Search

Supplementary Figure 1 from NEDD9 Promotes Oncogenic Signaling in Mammary Tumor Development

Supplementary Figure 3 from NEDD9 Promotes Oncogenic Signaling in Mammary Tumor Development

Supplementary Table 1 from NEDD9 Promotes Oncogenic Signaling in Mammary Tumor Development

Supplementary Figure 2 from NEDD9 Promotes Oncogenic Signaling in Mammary Tumor Development

Large homozygous deletions of 9p21 that inactivate CDKN2A, ARF, and MTAP are common in a wide variety of human cancers. The role for CDKN2A and ARF in tumorigenesis is well established, but whether MTAP loss directly affects tumorigenesis is unclear. MTAP encodes the enzyme methylthioadenosine phosphorylase, a key enzyme in the methionine salvage pathway. To determine if loss of MTAP plays a functional role in tumorigenesis, we have created an MTAP-knockout mouse. Mice homozygous for a MTAP null allele (MtaplacZ) have an embryonic lethal phenotype dying around day 8 postconception. Mtap/MtaplacZ heterozygotes are born at Mendelian frequencies and appear indistinguishable from wild-type mice during the first year of life, but they tend to die prematurely with a median survival of 585 days. Autopsies on these animals reveal that they have greatly enlarged spleens, altered thymic histology, and lymphocytic infiltration of their livers, consistent with lymphoma. Immunohistochemical staining and fluorescence-activated cell sorting analysis indicate that these lymphomas are primarily T-cell in origin. Lymphoma-infiltrated tissues tend to have reduced levels of Mtap mRNA and MTAP protein in addition to unaltered levels of methyldeoxycytidine. These studies show that Mtap is a tumor suppressor gene independent of CDKN2A and ARF. [Cancer Res 2009;69(14):OF5961–8]

Supplementary Figures 1-3, Table 1 from Mice Heterozygous for Germ-line Mutations in Methylthioadenosine Phosphorylase (MTAP) Die Prematurely of T-Cell Lymphoma

In the past 3 years, altered expression of the HEF1/CAS-L/NEDD9 scaffolding protein has emerged as contributing to cancer metastasis in multiple cancer types. However, whereas some studies have identified elevated NEDD9 expression as prometastatic, other work has suggested a negative role in tumor progression. We here show that the Nedd9-null genetic background significantly limits mammary tumor initiation in the MMTV-polyoma virus middle T genetic model. Action of NEDD9 is tumor cell intrinsic, with immune cell infiltration, stroma, and angiogenesis unaffected. The majority of the late-appearing mammary tumors of MMTV-polyoma virus middle T;Nedd9−/− mice are characterized by depressed activation of proteins including AKT, Src, FAK, and extracellular signal-regulated kinase, emphasizing an important role of NEDD9 as a scaffolding protein for these prooncogenic proteins. Analysis of cells derived from primary Nedd9+/+ and Nedd9−/− tumors showed persistently reduced FAK activation, attachment, and migration, consistent with a role for NEDD9 activation of FAK in promoting tumor aggressiveness. This study provides the first in vivo evidence of a role for NEDD9 in breast cancer progression and suggests that NEDD9 expression may provide a biomarker for tumor aggressiveness. [Cancer Res 2009;69(18):7198–206]

Supplementary Figure Legends 1-3 from Mice Heterozygous for Germ-line Mutations in Methylthioadenosine Phosphorylase (MTAP) Die Prematurely of T-Cell Lymphoma

In mice, fetal/neonatal B-1 cell development generates murine CD5+ B cells (B1a) with autoreactivity. We analyzed B1a cells at the neonatal stage in a VH11/D/JH knock-in mouse line (VH11t) that generates an autoreactive antiphosphatidylcholine BCR. Our study revealed that antiphosphatidylcholine B1a cells develop in liver, mature in spleen, and distribute in intestine/colon, mesenteric lymph node (mLN), and body cavity as the outcome of B-1 cell development before B-2 cell development. Throughout life, self-renewing B-1 B1a cells circulate through intestine, mesenteric vessel, and blood. The body cavity–deposited B1a cells also remigrate. In old age, some B1a cells proceed to monoclonal B cell lymphocytosis. When neonatal B-1 B1a cells express an antithymocyte/Thy-1 autoreactivity (ATA) BCR transgene in the C.B17 mouse background, ATA B cells increase in PBL and strongly develop lymphomas in aging mice that feature splenomegaly and mLN hyperplasia with heightened expression of CD11b, IL-10, and activated Stat3. At the adult stage, ATA B cells were normally present in the mantle zone area, including in intestine. Furthermore, frequent association with mLN hyperplasia suggests the influence by intestinal microenvironment on lymphoma development. When cyclin D1 was overexpressed by the Eμ-cyclin D1 transgene, ATA B cells progressed to further diffused lymphoma in aged mice, including in various lymph nodes with accumulation of IgMhiIgDloCD5+CD23−CD43+ cells, resembling aggressive human mantle cell lymphoma. Thus, our findings reveal that early generated B cells, as an outcome of B-1 cell development, can progress to become lymphocytosis, lymphoma, and mantle cell lymphoma–like neoplasia in aged mice.

