Tonic B cell antigen receptor signals supply an NF-kappaB substrate for prosurvival BLyS signaling.

The survival of transitional and mature B cells requires both the B cell antigen receptor (BCR) and BLyS receptor 3 (BR3), which suggests that these receptors send signals that are nonredundant or that engage in crosstalk with each other. Here we show that BCR signaling induced production of the nonclassical transcription factor NF-kappaB pathway substrate p100, which is required for transmission of BR3 signals and thus B cell survival. The capacity for sustained p100 production emerged during transitional B cell differentiation, the stage at which BCR signals begin to mediate survival rather than negative selection. Our findings identify a molecular mechanism for the reliance of primary B cells on continuous BR3 and BCR signaling, as well as for the gradual resistance to negative selection that is acquired during B cell maturation.

BLyS inhibition eliminates primary B cells but leaves natural and acquired humoral immunity intact.

We have used an inhibiting antibody to determine whether preimmune versus antigen-experienced B cells differ in their requisites for BLyS, a cytokine that controls differentiation and survival. Whereas in vivo BLyS inhibition profoundly reduced naïve B cell numbers and primary immune responses, it had a markedly smaller effect on memory B cells and long-lived plasma cells, as well as secondary immune responses. There was heterogeneity within the memory pools, because IgM-bearing memory cells were sensitive to BLyS depletion whereas IgG-bearing memory cells were not, although both were more resistant than naïve cells. There was also heterogeneity within B1 pools, as splenic but not peritoneal B1 cells were diminished by anti-BLyS treatment, yet the number of natural antibody-secreting cells remained constant. Together, these findings show that memory B cells and natural antibody-secreting cells are BLyS-independent and suggest that these pools can be separately manipulated.

B cells and aging: balancing the homeostatic equation.

The interplay of selective and homeostatic processes dominates the behavior of B lineage subsets following B cell antigen receptor (BCR) expression, and extends to determinants of immune response quality and the persistence of immunologic memory. A key concept emerging from these considerations is that primary events acting upstream of mature B lymphocyte pools can profoundly impact downstream populations as the system attempts homeostatic adjustments. Since, advancing age is accompanied by profound changes in B cell generation and homeostasis, establishing the relative contributions of primary lesions versus compensatory homeostatic processes is critical to understanding these perturbations. Exploration of this problem requires an understanding of: (1) the identity, dynamics, and progenitor/successor relationships of marrow and peripheral B cell subsets; (2) the nature and interactions of selective and homeostatic processes acting in these subsets; (3) how these change with age. Our data show that BLyS and its receptors mediate peripheral B cell homeostasis, and that the size, dynamics and behavior of all B cell subsets influenced by B Lymphocyte Stimulator change with age. These findings suggest that homeostatic processes mediated through B Lymphocyte Stimulator are altered with age, and that these perturbations may primarily reflect compensatory homeostatic adjustments to upstream reductions in B cell generation.

Space, selection, and surveillance: setting boundaries with BLyS.

The BLyS family of ligands and receptors governs B cell homeostasis by controlling survival, differentiation, and lifespan. This family consists of multiple receptors and ligands, allowing independent regulation of different B cell subsets by varying the combination and levels of receptors expressed. Multiple downstream signaling pathways are implicated in these activities, reflecting this receptor complexity as well as cross-talk with other B cell signaling systems. BLyS levels are associated with multiple forms of humoral autoimmunity and can modulate tolerogenic elimination at the transitional checkpoint. BLyS responsiveness thus balances peripheral selection against cell numbers, providing an elastic system that varies selective stringency based on homeostatic demands.

Mouse plasmacytoid dendritic cells derive exclusively from estrogen-resistant myeloid progenitors.

Current models predict that mouse plasmacytoid dendritic cells (PDCs) derive from lymphoid progenitors. However, we show PDCs arise exclusively from common myeloid progenitors (CMPs) characterized by low-level expression of several lymphoid-associated genes, including a RAG2/GFP reporter transgene. This conclusion is supported by both adoptive transfer experiments and an estrogen treatment strategy that led to marked depletion of very early lymphoid progenitors without affecting RAG2/GFP(+) CMPs or the developmental kinetics, RAG-mediated recombinase activity, and cytokine production of PDCs. These data suggest that PDCs arise exclusively from early myeloid progenitors and that promiscuous low-level expression of lymphoid-associated genes is a general feature of PDC progenitors among CMPs.

B cell development and receptor diversity during aging.

Although it is clear that B cell genesis declines with age, the specifics of why this happens are largely unknown. Even less clear is how the age-related decline in B cell development might affect peripheral B cell function. Recent studies have investigated the impact of aging on both B cell genesis in the bone marrow and the resulting peripheral B cell repertoire. On the basis of these studies we propose a model in which the aging of very early B cell progenitors results in shifts in the peripheral B cell repertoire and, consequently, changes in mature B cell function.

Linking age-related defects in B lymphopoiesis to the aging of hematopoietic stem cells.

