Loss of a chromosomal region with synteny to human 13q14 occurs in mouse chronic lymphocytic leukemia that originates from early-generated B-1 B cells.

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.

Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation.

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(-)sIgM(high)), T2 (AA4(+)CD23(+)sIgM(high)), and T3 (AA4(+)CD23(+)sIgM(low)). 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.

TCR beta chain influences but does not solely control autoreactivity of V alpha 14J281T cells.

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.

Probability binning comparison: a metric for quantitating multivariate distribution differences.

BACKGROUND: While several algorithms for the comparison of univariate distributions arising from flow cytometric analyses have been developed and studied for many years, algorithms for comparing multivariate distributions remain elusive. Such algorithms could be useful for comparing differences between samples based on several independent measurements, rather than differences based on any single measurement. It is conceivable that distributions could be completely distinct in multivariate space, but unresolvable in any combination of univariate histograms. Multivariate comparisons could also be useful for providing feedback about instrument stability, when only subtle changes in measurements are occurring. METHODS: We apply a variant of Probability Binning, described in the accompanying article, to multidimensional data. In this approach, hyper-rectangles of n dimensions (where n is the number of measurements being compared) comprise the bins used for the chi-squared statistic. These hyper-dimensional bins are constructed such that the control sample has the same number of events in each bin; the bins are then applied to the test samples for chi-squared calculations. RESULTS: Using a Monte-Carlo simulation, we determined the distribution of chi-squared values obtained by comparing sets of events from the same distribution; this distribution of chi-squared values was identical as for the univariate algorithm. Hence, the same formulae can be used to construct a metric, analogous to a t-score, that estimates the probability with which distributions are distinct. As for univariate comparisons, this metric scales with the difference between two distributions, and can be used to rank samples according to similarity to a control. We apply the algorithm to multivariate immunophenotyping data, and demonstrate that it can be used to discriminate distinct samples and to rank samples according to a biologically-meaningful difference. CONCLUSION: Probability binning, as shown here, provides a useful metric for determining the probability with which two or more multivariate distributions represent distinct sets of data. The metric can be used to identify the similarity or dissimilarity of samples. Finally, as demonstrated in the accompanying paper, the algorithm can be used to gate on events in one sample that are different from a control sample, even if those events cannot be distinguished on the basis of any combination of univariate or bivariate displays. Published 2001 Wiley-Liss, Inc.

B cell development pathways.

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 V(H)-V(L) combinations.

Response by B cell precursors to pre-B receptor assembly: differences between fetal liver and bone marrow.

The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a longstanding puzzle in immunology. In the past few years it has become clear that production of mu heavy chain and subsequent assembly with surrogate light chain to form the pre-B cell receptor complex is critical to promote development of adult B cell precursors in mouse bone marrow. Recently we found that instead of promoting pre-B cell expansion as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver, providing a previously unrecognized mechanism for alteration of the B cell repertoire with age. The consequence is very distinct primary repertoires for development of fetal B1 cells and adult bone marrow B2 cells.

B-cell commitment, development and selection.

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.

Development and function of B-1 cells.

Results from immunoglobulin-transgenic mice and BCR-mutant mice have been widely interpreted in recent years as supporting a simple 'activation' model for the origin of CD5+/B-1 B cells. However cell transfer experiments over 10 years ago and recent work investigating pre-BCR signaling suggest striking differences between B cell development in fetal liver and adult bone marrow, lending support for a 'lineage' model that we favor. Recent progress has been made relating to the development and function of the CD5+/B-1 B cell subpopulation in mice; the data can be viewed in the context of the generation of this subpopulation by a distinctive fetal B cell developmental process.

A VH11V kappa 9 B cell antigen receptor drives generation of CD5+ B cells both in vivo and in vitro.

B lymphocytes can be divided into different subpopulations, some with distinctive activation requirements and probably mediating specialized functions, based on surface phenotype and/or anatomical location, but the origins of most of these populations remain poorly understood. B cells constrained by transgenesis to produce an Ag receptor derived from a conventional (B-2) type cell develop a B-2 phenotype, whereas cells from mice carrying a B-1-derived receptor acquire the B-1 phenotype. In this study transgenic enforced expression of a B cell receptor (mu/kappa) originally isolated from a CD5+ (B-1a) B cell generates B-1 phenotype cells in bone marrow cultures that show a distinctive B-1 function, survival in culture. Despite their autoreactivity, we find no evidence for receptor editing or that the paucity of B-2 cells is the result of tolerance-induced selection. Finally, Ca2+ mobilization studies reveal a difference between transgenic B-1 cells in spleen and peritoneal cavity, with cells in spleen much more responsive to anti-B cell receptor cross-linking. We discuss these results in terms of specificity vs lineage models for generation of distinctive B cell subpopulations.