CD79a and CD79b proteins associate with Ig receptors as integral signaling components of the B cell Ag receptor complex. To study B cell development in zebrafish, we isolated orthologs of these genes and performed in situ hybridization, finding that their expression colocalized with IgH-μ in the kidney, which is the site of B cell development. CD79 transgenic lines were made by linking the promoter and upstream regulatory segments of CD79a and CD79b to enhanced GFP to identify B cells, as demonstrated by PCR analysis of IgH-μ expression in sorted cells. We crossed these CD79-GFP lines to a recombination activating gene (Rag)2:mCherry transgenic line to identify B cell development stages in kidney marrow. Initiation of CD79:GFP expression in Rag2:mCherry+ cells and the timing of Ig H and L chain expression revealed simultaneous expression of both IgH-μ– and IgL-κ–chains, without progressing through the stage of IgH-μ–chain alone. Rag2:mCherry+ cells without CD79:GFP showed the highest Rag1 and Rag2 mRNAs compared with CD79a and CD79b:GFP+ B cells, which showed strongly reduced Rag mRNAs. Thus, B cell development in zebrafish does not go through a Raghi CD79+IgH-μ+ pre–B cell stage, different from mammals. After the generation of CD79:GFP+ B cells, decreased CD79 expression occurred upon differentiation to Ig secretion, as detected by alteration from membrane to secreted IgH-μ exon usage, similar to in mammals. This confirmed a conserved role for CD79 in B cell development and differentiation, without the requirement of a pre–B cell stage in zebrafish.

Hayakawa et al. show that distinctive B-lineage progression from B-1 development allows for generation of B1a cells with restricted BCRs and self-renewal capacity, both contributing to potential for CLL progression., In mice, generation of autoreactive CD5+ B cells occurs as a consequence of BCR signaling induced by (self)-ligand exposure from fetal/neonatal B-1 B cell development. A fraction of these cells self-renew and persist as a minor B1 B cell subset throughout life. Here, we show that transfer of early generated B1 B cells from Eμ-TCL1 transgenic mice resulted in chronic lymphocytic leukemia (CLL) with a biased repertoire, including stereotyped BCRs. Thus, B1 B cells bearing restricted BCRs can become CLL during aging. Increased anti-thymocyte/Thy-1 autoreactive (ATA) BCR cells in the B1 B cell subset by transgenic expression yielded spontaneous ATA B-CLL/lymphoma incidence, enhanced by TCL1 transgenesis. In contrast, ATA B-CLL did not develop from other B cell subsets, even when the identical ATA BCR was expressed on a Thy-1 low/null background. Thus, both a specific BCR and B1 B cell context were important for CLL progression. Neonatal B1 B cells and their CLL progeny in aged mice continued to express moderately up-regulated c-Myc and down-regulated proapoptotic Bmf, unlike most mature B cells in the adult. Thus, there is a genetic predisposition inherent in B-1 development generating restricted BCRs and self-renewal capacity, with both features contributing to potential for progression to CLL.

CD5(+) B-cell origins and their predisposition to lymphoma are long-standing issues. Transfer of fetal and adult liver BM Pro-B cells generates B cells with distinct phenotypes: fetal cells generate IgM(high) IgD(low) CD5(+) , whereas adult cells IgM(low) IgD(high) CD5(-) . This suggests a developmental switch in B lymphopoiesis, similar to the switch in erythropoiesis. Comparison of mRNA and miRNA expression in fetal and adult Pro-B cells revealed differential expression of Lin28b mRNA and Let-7 miRNA, providing evidence that this regulatory axis functions in the switch. Recent work has shown that Arid3a is a key transcription factor mediating fetal-type B-cell development. Lin28b-promoted fetal development generates CD5(+) B cells as a consequence of positively selected self-reactivity. CD5(+) B cells play important roles in clearance of apoptotic cells and in protective immune responses, but also pose a risk of progression to leukemia/lymphoma. Differential Lin28b expression in fetal and adult human B-cell precursors showed that human B-cell development may resemble mouse, with self-reactive "innate-like" B cells generated early in life. It remains to be determined whether such human B cells have a higher propensity to leukemic progression. This review describes our recent research with CD5(+) B cells and presents our perspective on their role in disease.

Fluorescence-activated cell sorting (FACS)-purified pro-B cells from fetal liver and adult bone marrow generate B cells with distinct phenotypes: fetal cells generate few IgD(high) B cells and half express CD5, whereas adult cells generate mostly IgD(high) cells and few express CD5. These results led us to propose a model of a developmental switch in B lymphopoiesis, similar to the well-known switch in fetal to adult erythropoiesis. More recent global analysis of mRNA and microRNA expression comparing these two types of pro-B cells revealed differential expression of Lin28b and microRNAs from the Let-7 family, indicating that this regulatory axis plays a role in the switch. Further analysis has provided data supporting this model, implicating Arid3a as a key transcription factor in mediating fetal-type B cell development. Function of this regulatory axis in human B lineage precursors may also explain the predominance of CD5(+) B cells in cord blood. We suggest that Lin28b-promoted B cell development generates many cells expressing CD5 as a consequence of positively selected self-reactivity. While such cells serve a useful role in clearance of senescent cells and in certain immune responses, they also carry the risk of progression to leukemia/lymphoma later in life.

Expression of a germline VH3609/D/JH2 IgH in mice results in the generation of B1 B cells with anti-thymocyte/Thy-1 glycoprotein autoreactivity by coexpression of Vk21-5/Jk2 L chain leading to production of serum IgM natural autoantibody. In these same mice, the marginal zone (MZ) B cell subset in spleen shows biased usage of a set of Ig L chains different from B1 B cells, with 30% having an identical Vk19-17/Jk1 L chain rearrangement. This VH3609/Vk19-17 IgM is reactive with intestinal goblet cell granules, binding to the intact large polymatrix form of mucin 2 glycoprotein secreted by goblet cells. Analysis of a μκ B cell AgR (BCR) transgenic (Tg) mouse with this anti–goblet cell/mucin2 autoreactive (AGcA) specificity demonstrates that immature B cells expressing the Tg BCR become MZ B cells in spleen by T cell–independent BCR signaling. These Tg B cells produce AGcA as the predominant serum IgM, but without enteropathy. Without the transgene, AGcA autoreactivity is low but detectable in the serum of BALB/c and C.B17 mice, and this autoantibody is specifically produced by the MZ B cell subset. Thus, our findings reveal that AGcA is a natural autoantibody associated with MZ B cells.

