Richard R Hardy, PhD
Office Phone: 215-728-2469
Lab Phone: 215-728-2463
Pre-BCR Signaling by Fetal- and Adult-Type Ig Heavy ChainsSusan Shinton, Lingjuan Tang & Yue-Sheng Li
In mouse bone marrow, pre-B cells experience a proliferative burst that is critically dependent on the assembly of newly generated immunoglobulin (Ig) heavy chain with a pair of B lineage specific peptides constituting a "surrogate light chain" (see Figure: Structure of the pre-B cell receptor (pre-BCR)). These peptides constitute part of a signaling complex that is referred to as the "pre-B cell receptor" (pre-BCR), in analogy to the B cell receptor (BCR) that mature B cells utilize to recognize foreign antigens.
Expression of the pre-BCR by association of newly formed Ig heavy chains with surrogate light chain induces increased cell proliferation, resulting in clonal expansion of preB cells (Figure: Pre-BCR formation results in a proliferative burst) . Failure to assemble a pre-BCR, as in SCID or Rag-null mice, results in a block in B cell development (Figure: Progression of pro-B cells (blue) to pre-B (red) requires the pre-BCR).
We have complemented this defect by expression of Ig heavy chain transgenes, (right-most panel in Figure: Progression of pro-B cells (blue) to pre-B (red) requires the pre-BCR), most recently by retroviral mediated gene transfer. During the course of this work we found that fetal B cell precursors respond differently to high levels of pre-BCR, exiting from the cell cycle, rather than proliferating. In our current work, we are generating libraries of VDJ segments from fetal liver and bone marrow of adult animals; inserting these into a heavy chain expression construct inside a retrovirus.
This allows us to investigate the extent of pre-BCR signaling by fetal-type and adult-type heavy chains with well-characterized differences in their VDJ segments expressing these heavy chains in cell lines and also in primary cells isolated from recombination-deficient animals. We are particularly interested in analyzing Ig heavy chain VDJs from B-1 B cells, largely generated during fetal/neonatal life.Top
B Cell Development in a VH11 Knock-In MouseSusan Shinton
Our previous work showed that certain Ig heavy chains failed to generate a normal pre-BCR signal, as assessed by changes in gene expression after transfection into a pro-B cell line. One such heavy chain, VH11, is of particular interest because it accounts for a significant fraction of the Ig heavy chain repertoire of the CD5+('B-1") B cell pool.
In order to carefully assess the behavior of B cells rearranging VH11 in a physiological context, we generated a VH11 "knock-in" mouse, where a rearranged VH11-D-JH1 replaces the normal germline JH locus. Analysis of this mutant mouse reveals a high level of VH replacement and loss of the VH11 transgene, as might be expected by weak pre-BCR function (failure to suppress Rag activity appears to allow other VH genes to replace VH11). Many of these new VH heavy chains mediate effective pre-BCR signaling, which induces progression to the B cell stage, populating the knock-in mouse with non-VH11+ B cells. Interestingly, in fetal liver, it appears that VH11 is less handicapped, and so contributes more effectively to the newly formed B cell pool. Weak pre-BCR association, together with preferential association Ig VH11-μ with only a few Ig light chains, results in bottlenecks in development of VH11+ B cells, limiting their generation to fetal/neonatal life (Figure: Model for VH11Vk9 natural autoantibody B cell generation).Top
The Earliest B Lineage Stages in Bone MarrowZhou & Susan Shinton
We have carried out a thorough analysis of the earliest stages in B cell development in mouse bone marrow, determining the potential of cells at every stage to develop into alternate lineages; as well as characterizing the extent of DJ rearrangement at the single cell level; and quantitating the expression of a set of genes critical in this pathway. Three of these stages, MLP, CLP, and pre-Pro-B (A in image to the left), are shown in Figure: B lineage stages identified by changes in cell surface protein expression, which diagrams the progression of hematopoietic stem cells (HSC) to newly formed (NF) and mature follicular (Fo) B cells (F in image to the left). Some of the cell surface proteins used for distinguishing these stages are shown with line thickness indicating relative expression. In addition, ELP indicates the "Early Lymphoid Progenitor" recognized by expression of a Rag1-GFP reporter.
