REGULATED B-CELL UNRESPONSIVENESS AND TOLERANCE

点击翻译按钮获取中文摘要

We have previously defined two sIgM-coupled signaling differences between mature and immature stage B cells which operate to maintain the unresponsiveness characteristic of immature B cells. We wish to continue to define the molecular basis for these signalling differences and to extend these studies to evaluate whether these specific signalling phenotypes characterize all populations of mouse immature B cells. Comparisons will be made between sIgM+, sIgD- (immature) B cells from the fetal liver, neonatal bone marrow, neonatal spleen, and adult bone marrow and mature B cells from adult spleen. Besides allowing a comprehensive evaluation of the molecular basis for activation unresponsiveness by different populations of immature-stage B cells, we expect to discern associations between the anatomic site of origin, the developmental state of the B cell, and the ontogeny of the animal. In addition, we will generate transgenic mice which express the EGR-1 protein at the pre-B and immature B cell stage. In vivo humoral immune responses as well as auto-antibody production will be evaluated in order to determine the effect of egr-l expression on tolerance sensitivity in these animals. In addition, immature B cells isolated from these transgenic animals will be tested for qualitative differences in sIgM-coupled signaling in vitro in order to determine the effect on the activation unresponsiveness intrinsic to immature B cells from non-transgenic animals. Finally, we will characterize the 56 kDa mu-associated protein associated with the transmembrane signalling defect that typifies immature B cells from the neonatal mouse spleen. p56 will be isolated by two-dimensional SDS-PAGE and microsequenced. This information will be utilized to generate anti-p56 peptide antibodies and cDNA probes to evaluate intrinsic and associated kinase activity, associations with other sIgM complex members, and cloning and sequencing of the p56 cDNA in order to evaluate structural homologies to other signalling molecules. These studies represent a comprehensive analysis of the molecular basis of developmentally-associated unresponsiveness and tolerance sensitivity to antigen receptor signaling in the B lymphocyte.