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Abstract: Flow cytometry is a powerful tool for quantitative biology because it can perform single-cell analysis of large cell populations using multiple parameters. Results are often visualized as a two-dimensional scatter or contour plot. Because these plots can be relatively diffuse, it is not always straightforward to discern a relationship between measured parameters. We have demonstrated that quantitative trends can be fit to the single-cell data generated from a heterogeneous population. We engineered Abelson virus-transformed pre-B cells to express a broad range of oncogenic Ras levels. Instead of individual cultures with individual expression levels, a continuous range of levels was expressed by different cells in one heterogeneous culture. We then stained cells for downstream Erk phosphorylation to monitor MAPK signaling or employed an E2F-responsive genetic reporter to monitor cell-cycle activity. Subsequent analysis by flow cytometry and locally weighted scatterplot smoothing (LOWESS) revealed that increasing Ras oncogene expression led to increasing MAPK signaling. In contrast, E2F activity peaked at an optimal, intermediate level of Ras. To make this analytical method widely available to others, we have provided a software application that performs LOWESS on any two-parameter population data collected by flow cytometry. [Copyright &y& Elsevier]

Abstract: The production of B cells and the primary antibody repertoire in mammalian species other than rodents or man appears to depend on gut-associated lymphoid tissue. Bovine B cells are generated in ileal Peyer''s patch from late gestational to juvenile age. However, little is known about where and when the bona fide B lymphopoiesis takes place. We analyzed bovine fetuses for signs of ongoing B lymphopoiesis using a combination of immunohistochemistry, flow cytometry, real-time quantitative PCR and RNA in situ hybridization. In fetal bone marrow and lymph node, we could demonstrate pre-B like cells positive for intracellular Ig μ but negative for membrane IgM. Strong expression of immunoglobulin lambda-like polypeptide 1 and recombination activating genes was also detected in the same tissues. Similar analyses did not reveal pre-B like cells in the corresponding adult tissues. These results suggest that bovine fetal bone marrow and lymph node support B lymphopoiesis via a pre-B cell like stage before and in parallel to the development of the ileal Peyer''s patch. [Copyright &y& Elsevier]

Abstract: The pre-B lymphoma in SL/Kh mice is a polygenic trait involving a number of host genes. In prelymphoma-stage bone marrow, transient pre-B cell expansion is induced by a host locus, Bomb1, and later followed by the emergence of a monoclonal population with a similar phenotype. To determine whether these pre-B cells represent precursors of lymphomas, we generated a congenic strain, NFS.SL/Kh-Bomb1 mice, by marker-assisted backcrossing to NFS. The congenic mice showed pre-B cell expansion, but pre-B lymphomas were not observed, even after 1 year of observation, irrespective of murine leukemia virus inoculation. Disturbed early B cell differentiation per se is not sufficient for SL/Kh lymphomagenesis. [Copyright &y& Elsevier]

Abstract: Rearrangements in reading frame 2 promote the expression of a truncated heavy chain, the Dμ protein. Dμ can assemble into a pre-B cell receptor like complex that appears to induce a subset of signals elicited by full length μ, but cannot promote the pro-B to pre-B cell transition of Rag−/− B cells. In order to determine if this could stem from an impaired survival signal not properly induced by the Dμ protein, we introduced Bcl-2 into Dμ-transgenic, Rag2−/− mice. Despite the fact that the Bcl-2 transgene expression promoted some increase in the fraction of CD43− B cells, an identical increase was also observed in Rag2−/− mice. Moreover, whereas in μ-transgenic Rag2−/−Bcl-2+ mice, CD2 and CD25 expression were up regulated and c-Kit was down regulated, these markers were unaltered in Dμ-transgenic Rag2−/− Bcl-2+ mice compared to Rag2−/− Bcl-2+ mice, indicating that Dμ cannot support pre-B cell maturation despite extended survival of B cell precursors by Bcl-2. In addition, we observed that in Dμ-transgenic recombination competent mice, the Dμ induced partial block is permissive for marginal zone B cell development whereas the formation of follicular B cells is severely reduced. While the Dμ protein is expressed in peripheral B cells escaping the block, only a minor fraction of Dμ is exposed to the outer cell surface. [Copyright &y& Elsevier]

Abstract: Senescent mice show decline in B lymphopoiesis marked by reduced pre-B cells. Analysis of bone marrow from aged (∼2 years old) BALB/c mice indicates that, in senescence, an increased proportion of immature B cells exhibit a CD43/S7+ surface phenotype. This results from continued production of new CD43/S7+ B cells in aged mice from their limited pre-B cell pool while production of CD43/S7- immature B cells is highly reduced. CD43/S7 is ordinarily observed on a minor subset of immature B cells in young mice and is indicative of their partial activation. Senescent immature B cells, both ex vivo and derived in vitro, also demonstrate increased expression of VhS107 concomitant with CD43/S7. These alterations in the phenotype and Vh repertoire among senescent immature B cells likely originate prior to surface Ig expression. In aged mice with depleted pre-B and immature B cells in vivo, pre-B and immature B cells exhibited increased apoptosis in vitro. Dexamethasone-induced apoptosis among B lineage cells in young adult mice also resulted in pre-B cell loss and increased expression of CD43/S7 and VhS107 among immature B cells similar to that observed spontaneously in aged mice. These results suggest that old age, possibly due to increased apoptosis, results in loss of pre-B cells and alterations in the phenotype and Vh repertoire of newly derived B cells. [Copyright &y& Elsevier]

