In this renewal application we seek continued support to examine the function of the pre-B cell receptor (pre- BCR) in regulating the proliferation and differentiation of pre-B cells, with a particular focus on distinctions in this process between fetl and adult B cell development. Building on miRNA analysis done in the preceding funding period that identified let-7b as absent from fetal B cell progenitors, we will determine the consequences for pre-BCR signaling when this microRNA is expressed in fetal liver progenitors by retroviral transduction (Aim 1a). We will also carry out complementary experiments using bone marrow precursors by expression of Lin28 that will down-regulate Let-7 and make them more fetal-like. In a second part of this aim, we will alter key downstream components of the pre-BCR signaling pathway by using mutant mice and by employing shRNA knockdown (Aim 1b). The effects of these perturbations will be assessed by PhosFlow, measuring signaling protein phosphorylation and also by determining changes in mRNA expression by quantitative PCR. This work will establish key signals that distinguish fetal and adult pre-BCR selection. In the prior period we also developed an assay for measuring the extent of pre-BCR association;now we will apply this assay to model the pre-BCR (Aim 2), taking advantage of a unique set of VHJ558 heavy chain VDJ segments that show different levels of association with surrogate light chain. This will enable us to determine critical CDR3 contacts with surrogate light chain that mediate pre-BCR selection. We will capitalize on our recent experience applying zinc finger nuclease technology to engineer DNA loci in mice by altering the mouse surrogate light chain locus to resemble that in humans, testing the consequences on CDR3 structure of heavy chains selected (Aim 3). This will provide definitive data showing the role of pre-BCR selection in B cell repertoire formation. Finally, we will determine whether, as in mouse, Pre-BCR selection in humans fetal development optimally requires heavy chains with weaker association for surrogate light chain, establishing a primary BCR repertoire with a self-reactive bias. We will do this by testing heavy chains cloned from human cord blood for pre-BCR assembly ((Aim 4), comparing the frequency of low-associating heavy chains with results already obtained in an analysis of heavy chains from peripheral blood and chronic lymphocytic leukemia B cells. These studies combine state-of-the-art flow cytometry with powerful in vitro culture systems and sophisticated manipulation of gene expression to dissect pre-BCR signaling. Our work will shed light on a critical stage in B cell development, pre-BCR selection, contrasting its modes during fetal and adult time, with implications in the area of stem cell transplantation, neonatal immunity, and the origins of B cell subsets. Understanding the fetal generation of B1/CD5+ B cells, with a surface phenotype similar to chronic lymphocytic leukemia, and dissecting pre-BCR signaling pathways, where disregulation can lead to pre-B acute lymphocytic leukemia, will lead to important insights into their origins and growth, likely resulting in improved therapies for these diseases.
This project addresses issues relating to the control of growth of B cell precursors, an important issue for human health, as changes that alter the normal function of this process can result in the most prevalent childhood leukemia, pre-B acute lymphocytic leukemia. This project also studies development of a type of natural autoreactive B cell that appears particularly susceptible to genetic changes that result in uncontrolled growth in aged individuals, resulting in the most common leukemia of individuals over 65, chronic lymphocytic leukemia. Information gained during the course of this project will increase our understanding of the origins and growth of these types of cells, potentially aiding in the design o rational therapies for these diseases of children and the aged.