bioRxiv2025
Intestinal immunity defends against enteric pathogens, mediates symbiotic relationships with the resident microbiota, and provides tolerance to food antigens, safeguarding critical nutrient absorption and barrier functions of this mucosal tissue. Despite the abundance of tissue resident activated T cells, their contributions to these various roles remains poorly understood. Here, we identify a dominant population of IL-10 producing, T-bet expressing CD4+ Tr1 T cells, residing in the small intestinal lamina propria at homeostasis. Remarkably, these intestinal Tr1 cells emerge at the time of weaning and accumulate independently of the microbiota displaying similar abundance, function and TCR repertoire under germ-free conditions. Instead, the small intestinal T-bet+ Tr1 program is driven and shaped by dietary antigens, and accumulates in a cDC1-IL-27 dependent manner. Upon activation, these cells robustly express IL-10 and multiple inhibitory receptors, establishing a distinct suppressive profile. Altogether, this work uncovers a previously unappreciated dominant player in homeostatic small intestinal immunity with the potential to play critical suppressive roles in this tissue, raising important implications for the understanding of immune regulation in the intestine. Significance StatementEstablishing immunological tolerance to self and environmental antigens is critical to preserve tissue homeostasis and function. In the intestine, both dietary and microbiota derived antigens are routinely encountered by the immune system, which deploys a variety of mechanisms to maintain tolerance to these innocuous antigens. Understanding how immunological tolerance is established is critical, a when this process goes awry it can lead to severe inflammatory and autoimmune diseases such as food allergy and inflammatory bowel disease. However, how tolerance is established in the intestine is still poorly understood. In this study we describe a novel dominant T cell population in the small intestine shaped by dietary components with the potential to play important roles in immune tolerance at this site. back # Introduction Barrier surfaces such as the gut and skin represent the first line of defense against the environment. These organs must strike a delicate balance between providing protection against environmental and infectious agents, maintaining tissue function, and establishing a homeostatic symbiotic relationship with resident microbes collectively known as the microbiota (1). The immune system plays a critical role in establishing these dynamic and carefully regulated relationships, as evidenced by the large number of immune cells present at these sites. Of particular note, activated T cells are very abundant at barrier tissues, where they orchestrate immune effector functions geared towards these varied tasks (1, 2). In the small intestine, the intraepithelial compartment harbors innate like natural CD8aa IELs, many of which are self reactive; as well as CD4CD8aa and CD8ab IELs responding to dietary and microbial antigens (3). The underlying lamina propria (SILP) harbors predominantly CD4 T cells, which participate in responses to commensal-derived and dietary antigens (2, 4). Despite the abundance of small intestinal CD4 T cells, only a handful of cognate immune interactions focusing on Type 17 and T regulatory helper subsets have been described. Thus, whether immune responses in this tissue are truly limited to a small number of antigenic triggers and effector functions remains to be fully elucidated. The small number of gut homeostatic CD4 T cell responses described thus far have been shown to primarily respond to specific commensal bacteria or dietary antigens (1, 2, 5-8): Among other examples, SFB induces cognate Th17 cells in the small intestine (9, 10), a consortium human commensal bacteria induces CD8b cells in the colon (11), and Akkermansia muciniphila indices TFH and other effector cells in the Peyers patches and lamina propria, respectively (12). Furthermore most regulatory T cells in the colon are induced in response to commensal or pathobiont species at homeostasis, providing critical regulatory functions (13, 14). Cognate immune responses to SFB help contain this commensal species in the intestine (15), but also have systemic impacts on the susceptibility to autoimmune disease (16, 17). Interestingly, despite presenting a classical Th17 effector profile, a subset of SFB-induced Th17 cells possess IL-10 secretion capabilities and suppress cognate immune responses without the expression of Foxp3 (18), suggesting immunoregulatory functions reminiscent of Tr1 cells. Whether these competing capabilities are unique to SFB-specific immune responses or a general hallmark of small intestinal immunity remains unknown. The description of SFB-specific Tr1-like cells in the small intestine was surprising, as this CD4 T cell subset, characterized by abundant IL-10 secretion in the absence of Foxp3 expression, has only been described in the context of chronic antigen stimulation, such as chronic infection or cancer (19). The Tr1 cell program is controlled by a variety of transcription factors and upstream signaling pathways, including IL-27 signaling, MAF and AHR (20). AHR-ligands are abundant in the intestine, and MAF is a hallmark of other regulatory commensal-specific responses (21, 14). Furthermore, IL-27, which can induce both proinflammatory and immunoregulatory functions, is abundant in the small intestine (22, 23). This raises the possibility that the Tr1 program is a more general feature of small intestinal immunity, not uniquely restricted to SFB-specific responses. In this study we explore the breadth of CD4 T cell responses in the small intestine, and uncover a previously uncharacterized CD4T-bet T cell immune response that is dominant in this tissue. Unexpectedly, these SILP CD4T-bet T cells are independent of the microbiota, maintaining a similar functional profile and shared antigen specificities in germ-free conditions. Instead, we reveal that dietary components drive the accumulation, function, and clonal selection of this T cell population. Finally, we show that, contrary to classical Th1 cells, SILP CD4T-bet T cells adopt a Tr1 immunoregulatory functional program during activation, suggesting that this is a general feature of CD4 T cell immunity in the small intestine wired towards immune regulation and tissue homeostasis.