Interferon regulatory factor 4 (IRF4) is a critical transcriptional regulator of B-cell development and function. A recent genome-wide single-nucleotide polymorphism (SNP) association study identified IRF4 as a major susceptibility gene in chronic lymphocytic leukemia (CLL). Although the SNPs located in the IRF4 gene were linked to a downregulation of IRF4 in CLL patients, whether a low level of IRF4 is critical for CLL development remains unclear. In rodents, CLL cells are derived from B1 cells whose population is dramatically expanded in immunoglobulin heavy chain Vh11 knock-in mice. We bred a Vh11 knock-in allele into IRF4-deficient mice (IRF4(-/-)Vh11). Here, we report that IRF4(-/-)Vh11 mice develop spontaneous early-onset CLL with 100% penetrance. Further analysis shows that IRF4(-/-)Vh11 CLL cells proliferate predominantly in spleen and express high levels of Mcl-1. IRF4(-/-)Vh11 CLL cells are resistant to apoptosis but reconstitution of IRF4 expression in the IRF4(-/-)Vh11 CLL cells inhibits their survival. Thus, our study demonstrates for the first time a causal relationship between low levels of IRF4 and the development of CLL. Moreover, our findings establish IRF4(-/-)Vh11 mice as a novel mouse model of CLL that not only is valuable for dissecting molecular pathogenesis of CLL but could also be used for therapeutic purposes.

Background The relationship between chemotherapy-related toxicities and prognosis is unclear. Previous studies have examined the association of myelosuppression parameters or neuropathy with survival and reported conflicting results. This study aims to investigate 13 common chemotherapy toxicities and their association with relapse-free survival and breast cancer-specific survival. Methods Chemotherapy-related toxicities were collected prospectively for 6,248 women with early-stage breast cancer from four randomised controlled trials (NEAT; BR9601; tAnGo; Neo-tAnGo). Cox proportional-hazards modelling was used to analyse the association between chemotherapy-related toxicities and both breast cancer-specific survival and relapse-free survival. Models included important prognostic factors and stratified by variables violating the proportional hazards assumption. Results Multivariable analysis identified severe neutropenia (grades ≥3) as an independent predictor of relapse-free survival (hazard ratio (HR) = 0.86; 95 % confidence interval (CI), 0.76–0.97; P = 0.02). A similar trend was seen for breast cancer-specific survival (HR = 0.87; 95 % CI, 0.75–1.01; P = 0.06). Normal/low BMI patients experienced more severe neutropenia (P = 0.008) than patients with higher BMI. Patients with fatigue (grades ≥3) showed a trend towards reduced survival (breast cancer-specific survival: HR = 1.17; 95 % CI, 0.99–1.37; P = 0.06). In the NEAT/BR9601 sub-group analysis by treatment component, this effect was statistically significant (HR = 1.61; 95 % CI, 1.13–2.30; P = 0.009). Conclusions This large study shows a significant association between chemotherapy-induced neutropenia and increased survival. It also identifies a strong relationship between low/normal BMI and increased incidence of severe neutropenia. It provides evidence to support the development of neutropenia-adapted clinical trials to investigate optimal dose calculation and its impact on clinical outcome. This is important in populations where obesity may lead to sub-optimal chemotherapy doses. Electronic supplementary material The online version of this article (doi:10.1186/s12916-015-0547-5) contains supplementary material, which is available to authorized users.

A common feature of B-cell chronic lymphocytic leukemia (CLL) is chromosomal loss of 13q14, containing the miR15a/16-1 locus controlling B-cell proliferation. However, CLL etiology remains unclear. CLL is an adult leukemia with an incidence that increases with advancing age. A unique feature of CLL is biased B-cell antigen receptor (BCR) usage, autoreactivity with polyreactivity and CD5 expression, all suggest a role for the BCR in driving CLL pathogenesis. Among human CLLs, BCRs autoreactive with non-muscle myosin IIA (AMyIIA) are recurrent. Here we identify an unmutated AMyIIA BCR in mouse, with distinctive CDR3 segments capable of promoting leukemogenesis. B cells with this AMyIIA BCR are generated by BCR-dependent signaling during B-1 fetal/neonatal development with CD5 induction, but not in adults. These early-generated AMyIIA B-1 B cells self-renew, increase during aging and can progress to become monoclonal B-cell lymphocytosis, followed by aggressive CLL in aged mice, often with the loss of a chromosomal region containing the miR15a/16-1 locus of varying length, as in human CLL. Thus, the ability to generate this defined autoreactive BCR by B-1 B cells is a key predisposing step in mice, promoting progression to chronic leukemia.

