Molecular Basis of Human VH Gene Replacement

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DESCRIPTION(provided by applicant): The candidate is a molecular biologist who wishes to establish an independent research career in the molecular immunology field. Enhanced training under Dr. Max D. Cooper?s supervision in immunology is proposed for the initial two or three years in order to achieve research independence within the award period. The proposal will focus on the molecular basis and regulatory mechanism of human VH gene replacement and its contribution to normal B cell development, B cell responses, and rheumatoid arthritis in particular. The hypothesis builds on an analysis of a clonal human cell line, EU12, that undergoes continuous in vitro differentiation from proB (CD34+) to preB (CD34 mu SLC+) then to B cells (CD34 mu+LC) and generates intraclonal diversity through serial VH replacements. The ongoing VH replacement in the EU12 cells is verified by the detection of VH replacement excision circles and double stranded DNA breaks at the cryptic RSS sites. (Aim 1) The EU12 cells will be used as an experimental model to dissect the molecular basis for the VH replacement. Purified RAG 1, RAG 2, and HMG 1 proteins will be used in in vitro studies to define the function of the cryptic RSS site. (Aim 2) Using the methods and principles derived from the study of the EU 12 model, normal human bone marrow and tonsillar germinal center B lineage cells will be used to investigate the occurrence and stage(s) of VH replacement in humans. (Aim 3) The regulatory mechanism of VH replacement will be investigated using the EU12 model through evaluating the effects of. (i) modulating surrogate light chain (lamda 5/14. 1) or conventional light chain (kappa and lamdaX) expression, (iii) ligating cell surface receptors (pre BCR, BCR, and CD40), and (iii) stimulating with cytokines (IL 1 beta, TFNaphla, IL 6, and IL 7) on VH replacement. (Aim 4) Determine if VH replacement occurs in synovial B cells thereby contributing to the generation of autoantibodies and to search for potential VH replacement stimuli in rheumatoid arthritis synovial tissue.