The intestinal immune system maintains tolerance to commensal bacteria and self-antigens while defending against pathogens. This study investigates how the spatial organization of immune cells in the gut influences the function of regulatory T cells (Treg cells), particularly in the context of tolerance and inflammation. Using in vivo live imaging, photo-activation-guided single-cell RNA sequencing, and spatial transcriptomics, the researchers tracked the behavior of T cells reactive to Helicobacter hepaticus (Hh) in the gut. They found that the lamina propria (LP) is the key microenvironment that supports the function of effector Treg (eTreg) cells, while embedded lymphoid aggregates do not. eTreg cells are stable once they establish their niche, but inflammation disrupts this compartmentalization, leading to the dominance of CD103+ SIRPα+ dendritic cells in the LP. The study identifies a putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the LP, and identifies receptor-ligand pairs that may govern this interaction. The results reveal a spatial mechanism of tolerance in the LP and demonstrate how understanding local interactions can contribute to the development of new tolerance-inducing therapies. The study also shows that the LP is a key site for the production of IL-10 by Treg cells, which is critical for maintaining tolerance. In contrast, inflammation disrupts this process, leading to the recruitment of CD103+ SIRPα+ dendritic cells into the LP, which may contribute to the breakdown of tolerance. The study highlights the importance of the LP as a unique microenvironment that supports the function of eTreg cells and their interactions with macrophages, which are essential for maintaining intestinal homeostasis. The findings suggest that the spatial organization of immune cells in the gut plays a critical role in the regulation of immune responses and the maintenance of tolerance.The intestinal immune system maintains tolerance to commensal bacteria and self-antigens while defending against pathogens. This study investigates how the spatial organization of immune cells in the gut influences the function of regulatory T cells (Treg cells), particularly in the context of tolerance and inflammation. Using in vivo live imaging, photo-activation-guided single-cell RNA sequencing, and spatial transcriptomics, the researchers tracked the behavior of T cells reactive to Helicobacter hepaticus (Hh) in the gut. They found that the lamina propria (LP) is the key microenvironment that supports the function of effector Treg (eTreg) cells, while embedded lymphoid aggregates do not. eTreg cells are stable once they establish their niche, but inflammation disrupts this compartmentalization, leading to the dominance of CD103+ SIRPα+ dendritic cells in the LP. The study identifies a putative tolerogenic interaction between CD206+ macrophages and eTreg cells in the LP, and identifies receptor-ligand pairs that may govern this interaction. The results reveal a spatial mechanism of tolerance in the LP and demonstrate how understanding local interactions can contribute to the development of new tolerance-inducing therapies. The study also shows that the LP is a key site for the production of IL-10 by Treg cells, which is critical for maintaining tolerance. In contrast, inflammation disrupts this process, leading to the recruitment of CD103+ SIRPα+ dendritic cells into the LP, which may contribute to the breakdown of tolerance. The study highlights the importance of the LP as a unique microenvironment that supports the function of eTreg cells and their interactions with macrophages, which are essential for maintaining intestinal homeostasis. The findings suggest that the spatial organization of immune cells in the gut plays a critical role in the regulation of immune responses and the maintenance of tolerance.