Fc receptors for IgG (Fc?R) are essential for mediating the effector properties of IgGs in vivo, as well as regulating the? afferent response through the engagement of immune complexes with B cells and dendritic cells. Perturbations in expression and signaling of these receptors can? contribute to the loss of tolerance and immunopathology observed in autoimmune diseases such as lupus and rheumatoid arthritis. While previous studies utilizing? gene disruptions have revealed the overall function of these receptors, a detai? led analysis of the contribution of individual cellular populations with their specific FcR expression and binding properties is, as yet, lacking. 1) We will address the functional contribution of specific receptors on discrete lymphoid and? myeloid cells using conditionally floxed alleles of the four murine Fc?Rs and the common ? chain in B6 ES cells. RIIB deletion in mature B cells, germinal center B cells, plasma cells and dendritic cells will determine their respective contributions to the RIIB mediated regulation of IgG in wild-type and autoimmune backgrounds. The contribution of macrophage/neutrophils, mast cells and NK cells to IgG mediated cytotoxicity and immune complex triggered inflammation will be characterized in strains conditionally deleted for RI, RIII, RIV or the common ? subunit. 2) Extrapolation of these results to the human system will be addressed by the generation of fully humanized FcR mouse lines, in which all endogenous murine FcR expression is ablated and human FcR expression is obtained in a cellular pattern appropriate for the human FcRs. These lines will be characterized in models of human IgG mediated cytotoxicity and immune complex inflammation for specific IgG subclasses and modified human FCs. 3) A detailed analysis of the activation and inhibitory FcR ligand binding domains will be evaluated using a high-throughput, yeast display approach to identify those specific amino acid substitutions that contribute to the IgG specificity and affinity of RIIB and RIV for IgG1 and 2a subclasses. These directed FcR mutants will be characterized for their ligand binding properties, cellular signaling responses and in vivo function in IgG mediated cytotoxicity to human disease The studies outlined in this proposal will provide a rational basis for the re-engineering of therapeutic IgG antibodies for the treatment of neoplastic and inflammatory diseases and for their evaluation in vivo in an animal model that recapitulates the human system. ?
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