B cell genesis declines with age, but at what stage and why remains unclear. Previous studies attribute the decline in B cell production in aged mice to both environmental and cell-intrinsic defects that impact mid-to-late stream B cell precursors. However, mounting evidence suggests that the aging process may also negatively affect the earliest phases of B cell development. We review past studies on the B cells and aging question, discuss recent data suggesting that age-associated defects in B cell development reflect deficiencies in hematopoietic stem cell-proximal progenitor pools, and provide an integrative model that will hopefully facilitate further studies into this complex problem.

The aging of early B-cell precursors.

B-cell genesis in the bone marrow declines with advancing age. In this review, we discuss our current understanding of why B-cell production rates decline with age with a special emphasis on why age-related factors might target very early lymphoid precursors. We consider the impact of aging on cytokine responsiveness and how current models for lineage relationships for very early B- and T-cell precursors might influence interpretations of experiments addressing age-associated declines in B- and T-cell differentiation. This discussion centers on the notion that aging affects events associated with the process by which hematopoietic stem cells are guided toward the B-cell pathway. Finally, we present a model in which the age-associated loss of early B-cell precursors is linked to suboptimal function of key transcriptional regulators of very early B-cell development.

B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors.

Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in approximately 5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.

Mice deficient for testis-brain RNA-binding protein exhibit a coordinate loss of TRAX, reduced fertility, altered gene expression in the brain, and behavioral changes.

Testis-brain RNA-binding protein (TB-RBP), the mouse orthologue of the human protein Translin, is a widely expressed and highly conserved protein with proposed functions in chromosomal translocations, mitotic cell division, and mRNA transport and storage. To better define the biological roles of TB-RBP, we generated mice lacking TB-RBP. Matings between heterozygotes gave rise to viable, apparently normal homozygous mutant mice at a normal Mendelian ratio. The TB-RBP-related and -interacting protein Translin-associated factor X was reduced to 50% normal levels in heterozygotes and was absent in TB-RBP-null animals. The null mice were 10 to 30% smaller than their wild-type or heterozygote littermates at birth and remained so to about 6 to 9 months of age, showed normal B- and T-cell development, and accumulated visceral fat. TB-RBP-null male mice were fertile and sired offspring but had abnormal seminiferous tubules and reduced sperm counts. Null female mice were subfertile and had reduced litter sizes. Microarray analysis of total brain RNA from null and wild-type mice revealed an altered gene expression profile with the up-regulation of 14 genes and the down-regulation of 217 genes out of 12,473 probe sets. Numerous neurotransmitter receptors and ion channels, including gamma-aminobutyric acid A receptor alpha1 and glutamate receptor alpha3, were strongly down-regulated. Behavioral abnormalities were also seen. Compared to littermates, the TB-RBP-null mice appeared docile and exhibited reduced Rota-Rod performance.

The decline in B lymphopoiesis in aged mice reflects loss of very early B-lineage precursors.

The primary age-related loss in B cell progenitors is thought to be at the pro- to pre-B cell transition. However, we show that the frequencies and absolute numbers of all progenitor populations for the B cell lineage, including B-lineage-committed pro-B cells and multipotent B-lymphoid progenitors, decline in aged C57BL/6 mice. Moreover, when derived from aged mice, lymphoid progenitors within every population examined exhibited suboptimal IL-7 responsiveness, demonstrating that age-associated suboptimal IL-7R signaling is a general property of all early B-lineage precursors. Collectively, these data indicate that aging results in a previously unappreciated decline in the earliest stages of B cell development.

Common lymphoid progenitors, early B-lineage precursors, and IL-7: characterizing the trophic and instructive signals underlying early B cell development.

Precursors for B lymphocytes develop from semirestricted lymphoid progenitors in the bone marrow. Here we review current knowledge on the cellular stages underlying early B cell development from multipotent progenitor cells, and discuss the factors implicated in the regulation of this process. In particular, we will focus on the role of cytokine receptor signaling in early lymphocyte ontogeny and lymphoid lineage commitment, with an emphasis on the role of interleukin- 7 (IL-7) in early lymphocyte development within the bone marrow microenvironment. We will also discuss recent evidence that lymphocytes and subsets of dendritic cells develop from a common pathway, speculating that IL-7 may regulate cell fate decisions in multipotent B/dendritic cell precursors by driving these cells to differentiate into B-lineage-committed cells.

Thymopoiesis independent of common lymphoid progenitors.

Early T lineage progenitors (ETPs) in the thymus are thought to develop from common lymphoid progenitors (CLPs) in the bone marrow (BM). We compared thymic ETPs to BM CLPs in mice and found that they differed in several respects. Thymic ETPs were not interleukin 7 (IL-7)-responsive and generated B lineage progeny with delayed kinetics, whereas BM CLPs were IL-7-responsive and rapidly generated B cells. ETPs sustained production of T lineage progeny for longer periods of time than BM CLPs. Analysis of Ikaros-deficient mice that exhibit ongoing thymopoiesis without B lymphopoeisis revealed near-normal frequencies of thymic ETPs, yet undetectable numbers of BM CLPs. We conclude that ETPs can develop via a CLP-independent pathway.

