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 μ 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 V_H11 μ 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 V_H genes toward fetal through early neonatal life.     

Multiple, conserved cryptic recombination signals in VH gene segments: detection of cleavage products only in pro B cells

Receptor editing is believed to play the major role in purging newly formed B cell compartments of autoreactivity by the induction of secondary V(D)J rearrangements. In the process of immunoglobulin heavy (H) chain editing, these secondary rearrangements are mediated by direct V_H-to-J_H joining or cryptic recombination signals (cRSs) within V_H gene segments. Using a statistical model of RS, we have identified potential cRSs within V_H gene segments at conserved sites flanking complementarity-determining regions 1 and 2. These cRSs are active in extrachromosomal recombination assays and cleaved during normal B cell development. Cleavage of multiple V_H cRSs was observed in the bone marrow of C57BL/6 and RAG2:GFP and μMT congenic animals, and we determined that cRS cleavage efficiencies are 30–50-fold lower than a physiological RS. cRS signal ends are abundant in pro–B cells, including those recovered from μMT mice, but undetectable in pre– or immature B cells. Thus, V_H cRS cleavage regularly occurs before the generation of functional preBCR and BCR. Conservation of cRSs distal from the 3′ end of V_H gene segments suggests a function for these cryptic signals other than V_H gene replacement.     

Early Function of Pax5 (BSAP) before the Pre-B Cell Receptor Stage of B Lymphopoiesis

The formation of the pre-B cell receptor (BCR) corresponds to an important checkpoint in B cell development that selects pro-B (pre-BI) cells expressing a functionally rearranged immunoglobulin μ (Igμ) heavy chain protein to undergo the transition to the pre-B (pre-BII) cell stage. The pre-BCR contains, in addition to Igμ, the surrogate light chains λ5 and VpreB and the signal transducing proteins Igα and Igβ. The absence of one of these pre-BCR components is known to arrest B cell development at the pre-BI cell stage. Disruption of the Pax5 gene, which codes for the B cell–specific activator protein (BSAP), also blocks adult B lymphopoiesis at the pre-BI cell stage. Moreover, expression of the mb-1 (Igα) gene and V_H-to-D_HJ_H recombination at the IgH locus are reduced in Pax5-deficient B lymphocytes ∼10- and ∼50-fold, respectively. Here we demonstrate that complementation of these deficiencies in pre-BCR components by expression of functionally rearranged Igμ and chimeric Igμ-Igβ transgenes fails to advance B cell development to the pre-BII cell stage in Pax5 (−/−) mice in contrast to RAG2 (−/−) mice. Furthermore, the pre-BCR is stably expressed on cultured pre-BI cells from Igμ transgenic, Pax5-deficient bone marrow, but is unable to elicit its normal signaling responses. In addition, the early developmental block is unlikely to be caused by the absence of a survival signal, as it could not be rescued by expression of a bcl2 transgene in Pax5-deficient pre-BI cells. Together, these data demonstrate that the absence of Pax5 arrests adult B lymphopoiesis at an early developmental stage that is unresponsive to pre-BCR signaling.     

Ordering of Human Bone Marrow B Lymphocyte Precursors by Single-Cell Polymerase Chain Reaction Analyses of the Rearrangement Status of the Immunoglobulin H and L Chain Gene Loci

CD19⁺CD10⁺ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, V_preB, recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for D_HJ_H and V_κJ_κ, V_κJ_κK^(de) and V_κK^(de) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34⁺V_preB⁺RAG-1⁺TdT⁺, D_HJ_H-rearranged, κL germline cycling pre-B I cells → CD34⁻V_preB⁺μH chain⁺ (pre-B receptor⁺) RAG-1⁻TdT⁻, V_HD_HJ_H-rearranged, κL germline, cycling pre-B II cells → CD34⁻V_preB⁻, intracytoplasmic μH chain⁺ (pre-B receptor⁻) RAG-1^+/− TdT⁻, V_HD_HJ_H-rearranged, mainly κL germline cycling pre-B II cells → CD34⁻V_preB⁻ intracytoplasmic μH chain⁺, RAG-1⁺TdT⁻, V_HD_HJ_H-rearranged, V_κJ_κ-rearranged, IgM⁻, resting pre-B II cells CD34⁺V_preB⁻, sIgM⁺, RAG-1⁺TdT⁻, V_HD_HJ_H- and V_κJ_κ-rearranged IgM⁺ immature B cells → CD34⁻, CD10⁻, sIgM⁺/sIgD⁺ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.     

