Crucial Role of Increased Arid3a at the Pre-B and Immature B Cell Stages for B1a Cell Generation.

The Lin28b+Let7- axis in fetal/neonatal development plays a role in promoting CD5+ B1a cell generation as a B-1 B cell developmental outcome. Here we identify the Let7 target, Arid3a, as a crucial molecular effector of the B-1 cell developmental program. Arid3a expression is increased at pro-B cell stage and markedly increased at pre-B and immature B cell stages in the fetal/neonatal liver B-1 development relative to that in the Lin28b-Let7+ adult bone marrow (BM) B-2 cell development. Analysis of B-lineage restricted Lin28b transgenic (Tg) mice, Arid3a knockout and Arid3a Tg mice, confirmed that increased Arid3a allows B cell generation without requiring surrogate light chain (SLC) associated pre-BCR stage, and prevents MHC class II cell expression at the pre-B and newly generated immature B cell stages, distinct from pre-BCR dependent B development with MHC class II in adult BM. Moreover, Arid3a plays a crucial role in supporting B1a cell generation. The increased Arid3a leads higher Myc and Bhlhe41, and lower Siglec-G and CD72 at the pre-B and immature B cell stages than normal adult BM, to allow BCR signaling induced B1a cell generation. Arid3a-deficiency selectively blocks the development of B1a cells, while having no detectable effect on CD5- B1b, MZ B, and FO B cell generation resembling B-2 development outcome. Conversely, enforced expression of Arid3a by transgene is sufficient to promote the development of B1a cells from adult BM. Under the environment change between birth to adult, altered BCR repertoire in increased B1a cells occurred generated from adult BM. However, crossed with B1a-restricted VH/D/J IgH knock-in mice allowed to confirm that SLC-unassociated B1a cell increase and CLL/lymphoma generation can occur in aged from Arid3a increased adult BM. These results confirmed that in fetal/neonatal normal mice, increased Arid3a at the pre-B cell and immature B cell stages is crucial for generating B1a cells together with the environment for self-ligand reactive BCR selection, B1a cell maintenance, and potential for development of CLL/Lymphoma in aged mice.

Early Generated B-1-Derived B Cells Have the Capacity To Progress To Become Mantle Cell Lymphoma-like Neoplasia in Aged Mice.

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.

NLR Nod1 signaling promotes survival of BCR-engaged mature B cells through up-regulated Nod1 as a positive outcome.

Although B cell development requires expression of the B cell antigen receptor (BCR), it remains unclear whether engagement of self-antigen provides a positive impact for most B cells. Here, we show that BCR engagement by self-ligand during development in vivo results in up-regulation of the Nod-like receptor member Nod1, which recognizes the products of intestinal commensal bacteria. In anti-thymocyte/Thy-1 autoreactive BCR knock-in mice lacking self-Thy-1 ligand, immunoglobulin light chain editing occurred, generating B cells with up-regulated Nod1, including follicular and marginal zone B cells with natural autoreactivity. This BCR editing with increased Nod1 resulted in preferential survival. In normal adult mice, most mature B cells are enriched for Nod1 up-regulated cells, and signaling through Nod1 promotes competitive survival of mature B cells. These findings demonstrate a role for microbial products in promoting survival of mature B cells through up-regulated Nod1, providing a positive effect of BCR engagement on development of most B cells.

Zebrafish B Cell Development without a Pre-B Cell Stage, Revealed by CD79 Fluorescence Reporter Transgenes.

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.

Early generated B1 B cells with restricted BCRs become chronic lymphocytic leukemia with continued c-Myc and low Bmf expression.

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.

Selection of natural autoreactive B cells.

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.

Perspectives on fetal derived CD5+ B1 B cells.

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.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

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.

A developmental switch between fetal and adult B lymphopoiesis.

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 by B-1 development can progress to chronic lymphocytic leukemia.

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.

Lin28b promotes fetal B lymphopoiesis through the transcription factor Arid3a.

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.

Natural anti-intestinal goblet cell autoantibody production from marginal zone B cells.

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.

A role for IRF4 in the development of CLL.

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.

The transcriptional landscape of αß T cell differentiation.

