As shown in Fig. 2A, the administration of CT caused notable changes in the expression of MHC-II and CD86 in LCs compared

with PBS administration, and these effects were primarily observed in the cell bodies. DC activation was also observed following local administration of a mixture of agonistic anti-CD40 and poly(I:C). Other surface markers such as CD40 were also expressed after local administration of CT but not with HEL or PBS (Supporting Information Fig. 3). Next, we assessed the consequences of local CTB inoculation compared with those of CT. As shown in Supporting Information Fig. 4A and B, CT induced a stronger degree of inflammation at the site of inoculation than CTB, which did not induce any overt inflammation. However, Ceritinib ic50 Sunitinib datasheet both CT and CTB induced expression of CD86 in LCs. To determine whether local administration of CT or CTB could induce the mobilization of LCs, the presence of MHC-II+ Langerin+ cells in epidermal sheets was evaluated. As shown in Fig. 2B, there was no difference 90 min after inoculation; however, by 24 h after inoculation with both CT and CTB, the number of LCs was significantly reduced. We next examined whether the inoculation of CT or CTB affected the production of cytokines by epidermal

and dermal cells. As Fig. 2C shows, inoculation with either CT or CTB induced a significant increase in the levels of TGF-β below in dermal cells. Interestingly, the cells that

expressed high levels of TGF-β after CT or CTB inoculation were Langerin+ DCs in the dermis (Fig. 2D). The inoculation of CT or CTB reduced the expression of IL-6 and MCP-1 in dermal cells but did not affect the production of IL-10 or TNF-α (Supporting Information Fig. 5). These results indicate that CT and the CTB subunit induce important changes in the phenotype of ear DCs. Considering both the robust CD4+ T-cell proliferation and the changes observed in DCs that were induced by the inoculation of CT in the ear, we next evaluated the cytokine profile of HEL-specific CD4+ T cells 3 days after immunization with HEL plus CT or CTB. A significantly increased levels of IFN-γ and (to a lesser extent) IL-2, TNF-α and IL-17 were observed in HEL-re-stimulated T cells isolated from mice that were immunized in the ear with only 0.3 μg HEL in combination with 1 μg CT or CTB (Table 1). We could not detect production of either IL-4 or IL-5. Practically none of the evaluated cytokines were detected in HEL-re-stimulated T cells isolated from mice that had received HEL alone or PBS or in T cells that were not re-stimulated in vitro with HEL. For comparison, we immunized mice in the ear with 0.3 μg HEL in combination with a mixture of anti-CD40/poly(I:C), and the resulting production of all of the evaluated cytokines was similar to that following co-administration of HEL and CT (Table 1).