The intestinal immune system is adapted to maintain tolerance to commensal microbiota and self-antigens while defending against pathogens. The study investigates the cellular neighborhoods and spatial compartmentalization that shape microorganism-reactive T regulatory (Treg) cell function. Using in vivo live imaging, photo-activation-guided single-cell RNA sequencing, and spatial transcriptomics, the researchers followed T cells reactive to *Helicobacter hepaticus* in tolerance and inflammation settings. They found that the lamina propria (LP) niche, not embedded lymphoid aggregates, supports effector Treg (eTreg) cell function. eTreg cells are stable once their niche is established, but inflammation disrupts compartmentalization, leading to dominance of CD103+ SIRPa+ dendritic cells in the LP. The study identifies and validates a tolerogenic interaction between CD206+ macrophages and eTreg cells in the LP and reveals receptor-ligand pairs governing this interaction. These findings reveal a spatial mechanism of tolerance in the LP and suggest that understanding local interactions may contribute to next-generation tolerance-inducing therapies.The intestinal immune system is adapted to maintain tolerance to commensal microbiota and self-antigens while defending against pathogens. The study investigates the cellular neighborhoods and spatial compartmentalization that shape microorganism-reactive T regulatory (Treg) cell function. Using in vivo live imaging, photo-activation-guided single-cell RNA sequencing, and spatial transcriptomics, the researchers followed T cells reactive to *Helicobacter hepaticus* in tolerance and inflammation settings. They found that the lamina propria (LP) niche, not embedded lymphoid aggregates, supports effector Treg (eTreg) cell function. eTreg cells are stable once their niche is established, but inflammation disrupts compartmentalization, leading to dominance of CD103+ SIRPa+ dendritic cells in the LP. The study identifies and validates a tolerogenic interaction between CD206+ macrophages and eTreg cells in the LP and reveals receptor-ligand pairs governing this interaction. These findings reveal a spatial mechanism of tolerance in the LP and suggest that understanding local interactions may contribute to next-generation tolerance-inducing therapies.