A central theme of the current program project relies on the ability to target specific dendritic cell subsets in vivo to determine their functional properties in immunity and tolerance. In the past funding period a method was collectively developed that facilited this analysis through the use of specifically engineered monoclonal antibodies that recognized a DC-restricted surface marker and delivered a defined antigen to those cells. This method of DC targeting will be extensively utilized in all three projects in the next funding period to direct model antigens and authentic autoantigens to CD8+ and CDS- DC subsets in vivo. Monoclonal antibodies to DEC-205 and 33D1 will initially be modified to deliver antigens in vivo and further Fc engineered to direct binding of the antibody to either activation or inhibitory FcRs on the specific DC subset. During the past funding period efficient methods were developed to engineer the heavy chains of these antibodies for antigen delivery and selective FcR binding. Transient transfection of the modified heavy chain, together with the unmodified light chain into 293T cells resulted in expression of the chains, assembly of the tetrameric antibody molecule and its secretion into the medium. We propose to centralize these procedures into a core facility to provide each laboratory with the necessary reagents to carry out the experiments proposed. Such an approach will streamline the process of generating and expressing these modified and Fc engineered antibodies, reduce the redundancy of having each laboratory carry out these same procedures and insure reproducibility and quality. The facility will occupy 1,000 sq. ft of newly renovated space, fully equipped for mammalian cell culture propagation, protein isolation and purification.
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