A new langerin ؉ DC subset has recently been identified in murine dermis (langerin ؉ dDC), but the lineage and functional relationships between these cells and langerin ؉ epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC allowed viable LC to be easily distinguished from langerin ؉ dDC in skin and lymphoid tissue and ex vivo as well. Differential expression of EpCAM and langerin revealed the presence of at least 3 distinct skin DC subsets. We determined that LC and langerin ؉ dDC exhibit different migratory capabilities in vitro and repopulate distinct anatomic compartments in skin at different rates after conditional depletion in vivo. Langerin ؉ dDC, in contrast to LC, did not require TGF1 for development. Carefully timed gene gun immunization studies designed to take advantage of the distinct repopulation kinetics of langerin ؉ dDC and LC revealed that langerin ؉ dDC were required for optimal production of -galactosidase-specific IgG2a/c and IgG2b in the acute phase. In contrast, immunization via LC-deficient skin resulted in persistent and strikingly reduced IgG1 and enhanced IgG2a Ab production. Our data support the concepts that LC and langerin ؉ dDC represent distinct DC subsets that have specialized functions and that LC are important immunoregulatory cells. The presence of at least 3 functionally distinct skin DC subsets may have particular relevance for vaccines that are administered epicutaneously.EpCAM ͉ gene gun ͉ langerin ͉ TGF-beta T he remarkable phenotypic heterogeneity of DC, both between and within certain tissues, has been long recognized. To date, however, it has been possible to clearly relate DC phenotype to DC function in only a few instances, even in mice. For example, plasmacytoid DC are recognized as the primary source of virus-induced type I IFN (1), CD8␣ ϩ lymph node DC are largely responsible for cross-presentation of cell-associated antigen to CD8 T cells (2-4), and 33D1-reactive (DCIR2 ϩ ) splenic DC (as compared with CD205 ϩ DC) preferentially stimulate CD4 T cells (5). Epidermal Langerhans cells (LC) represent perhaps the most striking example of an extensively studied tissue DC subpopulation whose function is incompletely understood.LC have long been thought to play pivotal roles in initiating immunity by acquiring antigens that are encountered in skin, migrating to draining LN after activation, and stimulating antigen-specific T cells (6). However, recent studies suggested that LC do not function as essential antigen-presenting cells for anti-viral immune responses (2, 7) or for contact hypersensitivity reactions (8-12) in established murine models. Studies of LC have been challenging, in part, because readily detectable cell surface proteins that are constitutively expressed by epidermal LC and LC that have emigrated from epidermis have not been well recognized. The C-type lectin langerin has been regarded as a pathognomonic LC marker, but recent studies suggest that this protein is present in at le...