CD40 ligand (CD40L) is a T cell surface molecule that is expressed early and transiently after cell activation. The interaction between CD40L and the B cell molecule CD40 has profound effects on B cell survival and function, on T cell priming and recall proliferation, and on monocyte activation and function. The importance of CD40/CD40L interaction is evident in the X-linked hyper IgM Syndrome (XHIM-1), which results from mutations in CD40L and is characterized by recurrent bacterial and opportunistic infections as well as malignant and autoimmune disease. XHIM-1 is an attractive candidate for gene therapy because of the accessability of the affected tissues and the evidence from maternal carriers that only partial reconstitution is required for normal function. CD40L surface expression is highly regulated, limited essentially to activated CD4+ T cells. It is subject to positive regulation by CD28 co-stimulation and to negative regulation by interferon gamma and TGF- beta. CD40L gene expression is dependent on the binding of a complex of the calcineurin-dependent nuclear factor of activated T cell (NF-AT) family members and activation protein-1 (AP-1) members in the CD40L promoter. Our preliminary data indicate that a second calcium/ calmodulin-dependent signal, mediated by CaM kinase IV/Gr, augments activity of the CD40L promoter. We propose to study the molecular mechanisms regulating CD40L gene expression by examining: I. Surface molecules that regulate CD40L expression: TCR/CD3, CD40L, CD28, IFNgamma, and TGF-beta using reporter gene assays, mRNA expression by Northern blot analysis, and by CD40L surface expression. II. The role of signaling pathways activated by T cell surface molecules in CD40L gene expression: calcium/calcineurin, CaM kinase IV/Gr, protein kinase C, Ras family members, STAT proteins, and octamer binding proteins. III. Identify the nuclear factors that are activated by the signaling pathways and that regulate CD40L gene transcription using electromobility gel shift and DNA footprinting assays. Definition of the regulation of CD40L gene expression should provide deeper understanding of T cell and B cell biology and may allow for the rational design of a construct for gene therapy of XHIM-1.