The goal of this research is to connect our work on B220+ B lineage stages with the work of those that have identified earlier stages in lymphoid development. In addition, we have identified the cell stage, Multilineage Progenitor (MLP), in which genes involved in Ig recombination initially become activated, and cells can still efficiently develop into both myeloid and lymphoid lineages. Later stages, Common Lymphoid Progenitor (CLP) and pre-pro-B (A in image above) show increasing lymphoid restriction, culminating in B lineage restricted precursors at the CD19+ pro-B (B in image above) stage. Our gating scheme for sorting three early B lineage fractions is shown in Figure: Early B lineage cell stages analyzed for lineage potential (panel A). Three assays for assessing lineage potential are also shown in Figure: Early B lineage cell stages analyzed for lineage potential, (panels B-D). Our analysis supports a view of progressive B lineage specification, prior to absolute and irreversible B lineage commitment. It also provides a foundation for elucidation of gene networks that mediate this process.Top
Determining Gene Networks in B Cell DevelopmentZhou & Susan Shinton
The production of B lymphocytes from hematopoietic stem cells goes through a well-characterized series of cell stages, identified previously by flow cytometry. This development entails a highly orchestrated program of gene interactions; to probe this network, we are determining global gene profiles by microarray analysis (Figure: Cluster analysis of microarray data reveals a B lineage signature (B)). We plan to examine all the intermediate stages of developing cells we have resolved in bone marrow, identifying critical players at key checkpoints in this process. During the course of this work we will also determine how this process differs from that of generating distinctive B cells in fetal liver. Our high throughput analysis of gene expression in developing lymphocyte subsets may provide insights into the normal and abnormal development of the immune system, with relevance to disregulated growth, lymphoma and autoimmunity. Currently we are pursuing this work as part of the ImmGen Consortium.Top
B Cell Maturation in SpleenYue-Sheng Li & Susan Shinton
Most newly-formed B cells in bone marrow migrate to the spleen, where they progress through a sequence of transitional stages, undergoing negative (and potentially positive) selection before entering the functionally mature long-lived follicular B cell pool. We have described a procedure for identifying three transitional stages (T1-T3; Figure: Identifying transitional stages (T1-T3) in B cell maturation), prior to the follicular (Fo) B cell population, although there is some controversy concerning one of these (T3). We are currently applying single cell sequence analysis of heavy chain VDJs in order to assess the progressive selection of B cells.Top
B Cell Development in ZebrafishXingjun Liu
The zebrafish model organism is particularly suited for genome-wide mutational analysis, screening to identify genes critical for development of specific cell lineages. As part of such a mutational screen (in collaboration with Rhodes, Wiest, and Kappes), we are developing B lineage reporter transgenic zebrafish and also carrying out characterization of B cell development in embryonic and adult zebrafish.
Initially we characterized the expression of several genes important for the early stages of B cell development in mice, performing RT-PCR for Rag-1, Rag-2, EBF1, and Pax5 using cell fractions enriched for lymphoid lineage cells by sorting for Rag2-GFP+ or Lck-GFP+ cells from tissues of transgenic zebrafish lines (Figure: Gene expression in zebrafish cell fractions from thymus and kidney). As expected, we observed detectable signals for EBF1 and Pax5 in the GFP+ cells from head kidney (a known site for early B lineage cells in zebrafish), but not in GFP+ fractions from the thymus, predominantly a site for T cell development. In contrast, Rag-1 and Rag-2, important in both B and T lineage development were present at both sites. Thus EBF1 or Pax5 may serve as markers of developing B lineage cells. Our current work focuses on further characterization of the expression of these (and other) genes in zebrafish by in situ hybridization as well as development of GFP reporters using bacterial artificial chromosomes (BACs) transgenesis.Top