Developmental switches in chemokine response profiles during B cell differentiation and maturation.

Developing B cells undergo dramatic changes in their responses to chemoattractant cytokines (chemokines) and in expression of chemokine receptors. Bone marrow pre-pro-B cells (AA4.1(+)/natural killer 1.1(-) Fraction A cells) and cells capable of generating pro-B colonies in the presence of interleukin 7 and flt3 ligand migrate to thymus-expressed chemokine (TECK), a response lost in later stages of B cell development. B cell-attracting chemokine 1 (BCA-1) responses correlate with CXC chemokine receptor (CXCR)5 expression, are first displayed by a pro-B cell subset, are lost in pre-B cells, and then are regained just before and after egress from the marrow. All peripheral B cell subsets, including follicular and germinal center as well as marginal zone and peritoneal B1 B cells, respond to BCA-1, implying that responsiveness to this follicular chemokine is not sufficient to predict follicle localization. Responses to the CC chemokine receptor (CCR)7 ligands secondary lymphoid tissue chemoattractant (SLC) and macrophage inflammatory protein (MIP)-3beta, implicated in homing to lymphoid tissues, are upregulated before B cell exit from the marrow, but increase further in the periphery and are shared by all peripheral B cells. In contrast, responsiveness to MIP-3alpha and expression of CCR6 are acquired only after emigration to the periphery and during maturation into the recirculating B cell pool. Chemotaxis to stromal cell-derived factor 1alpha is observed at all stages of B cell differentiation. Thus, unique patterns of chemokine responses may help define developing B cell populations and direct their maturation in the marrow and migration to the periphery.

Functional consequences of the developmental arrest and follicular exclusion of anti-double-stranded DNA B cells.

Anti-dsDNA B cells are actively tolerized in nonautoimmune BALB/c mice, as manifested by their developmental arrest, follicular exclusion, and rapid turnover rate. Previously, we have documented changes in the maturation status and follicular localization of anti-dsDNA B cells in autoimmune-prone MRL (+/+ and lpr/lpr) mice. To determine whether these differences in developmental status and follicular localization affect the functional capacity of anti-dsDNA B cells, we have now compared their in vivo life spans and their responses to in vitro stimuli. Our study shows that although anti-dsDNA B cells from both BALB/c and MRL-+/+ mice are localized to the T/B interface, only those in BALB/c mice have a rapid turnover rate. Therefore, the immature status and not the exclusion from the B cell follicle correlates with a shortened life span. Interestingly, apoptotic anti-dsDNA B cells were not detected at the T/B interface in BALB/c mice, suggesting that they are not dying there. This study also demonstrates that anti-dsDNA B cells, regardless of maturation status or follicular localization, are able to proliferate and up-regulate the costimulatory molecule B7-2 in response to CD40 ligand and IL-4. Therefore, one of the critical in vivo differences between anti-dsDNA B cells in BALB/c and MRL-+/+ mice compared with MRL-lpr/lpr mice may be the availability of T cell help.

Notch1 expression in early lymphopoiesis influences B versus T lineage determination.

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.

Positive selection of natural autoreactive B cells.

Lymphocyte development is critically influenced by self-antigens. T cells are subject to both positive and negative selection, depending on their degree of self-reactivity. Although B cells are subject to negative selection, it has been difficult to test whether self-antigen plays any positive role in B cell development. A murine model system of naturally generated autoreactive B cells with a germ line gene-encoded specificity for the Thy-1 (CD90) glycoprotein was developed, in which the presence of self-antigen promotes B cell accumulation and serum autoantibody secretion. Thus, B cells can be subject to positive selection, generated, and maintained on the basis of their autoreactivity.

Commitment to the B lymphoid lineage occurs before DH-JH recombination.

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 essential for B lymphocyte survival and cell cycle progression.

c-Rel is a lymphoid-specific member of the NF-kappaB/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 alpha 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.

