B lymphopoiesis declines dramatically due to the aging process. In the mouse, senescence results in decreased pre-B-cell numbers, altered pro-B-cell function, and generation of an antibody repertoire which is critically different from that seen in young adult mice. The investigator hypothesizes that pro-B-cells from aged mice have altered expression of Rag-1, and Rag-2, and terminal deoxynucleotidyl transferase (TdT) enzymes, and, consequently, altered generation of the mu heavy chain VH repertoire. He further hypothesizes that altered generation and diversification of the VH repertoire contributes to diminished formation or dysfunction of the pre-B-cell receptor complex (mu/lambda-5/V-pre-B) and subsequent failure to recruit new pre-B-cells into mitosis. This would result in both low production of pre-B-cells and an altered specificity repertoire in aged mice. In order to test these hypotheses, he proposes in Specific Aim 1 to assess the regulation of transcription and protein expression of the pre-B-cell receptor proteins mu, lambda-5, and V-pre-B into pro-B-cells (CD43+CD25-B220+), newly formed pre-B-cells (CD43+CD25+B220+), and late stage pre-B-cells (CD43-CD25+B220+) isolated from aged (24 mo.) vs. young (3 mo.) BALB/c mice via fluorescence activated cell sorting. He will also assess the capacity of these proteins to assemble into surface membrane associated pre-B-cell receptors by utilizing surrogate light chain and pre-B-cell receptor complex specific monoclonal antibody staining and analysis of pre-B-cell receptor assembly and turn-over in A-MuLV pre-B-cell lines from young vs. aged mice. The capacity of aged pre-B-cell receptor expression to functionally affect the expression of Rag-1 and Rag-2; TdT; the protein tyrosine kinase Blk; and the expression of ^X5 itself will be assessed.
In Specific Aim 2, the pre-B-cell receptor dependent selection of the mu heavy chain repertoire will be evaluated in nascent pre-B-cells (CD43+CD25+B220+) vs. later (post-selection) pre-B-cells (CD43- CD25+B220+) from aged vs. young mice with particular emphasis on diversity in CDR3.
In Specific Aim 3, the importance of the bone marrow microenvironment of aged mice in dictating the changes observed in B lineage cell development will be investigated using both in vivo adoptive transfer models and in vitro stroma cell culture systems. The experiments in this application will provide a cellular and molecular basis for understanding the temporal loss of B lymphopoiesis and altered generation of VH repertoire during senescence.
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