Amoury and L

Amoury and L. induction and their redundancy during oral tolerance development. The peripheral immune system must maintain a balance between protective responses and tolerance. This equilibrium represents a major challenge for the mucosal surfaces, particularly the intestine, which is chronically exposed to both potentially pathogenic microbes and harmless dietary and commensal-derived antigens. Not surprisingly, several cellular and molecular mechanisms exist to ensure robust tolerance induction in the mucosae. Peripherally-induced Foxp3+ regulatory T cells (pTreg cells) are thought to be instrumental in the induction and maintenance of peripheral tolerance1, 2, 3, 4. Innocuous antigen exposure via mucosal surfaces efficiently induces pTreg cell differentiation from na?ve CD4+ T cells via a retinoic acid (RA)- and TGF–dependent process2, 5, 6, 7, 8. In turn, genetic loss-of-function strategies that target pTreg cells result in severe inflammatory phenotypes in the lungs and intestine 3, 4. Antigen presenting cells (APCs), including dendritic cells (DCs) and macrophages, have been ascribed critical roles in triggering pTreg cell differentiation6, 7, 8, 9, 10. In particular, intestinal APCs expressing the fraktalkine receptor CX3CR1 take up soluble luminal antigens 11, 12 and, under certain conditions, migrate to the mesenteric lymph nodes (mLNs) where they present antigens to na?ve T cells13. In addition, CX3CR1Cexpressing phagocytes appear to transfer antigens to neighboring migratory DCs11 and these DCs are believed to induce pTreg cell conversion after they migrate to the mLNs14, 15. Indeed, both lamina propria and mLN-derived DCs, particularly E integrin+ (CD103+) or DEC205+ DCs, produce high amounts of RA and TGF- and efficiently induce pTreg cells 1, 6, 7, 8, 16, 17, 18, 19. However, whether these pTreg cell-inducing APCs are also required for oral tolerance induction has not been investigated. Furthermore, because the strategies relying on cell surface markers utilized to date target multiple APC lineages, the exact nature and origin of APCs responsible for pTreg cell induction are still unclear. We demonstrate Galanthamine an essential role for pre-DCCderived classical dendritic cells (cDCs) for both pTreg cell and oral tolerance induction, while macrophages and monocyte-derived cells appear dispensable. Further, we identify a hierarchical pattern in pTreg Galanthamine cell-inducing capacity of mLN-derived cDC subsets, whereby dietary antigen mediated pTreg cell polarization is most dependent on migratory IRF8Cdependent CD11b? cDCs. Oral tolerance is intact, however, in absence of this cDC subset, highlighting robustness of the process and functional redundancy of cDCs. Results Systemic absence of cDCs leads to break in oral tolerance We first set out to determine whether the APCs required for induction of oral tolerance could be classified by one of the two major myeloid lineages (Supplementary Fig. 1a). We focused on the populations present in the mLNs, the major inductive sites of oral tolerance14. Macrophages were identified as Lin?MHCII+CD11c+CD64+ cells, and cDCs as Lin?MHCII+CD11c+CD64? cells (Fig. 1a)20. Within the cDCs, we distinguished between two resident MHCIIint populations, CD8+CD11blow versus CD8?CD11b+ and two migratory MHCIIhi populations, CD103+CD11b? versus CD103+CD11b+ Galanthamine (Fig. 1a). We first used a mouse model of TH1 delayed-type hypersensitivity (DTH) 9 to address whether a specific APC lineage is required for the induction phase of oral tolerance. Tolerance was assessed by measuring the cellular and humoral inflammatory immune response towards OVA in mice pre-exposed to oral ovaIbumin (OVA) or oral PBS as control and immunized with OVA in complete Freund’s adjuvant (CFA) (Fig. 1b). We targeted the macrophage-monocyte lineage using mice bearing the Cre recombinase gene under the promoter, and the diphtheria toxin receptor (DTR) gene preceded by a site-flanked stop cassette under control of the promoter (gene (promoter, the Galanthamine gene encoding integrin CD11c (here CD11cDTR mice)20, 22. PBS-fed and OVA-fed CD11cDTR mice HK2 showed similar ear swelling and serum anti-OVA antibody responses (Fig. 1c-e), suggesting lack of tolerance to OVA. These observations indicated that monocyte-macrophageCderived APCs are dispensable for oral tolerance induction, while Galanthamine pre-DCCderived cells are critical. Next, we assessed.