Human B Cell Differentiation in a Model System

Rutgers The St Univ Of Nj New Brunswick, New Brunswick NJ

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Abstract

DESCRIPTION (provided by applicant): We have identified a B-cell immunodeficiency that is distinguished by defective responses to CD4O and IL-4 signaling. B-cells from a young female patient (pt#l) have normal expression of CD4O but are clearly deficient in a subset of CD4O-mediated functions including early signals required for switch recombination. However, under specific in vitro conditions pt#1 B-cells can regain functional responsiveness and undergo switching to express downstream antibody classes or isotypes. Our preliminary data support a model whereby a signaling molecule or transcription factor in the CD4O signal transduction pathway leading to NF-kB activation is affected. This hypothesis is supported by our finding that pt#1 B-cells that are transformed by Epstein-Barr virus (EBV) do not express CD23 a cell surface molecule that is critically dependent on the viral latent infection membrane protein LMP)1 and the subsequent activation of NF-kB by this protein. Furthermore, LMP1 usurps the CD4O signaling pathway in order to maintain cell transformation. Surprisingly, the pt#1 EBV-transformed B-cells loose their transforming potential when grown in dilute culture conditions which also suggests that LMP1 activity is compromised. Thus, three related lines of data strongly suggest that the pt#1 defect is located in the CD4O signaling pathway that leads to NF-kB activation and the transcription of specific cellular genes involved in B-cell activation. We propose to use primary and transformed B-cells from pt#1 to characterize the underlying defect Leading to B-cell dysfunction. Efforts will be initially focused on the characterization of TRAF and NF-kB expression and function which are the most proximal and distal signaling events in the CD4O signaling cascade, respectively. The EBV-transformed pt#l B-cells will be used to analyze signaling of LMP1 via the CD4O pathway. Also, these cells will be used in transfection studies that are aimed at complimenting the pt#l defect and in the analysis of of NF-kappaB responsive promoters. Characterization of signaling pathways under the different growth conditions will provide information into the relationship between these signaling pathways and cell transformation. Finally, pt#l T-cells have subtle defects in helper function provided to control B-cells that manifest in inappropriate transcription of the heavy chain locus in response to CD4O signaling alone. Experiments are proposed to understand the basis of this functional defect.