Langerhans cells and CD11b+ dermal dendritic cells are involved in skin Treg generation

Abstract

Abstract Antigen applied to the skin can induce immune tolerance in naïve mice and can be used as epicutaneous immunotherapy (EPIT) to treat experimental food allergy. In previous unpublished work, we have shown that EPIT induces Tregs, including a population of intestinal-homing LAP+ Tregs that can suppress mast cell activation and anaphylaxis. In this extension of that work, our goal was to determine how dendritic cells (DCs) in the skin contribute to the induction of LAP+ Tregs. Epicutaneous antigen exposure of mice that have received DO11.10 T cells with Viaskin®-OVA patches for 48 h induced OVA-specific LAP+ Tregs to a similar extent in naïve and sensitized mice. Therefore all additional studies were done in naïve mice. FACS sorting of DC subsets from skin-draining lymph nodes of Viaskin®-treated mice and then co-cultured with DO11.10 T cells demonstrated that CD11b+ CD103− dermal DCs and CD11b+EpCAM+ Langerhans cells (LCs) were both capable of antigen capture and presentation to CD4+ T cells, as well as Treg generation. PDL2 was found on both subsets and effectively discriminated between cells with and without antigen capture and presentation capacity. PDL2+ DCs presented a more mature phenotype, with higher expression of MHCII, CD86 and CD80 than PDL2− DCs. PDL2+ LCs and PDL2+ CD11b+ DCs were sorted separately and expression of regulatory molecules (GM-CSF, IL-27, IL-10, TGF-b, ALDH2 and IDO) were assessed by RT-PCR. PDL2+ LCs uniquely expressed IL-27, while PDL2+ CD11b+ DCs were enriched for ALDH2, which was then confirmed by flow cytometry. In vivo depletion of LCs using anti-CSF1 did not influence generation of Tregs in vivo. These results indicate that induction of Tregs through the skin is not restricted to one DC subset.