The role of hedgehog (Hh) signaling in B lymphopoiesis has remained unclear. We observed that the proliferation of pro-B cells in stromal cocultures was impaired by interruption of Hh signaling, prompting us to investigate whether the target of Hh antagonism was intrinsic or extrinsic to the B-lymphoid compartment. In the present study, using conditional deletion of the pathway activator gene Smo, we found that cell-autonomous Hh signaling is dispensable for B-cell development, B-lymphoid repopulation of the BM, and humoral immune function. In contrast, depletion of the Smo protein from stromal cells was associated with impaired generation of B-lymphoid cells from hematopoietic stem progenitor cells, whereas reciprocal removal of Smo from these cells had no effect on the production of B-cell progenitors. Depletion of Smo from stromal cells was associated with coordinate down-regulation of genes for which expression is associated with osteoblastoid identity and B-lymphopoietic activity. The results of the present study suggest that activity of the Hh pathway within stromal cells promotes B lymphopoiesis in a non–cell-autonomous fashion.

nology is an ideal field for the application of systems approaches, with its detailed descriptions of cell types (over 200 immune cell types are defined in the scope of the Immunological Genome Project (ImmGen)), wealth of reagents and easy access to cells. Thanks to the broad and robust approaches allowed by gene-expression microarrays and related techniques, the transcriptome is probably the only ‘-ome’ that can be reliably tackled in its entirety. Generating a complete perspective of gene expression in the immune system

The 3' regulatory region (3' RR) of the murine immunoglobulin heavy chain (IgH) locus contains multiple DNase I-hypersensitive (hs) sites. Proximal sites hs3A, hs1.2, and hs3B are located in an extensive palindromic region and together with hs4 are associated with enhancers involved in the expression and class switch recombination of IgH genes. Distal hs5, -6, and -7 sites located downstream of hs4 comprise a potential insulator for the IgH locus. In pro-B cells, hs4 to -7 are associated with marks of active chromatin, while hs3A, hs1.2, and hs3B are not. Our analysis of DNA methylation-sensitive restriction sites of the 3' RR has revealed a similar modular pattern in pro-B cells; hs4 to -7 sites are unmethylated, while the palindromic region is methylated. This modular pattern of DNA methylation and histone modifications appears to be determined by at least two factors: the B-cell-specific transcription factor Pax5 and linker histone H1. In pre-B cells, a region beginning downstream of hs4 and extending into hs5 showed evidence of allele-specific demethylation associated with the expressed heavy chain allele. Palindromic enhancers become demethylated later in B-cell differentiation, in B and plasma cells.

BCR editing in the bone marrow contributes to B cell tolerance by orchestrating secondary Ig rearrangements in self-reactive B cells. We have recently shown that loss of the BCR or a pharmacologic blockade of BCR proximal signaling pathways results in a global “back-differentiation” response in which immature B cells down-regulate genes important for the mature B cell program and up-regulate genes characteristic of earlier stages of B cell development. These observations led us to test the hypothesis that self-Ag-induced down-regulation of the BCR, and not self-Ag-induced positive signals, lead to Rag induction and hence receptor editing. Supporting this hypothesis, we found that immature B cells from xid (x-linked immunodeficiency) mice induce re-expression of a Rag2-GFP bacterial artificial chromosome reporter as well as wild-type immature B cells following Ag incubation. Incubation of immature B cells with self-Ag leads to a striking reversal in differentiation to the pro-/pre-B stage of development, consistent with the idea that back-differentiation results in the reinduction of genes required for L chain rearrangement and receptor editing. Importantly, Rag induction, the back-differentiation response to Ag, and editing in immature and pre-B cells are inhibited by a combination of phorbol ester and calcium ionophore, agents that bypass proximal signaling pathways and mimic BCR signaling. Thus, mimicking positive BCR signals actually inhibits receptor editing. These findings support a model whereby Ag-induced receptor editing is inhibited by BCR basal signaling on developing B cells; BCR down-regulation removes this basal signal, thereby initiating receptor editing.

Natural antibodies produced by CD5+ B1 B cells include anti-thymocyte autoantibody (ATA). Transgenic mice bearing the Ig-μ heavy chain of a prototypic ATA, V(H)3609Vκ21c, demonstrated a critical requirement for self-antigen in the accumulation of ATA B cells and production of high levels of serum ATA. Further work with ATA-μκ transgenic mice revealed that, while development of most B cells were blocked at an immature stage in spleen, some mature ATA B cells were present. ATA-μκ transgenic mice with varying levels of Thy-1 autoantigen showed a clear relationship between BCR crosslinking and B cell fate, with low levels generating marginal zone ATA B cells and complete antigen absence allowing maturation to follicular ATA B cells. Finally, different fates of developing ATA B cells encountering high levels self-antigen may be accounted for by variations in the response of newly formed B cells arising from foetal and adult development.

Fluorescence-activated cell sorting (FACS)-purified pro-B cells from fetal liver and adult bone marrow generate B cells with distinct phenotypes: fetal cells generate few IgD(high) B cells and half express CD5, whereas adult cells generate mostly IgD(high) cells and few express CD5. These results led us to propose a model of a developmental switch in B lymphopoiesis, similar to the well-known switch in fetal to adult erythropoiesis. More recent global analysis of mRNA and microRNA expression comparing these two types of pro-B cells revealed differential expression of Lin28b and microRNAs from the Let-7 family, indicating that this regulatory axis plays a role in the switch. Further analysis has provided data supporting this model, implicating Arid3a as a key transcription factor in mediating fetal-type B cell development. Function of this regulatory axis in human B lineage precursors may also explain the predominance of CD5(+) B cells in cord blood. We suggest that Lin28b-promoted B cell development generates many cells expressing CD5 as a consequence of positively selected self-reactivity. While such cells serve a useful role in clearance of senescent cells and in certain immune responses, they also carry the risk of progression to leukemia/lymphoma later in life.

