Summary Skin-resident dendritic cells (DC) are well positioned to encounter cutaneous pathogens and are required for the initiation of adaptive immune responses. There are at least 3 subsets of skin DC — Langerhans cells (LC), Langerin+ dermal DC (dDC), and classic dDC. Whether these subsets have distinct or redundant function in vivo is poorly understood. Using a Candida albicans skin infection model, we have shown that direct presentation of antigen by LC is necessary and sufficient for the generation of antigen-specific T helper-17 (Th17) cells but not for the generation of cytotoxic lymphocytes (CTL). In contrast, Langerin+ dDC are required for the generation of antigen specific CTL and Th1 cells. Langerin+ dDC also inhibited the ability of LC and classic DC to promote Th17 responses. This work demonstrates that skin-resident DC subsets promote distinct and opposing antigen-specific responses.

Langerhans cells (LCs) are skin-resident dendritic cells (DC) located in the epidermis that migrate to skin-draining lymph nodes during the steady state and in response to inflammatory stimuli. TGF-β1 is a critical immune regulator that is highly expressed by LCs. The ability to test the functional importance of LC-derived TGF-β1 is complicated by the requirement of TGF-β1 for LC development and by the absence of LCs in mice with an LC-specific ablation of TGF-β1 or its receptor. To overcome these problems, we have engineered transgenic huLangerin-CreER T2 mice that allow for inducible LC-specific excision. Highly efficient and LC-specific expression was confirmed in mice bred onto a YFP Cre reporter strain. We next generated huLangerin-CreER T2 × TGF-βRII fl and huLangerin-CreER T2 × TGF-β1 fl mice. Excision of the TGFβRII or TGFβ1 genes induced mass migration of LCs to the regional lymph node. Expression of costimulatory markers and inflammatory cytokines was unaffected, consistent with homeostatic migration. In addition, levels of p-SMAD2/3 were decreased in LCs from wild-type mice before inflammation-induced migration. We conclude that TGF-β1 acts directly on LCs in an autocrine/paracrine manner to inhibit steady-state and inflammationinduced migration. This is a readily targetable pathway with potential therapeutic implications for skin disease.skin immunology | pSMAD2/3 | tolerance

Understanding the function of Langerhans cells (LCs) in vivo has been complicated by conflicting results from LC-deficient mice. Human Langerin-DTA mice constitutively lack LCs and develop exaggerated contact hypersensitivity (CHS) responses. Murine Langerin-diptheria toxin receptor (DTR) mice allow for the inducible elimination of LCs and Langerin+ dermal dendritic cells (dDCs) after administration of diphtheria toxin, which results in reduced CHS. When Langerin+ dDCs have partially repopulated the skin but LCs are still absent, CHS returns to normal. Thus, LCs appear to be suppressive in human Langerin-DTA mice and redundant in murine Langerin-DTR mice. To determine whether inducible versus constitutive LC ablation explains these results, we engineered human Langerin-DTR mice in which diphtheria toxin ablates LCs without affecting Langerin+ dDCs. The inducible ablation of LCs in human Langerin-DTR mice resulted in increased CHS. Thus, LC-mediated suppression does not require their absence during ontogeny or during the steady-state and is consistent with a model in which LCs actively suppress Ag-specific CHS responses.

Langerhans cells (LC) are a subset of skin-resident DC that reside in the epidermis as immature DC where they acquire antigen. A key step in the life cycle of LC is their activation into mature DC in response to various stimuli including epicutaneous sensitization with hapten and skin infection with C. albicans. Mature LC migrate to the skin-draining LN where they present antigen to CD4 T cells and modulate the adaptive immune response. LC migration is thought to require the direct action of IL-1β and IL- 18 on LC. In addition, TLR-ligands are present in C. albicans and hapten sensitization produces endogenous TLR-ligands. Both could contribute to LC activation. We generated Langerin-Cre MyD88fl mice in which LC are insensitive to IL-1 family members and most TLR-ligands. LC migration in the steady-state, after hapten sensitization and after infection with C. albicans was unaffected. Contact hypersensitivity in Langerin-Cre MyD88fl mice was similarly unaffected. Interestingly, in response to C. albicans infection these mice displayed reduced proliferation of antigen-specific CD4 T cells and defective Th17 subset differentiation. Surface expression of co-stimulatory molecules was intact on LC but expression of IL-1β, IL-6, and IL-23 was reduced. Thus, sensitivity to MyD88-dependent signals is not required for LC migration but is required for the full activation and function of LC in the setting of fungal infection.

Ebola virus (EBOV) and Marburg virus are members of the family Filoviridae. Both are highly pathogenic and cause hemorrhagic fever, lethal in 90% of infected people. There is fear that the viruses can be used as bioterrorism agents. There are no approved vaccines, and intense effort is underway to discover drugs targeting these viruses. The EBOV genome encodes seven proteins, two of which have no known structures: RNA polymerase (L) and nucleoprotein (NP). NP is essential for packaging viral genomic RNA into the nucleocapsid. Other viruses also contain nucleoproteins, but only the Ebola and Marburg NP proteins contain two distinct domains. The C-terminal domain (Ct; ~100 residues) has no homologues; it acts as a hub for protein-protein interactions important for the assembly of the nucleocapsid and for the interaction with the VP40 matrix protein, embedded in the viral membrane. We obtained three distinct crystal forms of the Ct domain of NP from EBOV, and solved the structures using anomalous scattering from Se, and Molecular Replacement. High-quality NMR data were also collected. The models were refined at 1.6-2.0 Å resolution to R factors ~20%. The protein has a novel fold, with topology distantly related to the β-grasp fold. In spite of its small size, the Ct domain shows high melting temperature of ~60°C. Our efforts focus on the identification of how the C-terminal domain of NP binds to its partners. As part of an effort towards anti-filovirus drug discovery, proteins NP, VP24, VP35 and VP40 are being targeted for small molecule inhibition using a yeast-based phenotypic assay. Each protein, when expressed in budding yeast, produces a slow-growth phenotype. Chemical suppressors of the slow-growth phenotype will be identified and used in viral growth assays to confirm their antiviral activity. The structure of NP will be used to complement small molecule screening methods.