REGULATION OF B CELL DIFFERENTIATION IN COMMON VARIABLE IMMUNODEFICIENCY--CD40

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This proposal will determine the mechanism that account for the defective B-cell differentiation in a defined subset of subjects with Common Variable Immunodeficiency (CVI). Phenotypic, functional and molecular evidence provided by us and others supports the concept that the humoral immunodeficiency in a defined subset of CVI subjects reflects the failure of bone marrow emigrant B cells to be able to complete their developmental program in a stage specific fashion. Our overall hypothesis is that this developmental failure in CVI reflects the dysregulation of signals that control the alternative instructional programs for B cell apoptosis, growth or differentiation. Our ability, gained from studies undertaken as part of this Program Grant, to examine these CVI subjects B cells under specific conditions where they do or don not differentiate, now provides the opportunity to test this overall hypothesis. Furthermore, these studies have identified key triggers (e.g. CD40, IL-4R and CD20) that are implicated in the altered differentiation outcome in this subset of CVI patients. The FIRST AIM will determine how anti-CD40 plus IL-4 stimulation provides CVI B cells with the ability to ultimately differentiate. We will investigate CD40 plus IL-4 effects on the following B cell responses: a) prevention of apoptosis, b) enhancing growth or c) specifically driving differentiation under conditions that do and do not lead to differentiation. We expect, thereby, to elucidate the critical function CVI B cells require in order to be able to go on to differentiate. As part of this aim, differential mRNA screening using a novel PCR based approach will be employed to determine whether CD40 plus IL-4 induces new gene expression in CVI B cells, gene expression that is required for CVI B cells to undergo differentiation. The SECOND AIM will test the hypothesis that c-myc activity is responsible for preferential shunting of CVI B cells into non-differentiation pathways. We will measure the level of c-myc in our CVI patients' fresh cells and cell lines, test whether decreasing c-myc function rescue CVI B cells differentiative function (e.g. when stimulated by IL-4 plus CD40) and determine if enhanced expression of c- myc in normal B cells renders the cells differentiation defective. The THIRD AIM will determine the role of CD20 in the inhibition of differentiation in CVI B cells and if this is related to altering c-myc expression. We will employ agonist and antagonist CD20 mAbs to determine the effect of enhancing or inhibiting CD20 function on B cells ability to differentiate or proliferate and its relationship to myc expression. CD20 stimulation of myc will be blocked via cyclosporine A, FK506 and okadaic acid as this pathway is predicted to be maintaining CVI in a differentiation 'defective' state. We will also characterize the interrelationship between IL-4 plus CD40 driven CVI B cell differentiation and alterations in CD20 expression and function. By accomplishing the three aims outlined above, we intend to elucidate the mechanisms which lead to the failure of CVI B cells to proceed with physiologic differentiation in vivo and thereby determine the mechanism(s) accounting for the dysregulated B-cell differentiation in a subset of well defined CVI patients with intrinsic B cell abnormalities. This information will be important in ultimately designing therapeutic strategies as well as in providing for increased understanding of the normal human B cell development and differentiation.