Type 1 and type 2 dendritic cell subsets cooperate to maintain intestinal immune tolerance via integrin αvβ8-mediated TGF-β activation

ABSTRACT The gastrointestinal tract is a unique immunological environment where the host must balance tolerance to commensal microbes with defense against pathogens. A critical mechanism for maintaining this balance is the peripheral conversion of naïve T cells into regulatory T cells (pTregs), a process that depends on the TGF-β cytokine, which is produced in a latent form and must be activated. While the activation of latent TGF-β relies on the membrane-bound αvβ8 integrin, the precise cellular subset(s) responsible for this essential process have yet to be clearly defined. Conventional dendritic cells (cDCs), which migrate from the intestinal lamina propria to the gut-draining mesenteric lymph nodes (MLN), have long been considered the primary antigen-presenting cells (APCs) responsible for αvβ8-mediated TGF-β activation and pTreg induction. However, recent studies have challenged this paradigm by highlighting a new family of rare RORγt-expressing APCs, able to induce pTreg via αv integrins, raising questions about the in vivo role of cDCs in the maintenance of mucosal immune homeostasis. Using a β8 integrin gene reporter mouse model ( Itgb8-IRES-tdTomato ) combined with single-cell profiling, we comprehensively mapped Itgb8 -expressing APCs in the MLN. We show that cDCs, in particular migratory type 1 (cDC1) and type 2 (cDC2), constitute the predominant Itgb8 TdTomato + cells, both in neonatal and adult mice. Through cDC subset-specific β8 knockout models, we demonstrate that both cDC1 and cDC2 are required for optimal pTreg generation. Loss of β8 integrin in either subset led to a partial reduction in pTreg, while combined deletion resulted in profound pTreg loss and spontaneous colitis. Importantly, these effects were independent of RORγt⁺ APC populations, including ILC3s and Thetis cells. These findings resolve longstanding questions about the identity of key APCs driving pTreg induction in the MLNs. They demonstrate that cDC1 and cDC2 are non-redundant, essential mediators of pTreg induction and intestinal immune tolerance. Although different populations of RORγt⁺ APCs may contribute in specific contexts, such as early development, infection, or in the prevention of allergic disease, cDCs remain one of the primary guardians of intestinal immune homeostasis in response to microbiota.