepaticus infection, using a colonic IEC organoid-CD4 + T cell co-culture system, we demonstrate that IECs can activate antigen-specific CD4 + T cells in an MHC II-dependent manner, modulating both regulatory and effector Th cell subsets. Furthermore, we assessed adoptively transferred H. hepaticus -specific CD4 + T cells during intestinal inflammation in vivo and report that IEC MHC II expression dampens pro-inflammatory effector Th cells. Our findings indicate that IECs can function as non-conventional antigen presenting cells and that IEC MHC II expression fine-tunes local effector CD4 + T cell responses during intestinal inflammation.

The intestinal immune system is highly adapted to maintaining tolerance to the commensal microbiota and self-antigens while defending against invading pathogens1,2. Recognizing how the diverse network of local cells establish homeostasis and maintains it in the complex immune environment of the gut is critical to understanding how tolerance can be re-established following dysfunction, such as in inflammatory disorders. Although cell and molecular interactions that control T regulatory (Treg) cell development and function have been identified3,4, less is known about the cellular neighbourhoods and spatial compartmentalization that shapes microorganism-reactive Treg cell function. Here we used in vivo live imaging, photo-activation-guided single-cell RNA sequencing5-7 and spatial transcriptomics to follow the natural history of T cells that are reactive towards Helicobacter hepaticus through space and time in the settings of tolerance and inflammation. Although antigen stimulation can occur anywhere in the tissue, the lamina propria-but not embedded lymphoid aggregates-is the key microniche that supports effector Treg (eTreg) cell function. eTreg cells are stable once their niche is established; however, unleashing inflammation breaks down compartmentalization, leading to dominance of CD103+SIRPα+ dendritic cells in the lamina propria. We identify and validate the putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the lamina propria and identify receptor-ligand pairs that are likely to govern the interaction. Our results reveal a spatial mechanism of tolerance in the lamina propria and demonstrate how knowledge of local interactions may contribute to the next generation of tolerance-inducing therapies.

The intestinal immune system must maintain tolerance to commensal microbiota and self antigens whilst defending against invading pathogens. Recognising how homeostasis is established and maintained in a complex immune environment such as the gut is critical to understanding how to re-establish tolerance once broken in inflammatory disorders. Peripherally induced regulatory T cells (Tregs) play a key role in homeostasis. In intestinal tissue, Tregs work in concert with a diverse network of cells but which cellular interactions occur to instruct Treg adaptation and acquisition of distinct Treg suppressor function is not clear. We used two-photon in vivo live imaging and NICHE-seq [1] to deep phenotype Helicobacter hepaticus ( Hh )-specific Tregs with shared specificity but distinct spatially compartmentalised functions in the tissue. We show transcriptionally distinct central Treg (cTreg) and effector Treg (eTreg) populations in lymphoid versus gut tissue. The lamina propria (LP), and not embedded lymphoid aggregates (LA), is the key location of acquired immune suppressor eTreg function. Tregs recruited to the LP compartment are the dominant interacting cell type and acquired a more effector Treg profile with upregulation of Areg, Gzmb, Icos, Tigit, Tnfrsf4 (OX40), and Tnfrsf18 (GITR). We identify IL-1β + macrophages, CD206+ macrophages, and ILC2 in the LP niche as the key players governing Treg survival and function. In contrast, LA, dominated by interactions with ILC3s and populations of IL-6 + DCs, are equipped to tip the balance towards a pro-inflammatory response. By functionally isolating the gut tissue from secondary lymphoid organs, we show that eTregs maintain their phenotype in the context of inflammatory insult. Blocking their key effector molecule, IL-10, results in locally differentiated Th17 cell proliferation without overt inflammation due to local IL-10 independent mechanisms that constrain inflammation. Our results reveal a previously unrecognised spatial mechanism of tolerance, and demonstrate how knowledge of local interactions can guide cell function and potentially be manipulated for the next generation of tolerance-inducing therapies.

Intestinal epithelial cells (IECs) constitute the border between the vast antigen load present in the intestinal lumen and the mucosal immune compartment. Their ability to express antigen processing and presentation machinery evokes the question whether IECs function as non-conventional antigen-presenting cells. Major histocompatibility complex (MHC) class II expression by non-haematopoietic cells, such as IECs, is tightly regulated by the class II transactivator (CIITA) and is classically induced by IFN-γ. As MHC class II expression by IECs is upregulated under inflammatory conditions, it has been proposed to activate effector CD4+ T (Teff) cells. However, other studies have reported contradictory results and instead suggested a suppressive role of antigen presentation by IECs, through regulatory T (Treg)-cell activation. Recent studies investigating the role of MHC class II + exosomes released by IECs also reported conflicting findings of either immune enhancing or immunosuppressive activities. Moreover, in addition to modulating inflammatory responses, recent findings suggest that MHC class II expression by intestinal stem cells may elicit crosstalk that promotes epithelial renewal. A more complete understanding of the different consequences of IEC MHC class II antigen presentation will guide future efforts to modulate this pathway to selectively invoke protective immunity while maintaining tolerance to beneficial antigens.