B cells generated early during fetal/neonatal B-1 development in mice include autoreactive cells with detectable CD5 upregulation induced by B cell receptor (BCR) signaling (B1a cells). A fraction of B1a cells are maintained by self-renewal for life, with the potential risk of dysregulated growth and progression to chronic lymphocytic leukemia (CLL)/lymphoma during aging. In studies using the Eμ-hTCL1 transgenic mouse system, it became clear that this B1a subset has a higher potential than other B cell subsets for progression to CLL. We have generated several autoreactive germline BCR gene models to compare B cells generated under conditions of natural exposure to autoantigen. Analysis of the mice has been key in understanding the importance of the BCR and BCR signaling for generating different B cell subsets and for investigating the cellular origin of B-CLL.

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

Global gene profiling using the data we generated for the ImmGen Consortium allows us to address three issues in B cell function and development: (1) progression through transitional stages in spleen; (2) differences in signaling networks among mature B cell populations; and (3) variation in follicular-type (Fo) B cells with tissue location. For the first issue, we focused on the third transitional stage, T3, asking whether it was a pure population of anergic cells and concluding that only a small fraction of T3 is anergic. For the second, we clustered genes up- and downregulated distinctively in three mature populations, relating these gene sets to differences in B cell receptor signaling that distinguish these subsets. Finally, for the third issue, we identified differences in gene expression in the Fo B cell pool present in spleen, bone marrow, peritoneal cavity, and lymph nodes and found that peritoneal cavity Fo B cells were enriched for apoptosis-related genes compared with spleen, and that lymph node resident cells appeared most functionally mature. These analyses highlight the complexity of gene regulation in different maturation stages and subpopulations of B cells and also the richness of information in the ImmGen data sets.

CD5+ B cells have attracted considerable interest because of their association with self-reactivity, autoimmunity, and leukemia. In mice, CD5+ B cells are readily generated from fetal/neonatal precursors, but inefficiently from precursors in adult. One model proposed to explain this difference is that their production occurs through a distinctive developmental process, termed B-1, that enriches pre-B cells with novel germline VDJs and that requires positive selection of newly formed B cells by self-Ag. In contrast, follicular B cells are generated throughout adult life in a developmental process termed B-2, selecting VDJs that pair well with surrogate L chain, and whose maturation appears relatively independent of antigenic selection. In the present study, I focus on processes that shape the repertoire of mouse CD5+ B cells, describing the differences between B-1 and B-2 development, and propose a model encompassing both in the generation of functional B cell subpopulations.

Natural antibodies produced by CD5+ B-1 B cells include those with specificity for senescent erythrocytes (anti-BrMRBC, anti-PtC) and for thymocytes (anti-thymocyte autoantibody, ATA). Here we describe work from our laboratories studying two prototypic examples, V(H)11Vkappa9-encoded anti-BrMRBC and V(H)3609Vkappa21c-encoded ATA. Using V(H)11-mu transgenic mice, we discovered that certain natural autoantibodies utilize V(H) genes that are selected against in bone marrow B cell development, but not fetal liver, effectively restricting their generation to fetal/neonatal life. Studies with ATA-mu transgenic mice demonstrated a critical requirement for self antigen in the accumulation of B cells with this specificity and for the production of high levels of serum ATA. Finally, analysis of B cell development in ATA-mu kappa transgenic mice revealed two distinct responses by B cells to expression of this B cell receptor (BCR): most developing B cells in spleen of adult mice were blocked at an immature stage and only escaped apoptosis by editing their BCR to eliminate the ATA specificity; nevertheless, high levels of serum ATA were observed, indicating that some B cells differentiated to antibody-forming cells without altering their specificity. Thus, our studies reveal mechanisms for restricting the generation of B cells producing natural autoantibodies, demonstrate a key positive selection step in their development, and show that most developing B cells in adult mice bearing such specificities fail to reach a mature stage.

Cd79a (called mb-1 here) encodes the Ig-alpha signaling component of the B cell receptor. The early B cell-specific mb-1 promoter was hypermethylated at CpG dinucleotides in hematopoietic stem cells but became progressively unmethylated as B cell development proceeded. The transcription factor Pax5 activated endogenous mb-1 transcription in a plasmacytoma cell line, but could not when the promoter was methylated. In this context, early B cell factor (EBF), a transcription factor required for B lymphopoiesis, potentiated activation of mb-1 by Pax5. EBF and the basic helix-loop-helix transcription factor E47 each contributed to epigenetic modifications of the mb-1 promoter, including CpG demethylation and nucleosomal remodeling. EBF function was enhanced by interaction with the transcription factor Runx1. These data suggest a molecular basis for the hierarchical dependence of Pax5 function on EBF and E2A in B lymphocyte development.

Natural autoantibodies constitute a large portion of serum immunoglobulin M (IgM) and bridge the adaptive and innate immune systems, serving as a rapid response to common pathogens. Many arise from a distinctive subset of B cells, termed B-1, that express CDS. Here, we describe our studies with a representative CD5 + B-cell-derived natural autoantibody, the V H 11V κ 9 B-cell receptor (BCR) that binds a determinant on senescent erythrocytes. This specificity represents 5-10% of the CD5 + B-cell subset, with a large portion accounted for by two novel BCRs, V H 11V κ 9 and V H 12V κ 4. We have found that the development of B-lineage cells with a V H 11 rearrangement is surprisingly restricted at several crucial bottlenecks: (i) one of the most common V H 11 rearrangements generates a heavy-chain protein that only inefficiently assembles a pre-BCR, key for recombinase-activating gene downregulation/allelic exclusion and pre-B-clonal expansion; (ii) cells containing V H 11-μ chains lacking N-addition are favored for progression to the B-cell stage, eliminating most bone marrow V H 11 rearrangements; and (iii) only a subset of V κ -light chains combine with V H 11 heavy chain to foster progression to the mature B-cell stage. Together, these constrain V H 11 generation to fetal development and may favor production of B cells with the prototype V H 11V κ 9 BCR.

