Studies of human dendritic cells in the skin after antigen exposure

Abstract: Dendritic cells (DCs) act at the interface of innate and adaptive immunity. They are capable of inducing strong effector immune responses to invading pathogens, but also exert important functions in the maintenance of tolerance. The DCs of the skin are strategically located to be amongst the first cells to react to exposure to foreign substances and initiate immune responses, and increased understanding of these cells is critical for the development of new treatments of infections, allergic reactions and autoimmune disorders, as well as of new vaccine delivery strategies, targeting the skin. Studies of skin DC subsets are held back by technical difficulties in isolating the cells. In paper I of this thesis, we therefore aimed to develop a new DC purification method, using a skin graft mesher. This device is used clinically for expansion of skin graft tissue for transplantation. We found that by using the skin graft mesher the processing of skin was significantly accelerated, which had both practical and biological advantages. The DCs isolated from epidermis consisted of a uniform CD1a+ Langerin+ Langerhans cell population with high antigen uptake capacity. The DCs from dermis showed varied antigen uptake capacity and could be divided into three distinct populations based on CD1a expression. In addition, a skin explant model to study antigen uptake by DC subsets in situ was developed. We found that antigen injected into the skin explants was rapidly taken up, mainly by DCs in the dermis. This model can be a useful tool to study immediate responses to antigen exposure. However, since no recruitment of new cells into the tissue can occur, skin explants cannot illustrate all the components of innate immune responses. This prompted for the studies in paper II and III, where we took the approach to study infiltration of DCs to sites of antigen exposure in the skin in vivo. By using skin punch biopsies collected from standard skin antigen test sites like the tuberculin skin test (TST), we quantified recruited immune cells by immunohistochemical stainings of cryosections. We found that several DC subsets including plasmacytoid DCs (pDCs) accumulated in the dermis in the indurations induced in individuals with positive TSTs. In contrast, donor-matched saline injected skin did not show any DC recruitment, nor did TST sites of individuals lacking induration. The positive TST induration tissue was also associated with cell death and high expression of the antimicrobial peptide LL37, which together can provide a means for pDC activation and IFN! production. In line with this, IFN-inducible MxA was highly induced at the positive TST sites. By in vitro experiments, we found that pDCs were not as efficient as myeloid DC subsets to take up the TST antigens. Further, IFN! containing supernatants from pDC activated with LL37-DNA complexes reduced antigen uptake in myeloid DCs as well as decreased their capacity to activate T cells. Infiltrating pDCs in the TST reaction may thus have a regulatory effect upon the antigen processing and presentation functions of surrounding DC subsets, to limit potentially detrimental and excessive immune stimulation. TST and other antigen skin tests are used to monitor the integrity of the cellular immune system in HIV-1 infected individuals. To evaluate whether mobilization of DCs in response to antigen skin tests was impaired in HIV-1 infected individuals, we analyzed skin punch biopsies from skin test sites using mumps virus, Candida albicans or TST. Control skin biopsies showed that there was no difference in the number of skin-resident DCs between healthy and HIV-1 infected individuals. Also, multiple DC subsets infiltrated the dermis in response to injection with all antigens. However, the levels of DC infiltration correlated with the generally lower levels of memory T cells that accumulated at the antigen sites. A depleted or deficient T cell compartment could therefore lead to compromised DC recruitment and insufficient antigen presentation to T cells. Taken together, these studies contribute with new methods and data on the function and phenotypes of skin DCs, and their response to antigen administration in the skin in health and HIV-1 infection.