Cross-presentation of antigens by skin dendritic cells

Dendritic cells (DCs) are bone marrow-derived leukocytes. They are highly specialized for anti-gen presentation, in particular, for the initiation of primary immune responses. The specializations are manifold and are operative at different stages of the cell`s life cycle: antigen uptake and proces-sing at an immature stage, mostly in peripheral organs such as the skin; migratory capacity as DCs begin to differentiate (mature); T cell sensitizing capacity in a terminally mature state in the T cell areas of lymphoid organs. The immunogenic properties of DCs are beginning to be clinically exploited for the immunotherapy of tumors or autoimmune diseases. In the skin, DCs can be found in the epidermis as "Langerhans cells" and in the underlying dermis as "dermal dendritic cells". Antigens that encounter the host via the skin are efficiently taken up by these DCs. They process them intracellulary, associate the resulting peptides onto MHC class II molecules, translocate the MHC/peptide complexes to the cell surface and migrate from the skin, via afferent lymphatic vessels into the draining lymph nodes where they initiate an antigen-specific CD4 helper T cell response. This scenario is well established for the MHC class II pathway. Much less is known about how cutaneous DCs deal with antigens that are presented on MHC class I molecules to CD8 T cells. Two pathways exist. The first and classical pathway is operative for antigens that are synthetized or localized in the cytosol, such as viral antigens or tumor antigens. The second pathway is used when antigens are taken up exogenously (as may happen in the case of an uninfected DC in the midst of virally infected epithelial cells or a non-malignant DC in the midst of malignant tumor cells), yet peptides thereof are presented on MHC class I. This pathway is operative in the long-known phenomenon of "cross- priming". The skin is a very large organ that forms the border between the host and the threats of the outside world. Many viruses infect the cells of the skin and many tumors arise in the skin. We therefore hypothesize that DCs of the skin, that are positioned there as immunological sentinels, are efficient at generating immunogenic MHC class I / peptide complexes by the exogenous pathway. This hypothesis has not yet been addressed directly (i.e., with DCs isolated from the skin rather than in vitro generated skin DC equivalents). Systematic comparisons of the two types of skin DCs have not been performed, neither with regard to fundamental functional properties such as the immuno-stimulatory function for the development of cytotoxic T lymphocytes or the immunogenicity in vivo nor with specific regard to the exogenous pathway of antigen processing onto MHC class I. SPECIFIC AIM I: We propose to comparatively study in situ-derived Langerhans cells from the epidermis and dendritic cells from the dermis using isolation approaches that we have developed in our lab earlier. We will study the following topics: Phenotype (expression and intracellular distribution the machinery needed to produce MHC I/peptide complexes, i.e., transporters [TAP], proteasomes, ER-proteins, etc.); immunostimulatory capacity for the development of cytotoxic T lymphocytes in vitro and in vivo; processing capacity of skin DCs for endogenous antigens. SPECIFIC AIM II: The ability of DCs isolated from skin (in vitro) but also still within the skin (in situ) to process and present exogenous antigen on MHC class I will be studied in the mouse system using the ovalbumin model where many critical reagents are readily available. Data from the proposed studies will verify that epidermal and dermal DCs can present antigens on MHC I via the exogenous pathway, and if so, whether epidermal and dermal DCs are similar or different in efficacy and how this efficacy compares with DCs from other sources such as blood. This will have relevance not only for DC vaccination protocols but also for standard vaccinations into the skin as well as for better understanding of immune surveillance mechanisms in the skin.

Dendritic cells are highly specialized white blood cells for antigen presentation, in particular, for the initiation of primary immune responses. The specializations are manifold and are operative at different stages of the cell`s life cycle: antigen / pathogen uptake and processing at an immature stage, mostly in peripheral organs such as the skin; migratory capacity as dendritic cells begin to mature; T cell sensitizing capacity in a fully mature state in the lymphoid organs (lymph nodes). These properties lead to the generation of killer cells and help in the activation of phagocytic cells and antibody-producing cells, that is, the whole spectrum of immune defense mechanisms. The skin is a very large organ that forms the border between the host and the threats of the outside world. Many viruses infect the cells of the skin and many tumors arise in the skin. Antigens that encounter the host via the skin are efficiently taken up by epidermal Langerhans cells, the dendritic cells of the outermost layer of the skin. We therefore hypothesized that Langerhans cells, that are positioned there as immunological sentinels, are efficient at generating immunogenic molecular complexes from the pathogens by the so-called "exogenous pathway". This hypothesis has not yet been addressed previously with Langerhans cells proper. The data corroborated within the now terminated FWF project support the hypothesis in several important aspects. (1) Langerhans cells are indeed able to efficiently "cross-present" antigens. (2) This leads to the generation of killer cells (cytotoxic T lymphocytes) which may be harnessed for tumor defense. (3) These observations were not only made in vitro, but also in vivo, that is, in the living organism. (4) By means of unique antibody tools we were able to trace the migration of Langerhans cells from the skin to the lymph nodes. (5) We also succeeded in retrieving these antigen-loaded cells in the lymph nodes, which is an important precondition for further studies on the regulation of these processes. (6) Finally, we could show that the equipment of Langerhans cells with important receptors for antigen / pathogen uptake is defective in the period around birth and shortly after inflammatory episodes which helps explain the decline of immune protection of the skin in these periods. These data are not only important for the further development of tumor vaccination protocols using dendritic cells, but may also hold potential for the improvement of standard vaccination protocols as well as for the better understanding of fundamental immune surveillance mechanisms in the skin.