Considerable progress has been made over the past few years in determining the key molecular players that regulate the commitment and development of early B-lineage cells in mouse bone marrow (transcription factors, cytokines and their receptors). However, confusion remains as to the precise stages, surface phenotypes and lineage restrictions that are observed early in B-cell development. Increasingly powerful flow cytometry analysis of genetically altered animals (knockouts and transgenics) combined with sensitive functional assays may provide an answer, a ‘scorecard’ for the cell stages.

A natural serum autoantibody specific for the Thy-1 glycoprotein (anti-Thy-1 autoantibody [ATA]) is produced by B-1 cells that are positively selected by self-antigen. Here, using ATA micro kappa transgenic mice we show that cells with this B cell receptor are negatively selected during bone marrow (BM) development. In a Thy-1 null environment, BM ATA B cells progress to a normal follicular stage in spleen. However, in a self-antigen-positive environment, development is arrested at an immature stage in the spleen, concomitant with induction of CD5. Such cells are tolerant and short-lived, different from B-1. Nonetheless, ATA-positive selection was evident by self-antigen-dependent high serum ATA production, comprising approximately 90% of serum immunoglobulin M in ATA micro kappa mice. Splenectomy did not eliminate ATA production and transfer of tolerant splenic B cells did not induce it. These findings demonstrate that B-1 positive selection, resulting in the production of natural serum ATA, arises independently from the major pathway of BM B cell development and selection.

Zhou et al. demonstrate a requirement for the Let-7–Lin28b axis regulating a shift in development between fetal liver and bone marrow B lymphocyte progenitors in the generation of B1 versus B2 B cells. Specifically, the transcription factor Arid3a, induced by Lin28b and a target of Let-7 miRNA, is sufficient to recapitulate fetal B cell development from bone marrow progenitors., Mouse B cell precursors from fetal liver and adult bone marrow (BM) generate distinctive B cell progeny when transplanted into immunodeficient recipients, supporting a two-pathway model for B lymphopoiesis, fetal “B-1” and adult “B-2.” Recently, Lin28b was shown to be important for the switch between fetal and adult pathways; however, neither the mechanism of Lin28b action nor the importance of B cell antigen receptor (BCR) signaling in this process was addressed. Here, we report key advances in our understanding of the regulation of B-1/B-2 development. First, modulation of Let-7 in fetal pro-B cells is sufficient to alter fetal B-1 development to produce B cells resembling the progeny of adult B-2 development. Second, intact BCR signaling is required for the generation of B1a B cells from Lin28b-transduced BM progenitors, supporting a requirement for ligand-dependent selection, as is the case for normal B1a B cells. Third, the VH repertoire of Lin28b-induced BM B1a B cells differs from that of normal B1a, suggesting persisting differences from fetal progenitors. Finally, we identify the Arid3a transcription factor as a key target of Let-7, whose ectopic expression is sufficient to induce B-1 development in adult pro-B cells and whose silencing by knockdown blocks B-1 development in fetal pro-B cells.

Purpose: Associations between taxane-related sensory neuropathy (TRSN) and single-nucleotide polymorphisms (SNP) have previously been reported, but few have been replicated in large, independent validation studies. This study evaluates the association between previously investigated SNPs and TRSN, using genotype data from a study of chemotherapy-related toxicity in patients with breast cancer. Experimental Design: We investigated 73 SNPs in 50 genes for their contribution to TRSN risk, using genotype data from 1,303 European patients. TRSN was assessed using National Cancer Institute common toxicity criteria for adverse events classification. Unconditional logistic regression evaluated the association between each SNP and TRSN risk (primary analysis). Cox regression analysis assessed the association between each SNP and cumulative taxane dose causing the first reported moderate/severe TRSN (secondary analysis). The admixture likelihood (AML) test, which considers all SNPs with a prior probability of association with TRSN together, tested the hypothesis that certain SNPs are truly associated. Results: The AML test provided strong evidence for the association of some SNPs with TRSN (P = 0.023). The two most significantly associated SNPs were rs3213619(ABCB1) [OR = 0.47; 95% confidence interval (CI), 0.28–0.79; P = 0.004] and rs9501929(TUBB2A) (OR = 1.80; 95% CI, 1.20–2.72; P = 0.005). A further 9 SNPs were significant at P-value ≤ 0.05. Conclusion: This is currently the largest study investigating SNPs associated with TRSN. We found strong evidence that SNPs within genes in taxane pharmacokinetic and pharmacodynamic pathways contribute to TRSN risk. However, a large proportion of the inter-individual variability in TRSN remains unexplained. Further validated results from GWAS will help to identify new pathways, genes, and SNPs involved in TRSN susceptibility. Clin Cancer Res; 20(9); 2466–75. ©2014 AACR.

