Gut tissue-associated lymphoid structures include a heterogenous spectrum of lymphoid clusters of varying size and maturity, however little is known about their functional relevance. We hypothesise that lymphoid structure may promote gut homeostasis through a number of potential local mechanisms that is currently unclear. Firstly, signalling and subsequent repair of tissue injury is regulated by the alarmin IL-33, which has been shown to be expressed by non-immune cells within lymphoid structures. IL33 signals through its receptor ST2 which present on a subset of Tregs. Thus, IL-33 signalling to ST2+ Tregs within lymphoid structures may constitute one important pathway that promotes gut health. Secondly, lymphoid structures have been reported to act as regional hubs of immunity. Local priming of commensal-reactive T cells within lymphoid structures could promote the activation and function of T cells, inducing their tolerance towards intestinal microbiota. Using a combination of multi-parameter flow cytometry and histo-cytometry, we firstly characterised lymphoid structures of the murine gut, and report distinct differences in their architecture and complexity at different anatomical sites. We confirmed the presence of both IL-33-producing cells and ST2+ Tregs at close proximity to each other within lymphoid structures. However, ST2+ Tregs were not found to be spatially closer to IL-33-producing cells compared to the ST2- Treg subset during health and disease, indicating a close interaction may not be strictly necessary for optimal IL-33 cytokine function. Next, we employed a T cell receptor transgenic mouse model specific to the pathobiont Helicobacter hepaticus, and used advanced in vivo live imaging techniques to study the compartmentalised induction, phenotype and function of commensal-reactive T cells within lymphoid structures as compared to the surrounding lamina propria tissue. Our results show a spatial compartmentalisation of regulatory IL-10 production by Helicobacter hepaticus- specific Tregs in the lamina propria tissue, distinct from lymphoid structures. Unique transcriptional differences in dendritic cells at these distinct spatial sites may drive IL-10 production at some, but not other locations within the tissue microenvironments. Altogether, we highlight the complex cellular interactions that occur within intestinal tissue-associated lymphoid structures that promote gut health. We use advanced imaging techniques to further our understanding of the behaviour of T cells in response to microbial antigen in vivo and our findings show tissue-associated lymphoid structures and the lamina propria tissue support Treg functions in distinct ways.