The biology of chronic myelogenous leukemia:mouse models and cell adhesion.

Chronic myelogenous leukemia (CML) is a biphasic neoplasm of the bone marrow that is precipitated by the Philadelphia chromosome, a t(9;22) balanced translocation that encodes a constitutively activated nonreceptor tyrosine kinase termed P210(BCR-ABL). This oncoprotein has several intracellular functions; however, the most important effect of P210(BCR-ABL) leading to cell transformation is phosphorylation of signaling molecules through a constitutively active tyrosine kinase domain. Despite extensive knowledge of the structure and functional domains of BCR-ABL, its precise function in transformation is not known. Progress has been hampered, in part, by the lack of relevant CML models, as cell culture and in vitro assays do not mimic the pathogenesis of CML. Recently, there has been significant progress toward improving murine models that closely resemble human CML. This has allowed researchers to evaluate critical functions of BCR-ABL and has provided a model to test the efficacy of therapeutic medications that block these pathways. Our laboratory has developed two intersecting research programs to better understand the functioning of P210(BCR-ABL) in leukemogenesis. In one approach, we have developed a murine CML model by transferring HSCs that express BCR-ABL from a retroviral vector. All recipients develop a rapidly fatal MPD that shares several important features with CML. This model has been extremely useful for studying the function of BCR-ABL in the pathogenesis of CML. A second approach utilizes a quantitative cell detachment apparatus capable of measuring small changes in cell adhesion to investigate the mechanism by which P210(BCR-ABL) causes abnormal cell binding. Altered cell adhesion may contribute to the imbalance between proliferation and self-renewal in the hematopoietic progenitor compartment. To better understand the role abnormal adhesion may play in the development of leukemia, we have attempted to correlate the effects of functional P210(BCR-ABL) mutants in regulating adhesion and oncogenicity.

The earliest step in B lineage differentiation from common lymphoid progenitors is critically dependent upon interleukin 7.

Little is known about the signals that promote early B lineage differentiation from common lymphoid progenitors (CLPs). Using a stromal-free culture system, we show that interleukin (IL)-7 is sufficient to promote the in vitro differentiation of CLPs into B220(+) CD19(+) B lineage progenitors. Consistent with current models of early B cell development, surface expression of B220 was initiated before CD19 and was accompanied by the loss of T lineage potential. To address whether IL-7 receptor (R) activity is essential for early B lineage development in vivo, we examined the frequencies of CLPs and downstream pre-pro- and pro-B cells in adult mice lacking either the alpha chain or the common gamma chain (gamma(c)) of the IL-7R. The data indicate that although gamma(c)(-/-) mice have normal frequencies of CLPs, both gamma(c)(-/-) and IL-7R(alpha)(-/-) mice lack detectable numbers of all downstream early B lineage precursors, including pre-pro-B cells. These findings challenge previous notions regarding the point in B cell development affected by the loss of IL-7R signaling and suggest that IL-7 plays a key and requisite role during the earliest phases of B cell development.

The coiled-coil domain and Tyr177 of bcr are required to induce a murine chronic myelogenous leukemia-like disease by bcr/abl.

The bcr/abl fusion in chronic myelogenous leukemia (CML) creates a chimeric tyrosine kinase with dramatically different properties than intact c-abl. In P210 bcr/abl, the bcr portion includes a coiled-coil oligomerization domain (amino acids 1-63) and a grb2-binding site at tyrosine 177 (Tyr177) that are critical for fibroblast transformation, but give variable results in other cell lines. To investigate the role of the coiled-coil domain and Tyr177 in promoting CML, 4 P210 bcr/abl-derived mutants containing different bcr domains fused to abl were constructed. All 4 mutants, Delta(1-63) bcr/abl, (1-63) bcr/abl, Tyr177Phe bcr/abl, and (1-210) bcr/abl exhibited elevated tyrosine kinase activity and conferred factor-independent growth in cell lines. In contrast, differences in the transforming potential of the 4 mutants occurred in our mouse model, in which all mice receiving P210 bcr/abl-expressing bone marrow cells exclusively develop a myeloproliferative disease (MPD) resembling human CML. Of the 4 mutants assayed, only 1-210 bcr/abl, containing both the coiled-coil domain and Tyr177, induced MPD. Unlike full-length P210, this mutant also caused a simultaneous B-cell acute lymphocytic leukemia (ALL). The other 3 mutants, (1-63) bcr/abl, Tyr177Phe bcr/abl, and Delta(1-63) bcr/abl, failed to induce an MPD but instead caused T-cell ALL. These results show that both the bcr coiled-coil domain and Tyr177 are required for MPD induction by bcr/abl and provide the basis for investigating downstream signaling pathways that lead to CML.