Although immature/transitional peripheral B cells may remain susceptible to selection pressures before full maturation, the nature and timing of these selection events remain unclear. We show that correlated expression of surface (s) IgM (sIgM), CD23, and AA4 defines three nonproliferative subpopulations of immature/transitional peripheral B cells. We designate these populations transitional (T) 1 (AA4+CD23−sIgMhigh), T2 (AA4+CD23+sIgMhigh), and T3 (AA4+CD23+sIgMlow). Cells within all three subsets are functionally immature as judged by their failure to proliferate following sIgM cross-linking in vitro, and their rapid rate of turnover in vivo as assessed by 5-bromo-2′-deoxyuridine labeling. These labeling studies also reveal measurable cell loss at both the T1-T2 and T2-T3 transitions, suggesting the existence of multiple selection points within the peripheral immature B cell pool. Furthermore, we find that Btk-deficient (xid) mice exhibit an incomplete developmental block at the T2-T3 transition within the immature B cell pool. This contrasts markedly with lyn−/− mice, which exhibit depressed numbers but normal ratios of each immature peripheral B cell subset and severely reduced numbers of mature B cells. Together, these data provide evidence for multiple selection points among immature peripheral B cells, suggesting that the B cell repertoire is shaped by multiple unique selection events that occur within the immature/transitional peripheral B cell pool.

CD1d-dependent accumulation of alphabeta T cells bearing a canonical Valpha14Jalpha281 alpha-chain (Valpha14+ T cells) is thought to model positive selection of lipid-specific T cells, based on their ability to recognize CD1d-presented self glycolipid(s). However, it has been difficult to demonstrate self ligand specificity in this system, as most Valpha14+ T cells do not exhibit significant autoreactivity despite high reactivity to alpha-galactosylceramide presented by CD1d (alpha-GalCer/CD1d). To assess the role of TCRbeta chain in determining the alpha-GalCer/CD1d vs autoreactive specificity of Valpha14+ T cells, we conducted TCRalpha or TCRbeta chain transduction experiments. In this study we demonstrate, by combining different TCRbeta chains with the Valpha14 alpha-chain in retrovirally transduced T cell lines, that the Valpha14 alpha-chain plays a primary role, necessary but not sufficient for imparting alpha-GalCer/CD1d recognition. beta-Chain usage alone is not the sole factor that controls the extent of autoreactivity in Valpha14+ T cells, since transduction of TCRalphabeta chains from a high CD1d autoreactive Valpha14+ T cell line conferred the alpha-GalCer/CD1d specificity without induction of autoreactivity. Thus, heterogeneity of Valpha14+ T cell reactivity is due to both beta-chain diversity and control mechanism(s) beyond primary TCR structure.

B cell development is a highly regulated process whereby functional peripheral subsets are produced from hematopoietic stem cells, in the fetal liver before birth and in the bone marrow afterward. Here we review progress in understanding some aspects of this process in the mouse bone marrow, focusing on delineation of the earliest stages of commitment, on pre-B cell receptor selection, and B cell tolerance during the immature-to-mature B cell transition. Then we note some of the distinctions in hematopoiesis and pre-B selection between fetal liver and adult bone marrow, drawing a connection from fetal development to B-1/CD5+B cells. Finally, focusing on CD5+cells, we consider the forces that influence the generation and maintenance of this distinctive peripheral B cell population, enriched for natural autoreactive specificities that are encoded by particular germline VH-VLcombinations.

Background In multivariate distributions (for example, in 3- or more color flow cytometric datasets), it can become difficult or impossible to identify populations that differ between samples based only on a combination of univariate or bivariate displays. Indeed, it is possible that such differences can only be identified in “n”-dimensional space, where “n” is the number of parameters measured. Therefore, computer assisted identification of such differences is necessary. Such a method could be used to identify responses (i.e., by comparing cell samples before and after stimulation) in exquisite detail by allowing complete analysis of the collected data on only those events which have responded. Methods Multivariate Probability Binning can be used to compare different datasets to identify the distance and statistical significance of a difference between the distributions. An intermediate step in the algorithm provides access to the actual locations within the n-dimensional comparison which are most different between the distributions. Gates based on collections of hyper-rectangular bins can then be applied to datasets, thereby selecting those events (or clusters of events) that are different between samples. We term this process Frequency Difference Gating. Results Frequency Difference Gating was used in several test scenarios to evaluate its utility. First, we compared PBMC subsets identified by solely by immunofluorescence staining: based on this training data set, the algorithm automatically generated an accurate forward and side-scatter gate to identify lymphocytes. Second, we applied the algorithm to identify subtle differences between CD4 memory subsets based on 8-color immunophenotyping data. The resulting 3-dimensional gate could resolve cells subsets much more frequent in one subset compared to the other; no combination of two-dimensional gates could accomplish this resolution. Finally, we used the algorithm to compare B cell populations derived from mice of different ages or strains, and found that the algorithm could find very subtle differences between the populations. Conclusion Frequency Difference Gating is a powerful tool that automates the process of identifying events comprising underlying differences between samples. It is not a clustering tool; it is not meant to identify subsets in multidimensional space. Importantly, this method may reveal subtle changes in small populations of cells, changes that only occur simultaneously in multiple dimensions in such a way that identification by univariate or bivariate analyses is impossible. Finally, the method may significantly aid in the analysis of high-order multivariate data (i.e., 6-12 color flow cytometric analyses), where identification of differences between datasets becomes so time-consuming as to be impractical. Cytometry 45:56–64, 2001. Published 2001 Wiley-Liss, Inc.

Here we review three areas in B-cell development in the mouse, with a focus on relevance to B-1/CD5+ B cells. Multiparameter flow cytometry has allowed the dissection of intermediate stages of developing B cells, both in fetal liver and bone marrow. In the first area, we present recent work that has delineated a fraction of pre-pro-B cells, committed to the B lineage, but lacking any immunoglobulin rearrangements. Next, the role of the pre-B-cell receptor in B-cell repertoire selection has become clear in the past few years, but we present work suggesting that the action of this process during fetal life is different, resulting in selection of a very distinct repertoire compared with adult. Finally, we describe a new VH3609 antithymocyte Ig transgenic mouse model system that has provided the first definitive evidence for the role of self-antigen in development and maintenance of natural autoreactive B cells.

