Our goal is to define the role(s) of a novel chemotactic factor and its receptor in leukocyte homing and biology. The factor is a polypeptide attractant for cells that express an orphan G protein linked receptor, including immature human and mouse dendritic cells; and it has been tentatively identified as the product of a gene of unknown function. Preliminary results lead us to hypothesize that this chemoattractant/receptor pair may play a significant role in the trafficking of dendritic cells, key antigen processing and presenting cells that initiate and regulate immune responses in vivo. ? ? The protein product of the putative encoding gene will be expressed and purified, assessed to confirm interaction with the receptor, and assayed for its chemotactic activity for transfectants and its ability to attractin vitro differentiated and in vivo-derived dendritic cells and other leukocytes. Its structure will be determined in collaboration with T. Handel, Berkeley; and mutants will be generated to probe the functional role of key structural elements. Cell type and tissue expression and regulation of the attractant will be determined using quantitative RT-PCR and in situ hybridization to characterize mRNA levels, and generating monoclonal antibodies for immunohistochemistry. Ligand-Ig chimeras and anti-receptor Mabs will be generated and used to study the regulation of cell surface receptors during in vitro differentiation and maturation of monocyte or bone marrow-derived DC; and to characterize receptor expression by dendritic cell and other leukocyte subsets in vivo by flow cytometry. Receptor expression will be correlated with chemotactic responses to the ligand. Finally, the role of this novel chemoattractant/receptor combination in dendritic cell development, trafficking and function will be determined in studies employing inhibitory antibodies to the attractant, and receptor-deficient, gene -targeted mice. The effects of these interventions on dendritic cell recruitment, positioning and phenotype in normal and inflamed tissues; on induction and kinetics of immune responses; and on immune pathology in selected models of inflammation, will be determined. ? ? The proposed studies promise to define an important regulator of immune responses, and may suggest novel strategies to enhance immunity for vaccination, or to suppress immunity for transplantation, autoimmune and allergic diseases. ? ?
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