(B) Percentage of CD11b+cells among the four different DC subsets, irrespective of targeting. by the TLR7 agonist imiquimod. Complete removal of the site where ovalbumin-coupled anti-DEC-205 had been injected decreased endogenous cytotoxic responses against ovalbumin peptide-loaded target cells by 40-50%. Surprisingly, selective ablation of all Langerin+skin DCs in Langerin-Diphtheria-Toxin-Receptor knock-in mice did not affect WAY 170523 such responses, independent of the adjuvant chosen. Thus, in cutaneous immunization strategies where antigen is targeted to DCs, Langerin+skin DCs play a major role in transport of anti-DEC-205 mAb, although Langerinnegdermal DCs and CD8+DCs are sufficient to subsequent CD8+T cell responses. == INTRODUCTION == Dendritic cells (DCs) are critically involved in the generation of immunity induced by vaccines and pathogens (1). Cutaneous DC subsets include epidermal Langerhans cells (LCs), and dermal DCs, which subdivide into Langerinnegand Langerin+populations (2-5). These three DC subsets are positioned to take up intradermal vaccine, process it and carry it to the draining lymph nodes in order to stimulate antigen-specific T cells. Despite this, recent data has shed doubt on their immunogenic role in vivo (6-8). In particular, the contribution of CD8+DCs residing in draining lymph nodes has to be taken into account, because soluble antigens can reach them via the lymphatic flow (9) or by transfer from emigrating skin DCs (10). All DC subsets express C-type lectin receptors that facilitate uptake and processing of antigenic proteins (11). This ability has been exploited to improve immune responses by targeting antigens to DCs (12,13). The best-studied example is DEC-205/CD205, which is expressed at highest levels by dermal DCs, LCs and CD8+DCs (14-16). When protein antigens are coupled to anti-DEC-205 mAb and mice are immunized with these conjugates, endogenous T cell-dependent immune responses (17-19) are dramatically enhanced in vivo. This requires the concomitant administration of DC-activating agents, such as Toll-Like Receptor (TLR) ligands or agonistic anti-CD40 mAb. In many of the above-cited studies, immunisation with anti-DEC-205 conjugates was performed by injection into the subcutaneous tissue of the footpad. Despite extensive research performed with antibodies targeting DEC-205, only limited characterisation of the DC subsets involved in the induction of immune responses is available (17,20,21). We have previously reported that epidermal LCs and both subsets of dermal DCs are able to capture anti-DEC-205 mAb in situ, and that the model antigen ovalbumin (OVA) coupled to these mAb is presented by LCs to CD4+and CD8+transgenic T cells in vitro (16). Thus, we wished to complement these observations with additional studies in vivo on the transport of antigen within mAb targeting to DEC-205 and the subsequent development of endogenous immune responses. This appears important in view of the differential roles that epidermal LCs, dermal DCs, and lymph node-resident CD8+DCs seem to play (10,22,23). We compared WAY 170523 the contribution of these subsets in the transport of anti-DEC-205 targeting mAb and in the induction of antigen-specific, endogenous cytotoxic responses, in steady state and inflammation. Moreover, the role of Langerin+DC populations was specifically addressed by employing a mouse model allowing conditional depletion of Langerin-expressing cells (24). == MATERIALS AND METHODS == == Mice == Mice of inbred strain C57BL/6 and BALB/c were purchased from Charles River Laboratories (Sulzfeld, Germany) and used at 2 to 6 months of age. Langerin-DTR-EGFP mice were provided by Dr. B. Malissen, Marseille, France (25). All experimental protocols were approved by the Austrian Federal Ministry of Science and Research, Department for Genetic Engineering and Animal Experimentation (#66.011/16-II/106/2008). == Antibodies and reagents WAY 170523 == Targeting antibodies were detected with goat anti-rat immunoglobulins G (IgG; H+L) coupled with APC (BD-Pharmingen) for FACS KMT6A WAY 170523 analyses. For immunofluorescence in the murine dermis, we employed chicken anti-rat IgG coupled to Alexa Fluor 594 (Invitrogen) that limits background staining of dermal extracellular matrix. Anti-mouse LYVE-1 polyclonal antibody (rabbit IgG, Upstate Cell Signaling Solutions, Lake Placid, NY) was used to detect dermal lymphatic vessels, and was visualized with swine anti-rabbit Ig / FITC (Dako Cytomation A/S, Glostrup, Denmark). Phenotypical analyses of murine DCs were performed with mAb against MHC class II (anti-I-A/I-Ediverse, clone 2G9), CD11c (clone HL3), CD8 (clone Ly-2), CD103 (clone M290) (all from BD-Pharmingen), CD11b (clone M1/70; eBiosciences, San Diego, CA) and Langerin/CD207 mAb (clone 929F3; Dendritics, Lyon, France). When possible, viable cells were determined by exclusion of.