Notch receptors regulate fate decisions in many cells. One outcome of Notch signaling is differentiation of bipotential precursors into one cell type versus another. To investigate consequences of Notch1 expression in hematolymphoid progenitors, mice were reconstituted with bone marrow (BM) transduced with retroviruses encoding a constitutively active form of Notch1. Although neither granulocyte or monocyte differentiation were appreciably affected, lymphopoiesis was dramatically altered. As early as 3 weeks following transplantation, mice receiving activated Notch1-transduced BM contained immature CD4+CD8+ T cells in the BM and exhibited a simultaneous block in early B cell lymphopoiesis. These results suggest that Notch1 provides a key regulatory signal in determining T lymphoid versus B lymphoid lineage decisions, possibly by influencing lineage commitment from a common lymphoid progenitor cell.

Lineage commitment in B lymphopoiesis remains poorly understood due to the inability to clearly define newly committed B lineage progenitors and their multipotential descendants. We examined the potential of three recently described progenitor populations in adult mouse bone marrow to differentiate into each hematopoietic lineage. The earliest of these, termed fraction (Fr.) A0, exhibited myeloid, erythroid, and B and T lymphoid progenitor activity and included individual cells with myeloid/B lymphoid potential. In sharp contrast, two later populations, termed Frs. A1 and A2 and characterized by surface B220 expression and transcription of the germline immunoglobulin heavy chain (IgH) locus, lacked progenitor activity for all hematopoietic lineages except B lymphocytes. These observations, together with single cell polymerase chain reaction analysis showing a lack of DHJH rearrangements in each population and experiments showing identical precursor potentials when these populations were derived from recombination activating gene (Rag)-1−/− and JH−/− mice, demonstrate that commitment to the B lymphoid lineage occurs before and independently of VHDHJH recombination.

c-Rel is a lymphoid-specific member of the NF-κB / Rel family of transcriptional factors. To investigate the role of c-Rel in B lymphocyte function, we generated a c-Rel(− / −) mouse via a gene targeting approach. Although early lymphocyte development is normal in c-Rel(− / −) mice, there are significantly fewer B cells displaying a memory (IgM / IgD−) phenotype. Upon immunization, c-Rel(− / −) mice generate fewer B cells with a germinal center (PNAhi) phenotype. In vitro, c-Rel(− / −) B cells proliferate poorly upon ligation of their surface IgM or CD40 receptors or when stimulated with either lipopolysaccharide (LPS) or T cell help. Early molecular events that precede proliferation, such as increases in RNA synthesis as well as IL-2 receptor α chain expression, are greatly diminished in c-Rel(− / −) B cells. Furthermore, c-Rel(− / −) B cells are impaired in the ability to receive survival signals generated by anti-IgM or LPS. In contrast, CD40-mediated cell survival is normal in c-Rel(− / −) B cells, suggesting the involvement of a survival-signaling pathway that is independent of c-Rel. When c-Rel (− / −) B cells are co-stimulated with either anti-IgM and CD40 or LPS and CD40, they are rendered capable of progressing through the cell cycle. Finally, co-culture experiments suggest that the defects observed in c-Rel(− / −) B cells are intrinsic to the cell and can not be rescued through either cell-cell contact or addition of soluble factors. Thus, c-Rel is requisite for differentiation to the germinal center and memory B cells in vivo and is required for the transduction of survival and cell cycle progression signals mediated by anti-IgM and LPS in vitro. Furthermore, while c-Rel is involved in CD40-induced proliferation, it is apparently dispensable for the survival signals transduced by CD40.

Before the clinical onset of B-precursor lymphoblastic leukemia, Eμ-ret mice have an expansion of late pro-B cells (CD45R+CD43+CD24+BP-1+) within the bone marrow. To characterize the early effects of the transgene product on lymphopoiesis, we initially sequenced the Ig heavy chain (IgH) rearrangements within the late pro-B cells in 24-day-old Eμ-ret and transgene negative mice. In both mouse populations, the IgH rearrangements were polyclonal, predominately nonproductive, and exhibited similar V, D, and J gene usage. However, the frequency of N regions, a marker of postnatal lymphopoiesis, was notably different. At the VD junction, N regions were found in 25 of 25 (100.0%) rearrangements from transgene-negative mice compared with 12 of 36 (33.3%) rearrangements from Eμ-ret mice. At the DJ junction, N regions were found in 21 of 25 (84.0%) rearrangements from transgene negative mice compared with 4 of 36 (11.1%) rearrangements from Eμ-ret mice. Subsequently, we sequenced the clonal IgH rearrangements from 9 leukemias that developed in 10-to 38-week-old mice and found that 7 leukemias had a least 1 rearrangement that lacked N regions at the DJ junction. In addition, V replacement events were observed in the 1 leukemia studied in detail. Terminal deoxynucleotidyl transferase, the enzyme responsible for N region addition, was expressed at markedly lower levels in late pro-B cells from 7- to 10-day-old Eμ-ret mice compared with transgene-negative mice. Examination of fetal lymphopoiesis in Eμ-ret mice identified a relative increase in early (CD45R+CD43+CD24+BP-1−) and late pro-B cells and a decrease in more differentiated CD43− B-lineage cells. Fetal early pro-B cells from Eμ-ret mice proliferated threefold to fivefold greater but differentiated to a lesser extent than those from transgene negative mice when cultured in vitro with interleukin-7. These data suggest that the B precursor leukemias in adult Eμ-ret mice arise from the progeny of pro-B cells generated in utero.