Historically, there has been a widespread appreciation of the functional significance of interactions between CD40 ligand (CD40L, CD154) on activated CD4+ T cells and CD40 expressed on antigen presenting cells (APC) in both humoral and cell-mediated immunity. However, more recent discoveries have underscored the significance of CD40-CD40L interactions in inflammatory responses and autoimmune pathogenesis. The fact that CD40L is central to both appropriate and inappropriate immune responses underscores the importance of having a comprehensive understanding of pathways that regulate CD40L expression and how different levels of CD40 engagement affect immune responses. The overall goal of this proposal is to develop a mouse model of variegated CD40L expression by replacing the endogenous CD40L gene with a gene lacking the RNA stability element in the 3'untranslated region (UTR) of the CD40L transcript (CD40L-?5). Once the mouse is generated, experiments will test the effect of the CD40L-?5 mutation on the humoral immune response. In particular, B cell populations in the spleen, lymph node and bone marrow will be analyzed and compared to these cell populations in littermate controls. The expression of CD40L in the mutant mouse will also be analyzed in the T follicular helper T (Tfh) cells since these cells are the critical subset of T helper cells required for the germinal center (GC) response. Key findings from our lab that are pertinent to the current proposal include, 1) the identification of an activation-induced pathway of CD40L mRNA decay;2) the demonstration that this pathway is mediated by a PTB-containing complex (Complex I) binding to the CD40L mRNA at late times of activation;3) the characterization of the stability element in both human and mouse CD40L transcripts;and 4) the finding that PTB is required for appropriate distribution of CD40L RNA between the nucleus and cytoplasm from both activated and na?ve T cells. The proposed studies extend these preliminary and published findings by characterizing the PTB pathway of CD40L mRNA stability in an in vivo model. Results from these experiments will lead to a clearer understanding of factors controlling the expression of CD40L in humoral immunity and will uncover novel approaches for treating pathogen-related and autoimmune inflammatory diseases.
Effective immune responses to pathogens and vaccines critically depend on the production of high affinity antibodies by antigen-experienced B cells. This process of producing appropriate antibodies for a particular immune response occurs in response to interactions between CD40 expressed on B cells with CD40 ligand (CD40L, CD154) expressed on MHC-peptide activated CD4+ T helper (Th) cells. This application proposes to generate a unique mouse model of variegated CD40L expression with which to answer fundamental questions regarding the requirement for CD40L expression in B cell differentiation and antibody production throughout the course of an immune response.