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.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG015474-02
Application #
2899810
Study Section
Immunobiology Study Section (IMB)
Project Start
1998-04-01
Project End
2003-03-31
Budget Start
1999-04-01
Budget End
2000-03-31
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Miami School of Medicine
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146
Wilson, Emily L; King, Anne M; Sherwood, Erin M et al. (2005) Pre-B cell loss in senescence coincides with preferential development of immature B cells characterized by partial activation and altered Vh repertoire. Exp Gerontol 40:67-79
Riley, Richard L; Blomberg, Bonnie B; Frasca, Daniela (2005) B cells, E2A, and aging. Immunol Rev 205:30-47
Frasca, Daniela; Nguyen, Diep; Riley, Richard L et al. (2004) Effects of aging on DNA-binding activity of the E47 transcription factor in splenic B cells. Mech Ageing Dev 125:111-2
Frasca, Daniela; Van der Put, Elaine; Riley, Richard L et al. (2004) Reduced Ig class switch in aged mice correlates with decreased E47 and activation-induced cytidine deaminase. J Immunol 172:2155-62
Van der Put, Elaine; Frasca, Daniela; King, Anne M et al. (2004) Decreased E47 in senescent B cell precursors is stage specific and regulated posttranslationally by protein turnover. J Immunol 173:818-27
Frasca, Daniela; Van Der Put, Elaine; Riley, Richard L et al. (2004) Age-related differences in the E2A-encoded transcription factor E47 in bone marrow-derived B cell precursors and in splenic B cells. Exp Gerontol 39:481-9
Wilson, Emily L; Sherwood, Erin M; King, Anne M et al. (2003) A phenotypically distinct subset of immature B cells exhibits partial activation, increased survival, and preferential expression of VhS107. Eur J Immunol 33:3398-408
Frasca, Daniela; Nguyen, Diep; Van der Put, Elaine et al. (2003) The age-related decrease in E47 DNA-binding does not depend on increased Id inhibitory proteins in bone marrow-derived B cell precursors. Front Biosci 8:a110-6
Frasca, Daniela; Nguyen, Diep; Riley, Richard L et al. (2003) Decreased E12 and/or E47 transcription factor activity in the bone marrow as well as in the spleen of aged mice. J Immunol 170:719-26
Frasca, Daniela; Nguyen, Diep; Riley, Richard L et al. (2003) Effects of aging on proliferation and E47 transcription factor activity induced by different stimuli in murine splenic B cells. Mech Ageing Dev 124:361-9

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