The goal of this project is to further identify elements in CD4O signaling pathways that are potential drug targets for the enhancement of immune tolerance. This proposal exploits two novel findings from our laboratory. First, we observed CD4O activation by anti-CD4O or crosslinking of surface IgM in B cells triggers formation of complexes containing CD4O and the proto-oncogene product Cbl as well as the p85/p110 PI3-kinase. Second, we discovered a novel adapter protein (Grp1, an isoform of cytohesin-1) that specifically binds to phosphatidylinositol (3,4,5)P3 through its PH domain and to an integrin cytoplasmic domain through its Sec7 domain. We hypothesize that, in addition to the involvement of TRAF proteins, CD4O signaling (NFkB activation, B7 protein and ICAM upregulation) is regulated by Cbl/PI3 kinase complexes. Further, we postulate an acute activation of cell adhesion by CD4O through PI3-kinase-mediated activation of integrin function. We will test these hypotheses by identifying the domains on CD4O, Cbl, and PI3-kinase that mediate complex formation and prepare dominant inhibitory constructs and null mutants of these proteins that block their association in response to activation of CD4O. Cytohesin- I/Grp1 function will be ablated by expressing their dominant inhibitory' PH domains. Effects of such disruption of CD4O/Cbl signaling on upregulation of co-stimulatory molecules will be determined in cultured APCs and in B lymphocytes and macrophages of transgenic mice. Such animals and their B lymphocytes will be used in Projects I - 3 for tolerance induction to grafts. By discovering and then disrupting signaling elements in B lymphocytes that mediate co-stimulatory responses to CD4O and perhaps other receptors, we hope to provide a means of enhancing the tolerance induction that is observed in vivo by blockade of CD40 alone.