The differentiation of αßT cells from thymic precursors is a complex process essential for adaptive immunity. Here we exploited the breadth of expression data sets from the Immunological Genome Project to analyze how the differentiation of thymic precursors gives rise to mature T cell transcriptomes. We found that early T cell commitment was driven by unexpectedly gradual changes. In contrast, transit through the CD4(+)CD8(+) stage involved a global shutdown of housekeeping genes that is rare among cells of the immune system and correlated tightly with expression of the transcription factor c-Myc. Selection driven by major histocompatibility complex (MHC) molecules promoted a large-scale transcriptional reactivation. We identified distinct signatures that marked cells destined for positive selection versus apoptotic deletion. Differences in the expression of unexpectedly few genes accompanied commitment to the CD4(+) or CD8(+) lineage, a similarity that carried through to peripheral T cells and their activation, demonstrated by mass cytometry phosphoproteomics. The transcripts newly identified as encoding candidate mediators of key transitions help define the 'known unknowns' of thymocyte differentiation.

Identification of transcriptional regulators in the mouse immune system.

The differentiation of hematopoietic stem cells into cells of the immune system has been studied extensively in mammals, but the transcriptional circuitry that controls it is still only partially understood. Here, the Immunological Genome Project gene-expression profiles across mouse immune lineages allowed us to systematically analyze these circuits. To analyze this data set we developed Ontogenet, an algorithm for reconstructing lineage-specific regulation from gene-expression profiles across lineages. Using Ontogenet, we found differentiation stage-specific regulators of mouse hematopoiesis and identified many known hematopoietic regulators and 175 previously unknown candidate regulators, as well as their target genes and the cell types in which they act. Among the previously unknown regulators, we emphasize the role of ETV5 in the differentiation of γδ T cells. As the transcriptional programs of human and mouse cells are highly conserved, it is likely that many lessons learned from the mouse model apply to humans.

Positive and negative selection of natural autoreactive B cells.

Naturally occurring antibodies (NAbs) produced by CD5(+) B-1 B cells include those with specificity for thymocytes (anti-thymocyte autoantibody, ATA). Here we describe a prototypic example, encoded by an unmutated immunoglobulin µ/κ heavy chain/light chain. Studies with ATA-µ ("heavy chain only") 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 NAb. Furthermore, analysis of B-cell development in ATA-µκ ("heavy and light chain") transgenic mice revealed two distinct responses by B cells to expression of this B-cell receptor (BCR). (1) Most B cells developing from bone marrow of adult mice were blocked at an immature stage in spleen and only escaped apoptosis by editing their BCR to eliminate the ATA specificity. (2) Some B cells differentiated to antibody-forming cells without altering their specificity, produced high levels of serum ATA, and many ATA-secreting plasma cells were observed in spleen. Finally, examination of B-cell development and ATA NAb production in ATA-µκ transgenic mice with levels of Thy-1 autoantigen varying from very low to above physiologic reveals a clear relationship between BCR crosslinking by antigen and B-cell fate. Low levels of Thy-1 autoantigen resulted in diversion of ATA B cells into the marginal zone B-cell compartment, presumably because of reduced BCR signaling. Thus, our studies demonstrate a key positive selection step in the development of NAb-producing B cells and show that most of these cells in adult mice bearing such specificities fail to reach a mature stage. Importantly, because these specificities are isolated from B-1 B cells and, when expressed as transgenes, guide development into the B-1 or marginal zone B-cell pool, we identify these B cells as a major source of natural autoantibodies in mice.

Non-cell-autonomous hedgehog signaling promotes murine B lymphopoiesis from hematopoietic progenitors.

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.

Transcriptomes of the B and T lineages compared by multiplatform microarray profiling.

T and B lymphocytes are developmentally and functionally related cells of the immune system, representing the two major branches of adaptive immunity. Although originating from a common precursor, they play very different roles: T cells contribute to and drive cell-mediated immunity, whereas B cells secrete Abs. Because of their functional importance and well-characterized differentiation pathways, T and B lymphocytes are ideal cell types with which to understand how functional differences are encoded at the transcriptional level. Although there has been a great deal of interest in defining regulatory factors that distinguish T and B cells, a truly genomewide view of the transcriptional differences between these two cells types has not yet been taken. To obtain a more global perspective of the transcriptional differences underlying T and B cells, we exploited the statistical power of combinatorial profiling on different microarray platforms, and the breadth of the Immunological Genome Project gene expression database, to generate robust differential signatures. We find that differential expression in T and B cells is pervasive, with the majority of transcripts showing statistically significant differences. These distinguishing characteristics are acquired gradually, through all stages of B and T differentiation. In contrast, very few T versus B signature genes are uniquely expressed in these lineages, but are shared throughout immune cells.