The fetal origin of B-precursor leukemia in the E-mu-ret mouse.

Before the clinical onset of B-precursor lymphoblastic leukemia, E-mu-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 E-mu-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 Emicro-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 E-mu-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 E-mu-ret mice compared with transgene-negative mice. Examination of fetal lymphopoiesis in E-mu-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 Emicro-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 E-mu-ret mice arise from the progeny of pro-B cells generated in utero.

HD mice: a novel mouse mutant with a specific defect in the generation of CD4(+) T cells.

We have identified a spontaneous mutation in mice, which we term HD for "helper T cell deficient." This mouse is distinguished by the virtual absence of peripheral T cells of the CD4(+)8(-) major histocompatibility complex (MHC) class II-restricted T helper subset due to a specific block in thymic development. The developmental defect is selective for CD4(+)8(-) cells; the maturation of CD4(-)8(+) and gamma delta T cells is normal. The autosomal recessive mutation underlying the HD phenotype is unrelated to MHC class II, since it segregates independently of the MHC class II locus. Moreover, the HD phenotype is not caused by a defect of the CD4 gene. Bone marrow transfer experiments demonstrate that the defect is intrinsic to cells of the hematopoietic lineage, i.e., most likely to developing thymocytes themselves. The frequency of CD4(+)8(low) intermediate cells is markedly increased in HD mice, suggesting that class II-restricted thymocytes are arrested at this stage. This is the first genetic defect of its kind to be described in the mouse and may prove highly informative in understanding the molecular pathways underlying lineage commitment.

The evolution of B precursor leukemia in the Emu-ret mouse.

Emu-ret mice carrying an RFP/RET fusion gene under the transcriptional control of the immunoglobulin heavy chain enhancer develop B lineage leukemias/lymphomas. We have characterized B-cell development in these mice before the onset of clinical disease to determine the steps involved in leukemogenesis. Flow cytometry reveals that the CD45R+CD43(+)CD24(+)BP-1(+) late pro-B-cell population is markedly expanded in the bone marrow of 3- to 5-week-old Emu-ret mice. Compared with late pro-B cells from transgene-negative mice, Emu-ret late pro-B cells have a limited capacity to differentiate in interleukin (IL)-7 and a higher incidence of VDJ rearrangements, but a similar cell cycle profile. In contrast, CD45R+CD43(+)CD24(+)BP-1(-) early pro-B cells from 3- to 5-week-old Emu-ret mice, which also express the RFP/RET transgene, differentiate in IL-7 similarly to their normal counterparts. Furthermore, early pro-B cells from Emu-ret and transgene-negative mice have an identical pattern of growth inhibition when exposed to interferons (IFNs)-alpha/beta and -gamma, whereas, pro-B-cell leukemia lines derived from Emu-ret mice are markedly less sensitive to growth inhibition by these IFNs. In 13-week-old well-appearing Emu-ret mice, late pro-B cells upregulate CYCLIN D1 expression and downregulate CASPASE-1 expression in a pattern that correlates with the emergence of B precursor cells in the peripheral blood and the loss of other B lineage subsets in the bone marrow. Taken together, these results suggest that the expression of the RFP/RET transgene initially prevents the normal elimination of late pro-B cells with nonproductive rearrangements. Secondary events that simultaneously disturb the normal transcriptional regulation of genes involved in the control of the cell cycle and apoptosis may allow for subsequent malignant transformation within the expanded late pro-B-cell population.

A novel mechanism for B cell repertoire maturation based on response by B cell precursors to pre-B receptor assembly.

The expression of different sets of immunoglobulin specificities by fetal and adult B lymphocytes is a long-standing puzzle in immunology. Recently it has become clear that production of immunoglobulin mu heavy chain and subsequent assembly with a surrogate light chain to form the pre-B cell receptor complex is critical for development of B cells. Here we show that instead of promoting pre-B cell progression as in adult bone marrow, this complex inhibits pre-B cell growth in fetal liver. Curiously, we identify a fetal-associated VH11 mu heavy chain that allows continued pre-B proliferation in fetal liver. Interestingly, this heavy chain does not associate efficiently with a surrogate light chain, providing a previously unrecognized mechanism for skewing the expression of distinctive VH genes toward fetal through early neonatal life.