Ontogeny of Human Cutaneous Dendritic Cells

Due to an immature immune system, neonates respond poorly to conventional vaccines and certain microbes. This immaturity is caused, amongst other factors, by the impaired function of dendritic cells (DC). DC are key regulators of the immune response and are capable of promoting or suppressing T cell responses. Given the importance of these cells, the aim of this study is to investigate the ontogenetic aspects of cutaneous DC. The skin, and in particular the epidermis, is not only a physical barrier that protects the body from external threats, but it is also critically involved in immune reactivity. Key players in that regard are epidermal Langerhans cells (LC) and dermal dendritic cells (DDC). Both populations serve a sentinel function by detecting incoming skin pathogens and conveying information to T cells present in skin-draining lymph nodes. A focus of scientific interest in recent years has been the investigation of DC in adult skin in order to understand the pathogenesis of a variety of dermatologic disorders. Still, little is known about these cells in developing human skin. The first fetal liver- derived leukocytes that can be identified in embryonic epidermis (<10 weeks estimated gestational age (EGA)) differ substantially from those in the fetal (>10 weeks EGA) and adult epidermis. The cells are smaller, less dendritic and have a particular marker expression (CD36+ HLA-DR +/- CD1a - ). After 12 weeks EGA, a disappearance of these cells and a sudden increase of CD36 - HLA-DR + CD1a + Lag+ cells can be observed (=LC phenotype). Several possibilities exist to explain this phenomenon. Due to changes in the content and biological activities of cytokines in the developing skin, CD36 + cells may undergo a transformation within the epidermis involving de novo expression of surface molecules (e.g., CD1a). Alternatively, there may be an influx of CD36 - bone marrow-derived LC precursors into the epidermis and CD36 + cells may undergo apoptosis. The fact that the surge in CD1a reactivity at 11-12 weeks of gestation coincides with the initiation of bone marrow function raises the possibility that these two events are related. To assess whether CD36 + cells are indeed LC precursors, the cells will be purified and cultured (i) in the presence of cytokines known to generate LC from CD34 + precursors, (ii) with freshly-isolated adult epidermal cells that produce LC-promoting cytokines, and (iii) cell supernatants from adult epidermal cell cultures that promote LC maturation. After a certain time period, the cultures will be screened for cells with LC characteristics. In the event that no LC should become apparent, the aim is to assess whether autocrine factors with immunosuppressive qualities or extrinsic influences (e.g., keratinocytes) prevent maturation into LC. Antibodies to study the expression of important functional markers such as C-type lectin receptors on human cells have only recently become available and therefore the assessment of the expression on DC in the developing human skin is of great interest. These receptors are vital players in infectious and inflammatory diseases and eligible candidates for therapeutic targets. To unravel at which point cutaneous DC acquire such markers, LC and DDC in prenatal skin will be monitored over time. It is presumed that a lack and/or low expression of these proteins could, amongst other factors, explain the impaired immune competence and responsiveness to certain vaccines in neonates. Overall, knowledge concerning the phenotypic and functional repertoire and the differentiation capacity of prenatal DC will assist in improving our understanding of the level of immunocompetence in utero. Understanding the skin immune network is critically important for the future development of efficacious vaccines and immunotherapies against infectious agents and neoplastic diseases, and potentially for the development of "suppressive" immunotherapies to alleviate autoimmune disease.

The adult human skin is not only a physical barrier that protects the body from external threats but also harbours a variety of leukocytes providing immune surveillance and host defence. Antigen-presenting cells (APCs) such as epidermal Langerhans cells (LCs) and dermal dendritic cells (DDCs) are principal players orchestrating the decision between immunity and tolerance in adults. Given the importance of APCs and the scarce knowledge about their ontogeny in humans we aimed to investigate the quantitative, phenotypic and functional development of skin leukocytes and to analyze the factors controlling their immigration, proliferation and differentiation under homeostatic conditions. Comparing epidermal leukocytes from embryos and fetuses with adults revealed evidence of a stepwise progression of epidermal LC development. By the ninth week of gestation, scattered leukocytes are present in the epidermis and the dermis - likely attracted by the high levels of local chemokines. At that time, some of the epidermal leukocytes express CD1c, and only later during gestation acquire Langerin and CD1a, identifying them as mature LCs. As TGF-beta is critical for LC development and we found that the production of TGF-beta in developing human epidermis precede expression of these markers, our data confirm a possible role of this cytokine in the generation of the LC phenotype. With regard to APCs in the dermis we found a distinction between DDCs and macrophages already by the ninth week of gestation. Their numbers continuously increase as the developing skin generates an environment that promotes proliferation of skin resident-leukocytes and an influx of leukocytes from the circulation. Other leukocytes such as mast cells and T cells emerge at the end of the first trimester. To our surprise, already DCs from embryonic skin stimulate the proliferation of T cells in vitro, proving their functional capability. This potential may be dampened in situ by the high levels of the immunosuppressive cytokine IL-10 found to be present in embryonic tissues. Collectively, our data provide evidence that APCs in the skin are functional within the first trimester. Yet it is not ineptitude that keeps these cells in human embryonic skin from reacting to maternal tissues but they may be kept in check by a suppressive environment. Experimental evidence revealing the exact mechanism(s) behind APC migration, proliferation, and maturation in developing skin are to be addressed in the future.