The antibody-mediated humoral immune response has been shown to be depressed in aged humans and experimental I animals creating increased morbidity and mortality in the aged population. The pre-B-cell receptor (pBCR), composed of the surrogate light chain (SLC, lambda.5 and VpreB) and the immunoglobulin (Ig) mu (mu) heavy (H) chain is critical for formation of pre-B-cells and for variable region gene (VH) selection. These studies will focus on the molecular mechanisms responsible for the decline in pre-B-cells, SLC and transcription factors seen in aged mice. We hypothesize that the decrease in pre-B-cells is a direct result of a lower amount of the SLC produced in the pro-B/early pre-B-cell stages, and that this is regulated at the transcription level. This proposal will compare pro-B/pre-B-cells from aged vs. young mice for the presence and function of transcription factors known to regulate lambda5 and VpreB, accessibility of lambda5 and VpreB chromatin, whether the defects are intrinsic to B lineage cells, and attempt to reverse the aged phenotype in transgenic mice.
In Specific Aim I we will determine the regulation of transcription and transcription factors which affect SLC expression in senescent pro-B/pre-B-cells. Abelson Murine Leukemia Virus (A-MuLV) transformed pro-B/pre-B-cell lines will be prepared, and these, IL-7 expanded pro-B/pre-B-cells and ex vivo isolated (B220+ mu-) bone marrow cells from aged (>20 months) or young (2-4 months) mice will be used for this project. The mRNA stability and nascent transcripts of lambda5 and VpreB, the presence and function of transcription factors known (EBF and E47) and likely (Ikaros/Aiolos, Id) to be involved in lambda5/VpreB transcription regulation will be assayed by various methods including RT-PCR, electrophoretic mobility shift assay (EMSA), and transfection of lambda5/VpreB constructs. We will also measure the DNase I hypersensitivity and the histone acetylation status of the lambda5 and VpreB loci as a function of age.
In Aim 2 we will assay possible stromal influences on the transcription regulation of lambda5/VpreB. Young and aged stromal preparations will be used to assess their influence on the levels of lambda5/VpreB transcription regulation in pro-B/pre-B-cells in vitro, as well as in vivo adoptive transfer studies of young and/or old B lineage precursors into young or old recipients.
In Aim 3 we will prepare retroviral vectors and transgenic mice with lambda5/VpreB to attempt to rescue the aged B lineage phenotype. The experiments in this proposal will provide a molecular dissection of the abnormal regulation lambda5/VpreB which we hypothesize results in decreased pre-B-cells and an altered VH repertoire in aged mice.
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