Abstract: Introduction of a double-strand DNA break at the junction between a rearranging gene segment and its flanking recombination signal sequence (RSS) is the first step of V(D)J recombination. Such DNA breaks can be detected by either Southern blot hybridization or ligation-mediated PCR. While Southern blotting is easily quantifiable, it is often insufficiently sensitive and while LM-PCR is far more sensitive, it is poorly quantifiable. Reported here is a LM-qPCR assay which relies on real-time qPCR to provide an absolute measure of recombinase-mediated, or any other specific, double-strand DNA break in genomic DNA. The efficiency of the initial ligation reaction was found to be relatively low with just 3% of potential targets undergoing linker ligation. Using this assay, approximately 16% of murine bone marrow pre-B cells were determined to contain a dsDNA break adjacent to the Jκ1 gene segment. In addition, the kinetics of JΚκ1 dsDNA breaks in a temperature-sensitive cell line induced to recombine its κ locus was determined. [Copyright &y& Elsevier]

Senescence in murine models is associated with a reduction, albeit heterogeneous, in bone marrow pre-B cells. We have categorized aged BALB/c mice into two phenotypes based on their patterns of pre-B/pro-B cell loss. Each phenotype is characterized by distinct responses to the growth cytokine IL-7 and capacity for survival in vitro. A ‘moderate’ loss of late-stage pre-B cells (25–80%) coincided with decline in proliferation to rmIL-7. This was also associated with a decrease in the frequency of pro-B cells which increased phosphotyrosine content upon IL-7 stimulation, an indicator of early activation events. A ‘severe’ loss of pre-B cells (>80%) resulted in a reduced pro-B cell pool which retained normal activation and proliferative responses to IL-7. B cell precursors from aged mice with severe alterations in B lymphopoiesis displayed increased susceptibility to apoptosis in comparison to both aged mice with moderate B cell precursor loss and young mice. Conceivably, during senescence, aged mice may initially accumulate B cell precursors which are poorly responsive to IL-7. Progressively, these refractory B cell precursors may be eliminated via apoptosis; however, the remaining limited pool of B cell precursors retains the capacity to respond to IL-7 stimulation. [Copyright &y& Elsevier]

V(D)J recombination by the RAG1 and RAG2 protein complex in developing lymphocytes includes DNA double strand break (DSB) intermediates. RAG2 undergoes export from the nucleus and enrichment at the centrosome minutes following production of DSBs by genotoxic stress, suggesting that RAG2 participates in cellular responses to DSBs such as those generated during V(D)J recombination. To determine the effect of RAG2 expression on cell viability following DSB generation, we measured pre-B cells that expressed either full length (FL) wild-type RAG2, or a T490A mutant of RAG2 that has increased stability and fails to undergo nuclear export following generation of DSBs. Each RAG2 construct was labeled with GFP at the N-terminus. Compared to the T490A mutant, cells expressing FL RAG2 exhibited elevated apoptosis by 24 h following irradiation, and this coincided with a greater amount of Caspase 3 cleavage measured in cell lysates. Pre-B cells expressing either RAG2 protein exhibited similar increases in phospho-p53 levels following irradiation. Interestingly, FL RAG2-expressing cells exhibited elevated division relative to the T490A clone beginning ~24 h following irradiation, as well as an increased percentage of cells proceeding through mitosis, suggesting an improved rate of recovery following the initial burst in apoptosis. Altogether, these data show that FL RAG2, but not its stable nuclear export-defective T490A mutant, participates in pre-B cell decisions between apoptosis versus DNA repair and cell cycle progression following DNA damage. [ABSTRACT FROM AUTHOR]

Early B cell development is regulated by stage-specific transcription factors. PU.1, an ETS-family transcription factor, is essential for coordination of early B cell maturation and immunoglobulin gene (Ig) rearrangement. Here we show that RAG DNA double-strand breaks (DSBs) generated during Ig light chain gene (Igl) rearrangement in pre-B cells induce global changes in PU.1 chromatin binding. RAG DSBs activate a SPIC/BCLAF1 transcription factor complex that displaces PU.1 throughout the genome and regulates broad transcriptional changes. SPIC recruits BCLAF1 to gene-regulatory elements that control expression of key B cell developmental genes. The SPIC/BCLAF1 complex suppresses expression of the SYK tyrosine kinase and enforces the transition from large to small pre-B cells. These studies reveal that RAG DSBs direct genome-wide changes in ETS transcription factor activity to promote early B cell development. • RAG DNA breaks upregulate SPIC, which induces genome-wide changes in PU.1 activity • SPIC binds to gene-regulatory elements, resulting in loss of PU.1 at these regions • SPIC complexes with BCLAF1 to suppress transcription in response to RAG DNA breaks • SPIC/BCLAF1 inhibits SYK and promotes transition from large to small pre-B cells ETS-family transcription factors are key regulators of early B cell development. Soodgupta et al. show that RAG-induced DNA breaks generated during antigen receptor gene recombination activate a SPIC/BCLAF1 transcription factor complex that counters PU.1 activity and regulates gene expression changes to promote transition from large to small pre-B cells. [ABSTRACT FROM AUTHOR]