A Transgenic Marker for Mouse B Lymphoid Precursors

Three lines of transgenic mice have been generated which express human CD25 under the control of the 722-base pair region located immediately 5′ of the precursor (pre)–B cell–specific λ5 gene. All three strains express human CD25 in parallel to endogenous λ5 on pre–B cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human CD25 on B lineage cells of transgenic mice has allowed the identification of a new B220⁺CD19⁻λ5⁺ precursor of the B220⁺CD19⁺λ5⁺ c-kit⁺ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19⁻ precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19⁺ pre–BI cells are predominantly DJ_H rearranged. The results indicate that random integration of the 722-bp 5′ region of the λ5 gene into the mouse genome confers tissue and differentiation stage–specific expression of a transgene.     

Counterselection against Dμ Is Mediated through Immunoglobulin (Ig)α-Igβ

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (Dμ). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Igα-Igβ, whereas the mechanism for counterselection against Dμ has not been determined. We have examined the role of the Igα-Igβ signal transducers in counterselection against Dμ using mice that lack Igβ. We found that Dμ expression is not selected against in developing B cells in Igβ mutant mice. Thus, the molecular mechanism for counterselection against Dμ in pre-B cells resembles positive selection in that it requires interaction between mDμ and Igα-Igβ.     

Pre–B cell receptor–mediated cell cycle arrest in Philadelphia chromosome–positive acute lymphoblastic leukemia requires IKAROS function

B cell lineage acute lymphoblastic leukemia (ALL) arises in virtually all cases from B cell precursors that are arrested at pre–B cell receptor–dependent stages. The Philadelphia chromosome–positive (Ph⁺) subtype of ALL accounts for 25–30% of cases of adult ALL, has the most unfavorable clinical outcome among all ALL subtypes and is defined by the oncogenic BCR-ABL1 kinase and deletions of the IKAROS gene in >80% of cases. Here, we demonstrate that the pre–B cell receptor functions as a tumor suppressor upstream of IKAROS through induction of cell cycle arrest in Ph⁺ ALL cells. Pre–B cell receptor–mediated cell cycle arrest in Ph⁺ ALL cells critically depends on IKAROS function, and is reversed by coexpression of the dominant-negative IKAROS splice variant IK6. IKAROS also promotes tumor suppression through cooperation with downstream molecules of the pre–B cell receptor signaling pathway, even if expression of the pre–B cell receptor itself is compromised. In this case, IKAROS redirects oncogenic BCR-ABL1 tyrosine kinase signaling from SRC kinase-activation to SLP65, which functions as a critical tumor suppressor downstream of the pre–B cell receptor. These findings provide a rationale for the surprisingly high frequency of IKAROS deletions in Ph⁺ ALL and identify IKAROS-mediated cell cycle exit as the endpoint of an emerging pathway of pre–B cell receptor–mediated tumor suppression.The pre–B cell receptor promotes differentiation and proliferation signals in B cell precursor cells within the bone marrow (Rolink et al., 2000). It consists of an Ig μ heavy chain (μ chain; IGHM) coupled to the surrogate light chain with its two components, VpreB (VPREB1) and λ5 (IGLL1), which promote constitutive pre–B cell receptor signaling (van Loo et al., 2007; Fig. S1). Productive rearrangement of Ig V_H to DJ_H gene segments is a prerequisite for the expression of a functional μ chain, which is linked to the Igα and Igβ transmembrane signaling chains (Guo et al., 2000). The key components of the proximal pre–B cell receptor signaling cascade are assembled and stabilized by the linker protein SLP65 (or BLNK, BASH; Fu et al., 1998; Hayashi et al., 2000; Fig. S1). In the absence of SLP65, the function of the pre–B cell receptor is compromised and SLP65-deficient B cell precursors are arrested at the pre–B cell stage (Jumaa et al., 1999). In virtually all cases, B cell lineage acute lymphoblastic leukemia (ALL) is derived from B cell precursors that are arrested at pre–B cell receptor–dependent stages of development. Among ALL in adults, the Philadelphia chromosome (Ph) encoding the oncogenic BCR-ABL1 tyrosine kinase, represents the most frequent cytogenetic abnormality (∼25–30% of cases) and defines the ALL subset with the most unfavorable prognosis (Mancini et al., 2005). Ph⁺ ALL is also characterized by deletions of the IKZF1 (IKAROS) gene that are found in >80% of Ph⁺ ALL cases (Mullighan et al., 2008). IKZF1 deletions typically lead to the expression of dominant-negative IKAROS variants (e.g., IK6) that are characterized by loss of N-terminal zinc fingers that mediate DNA binding, whereas the C-terminal dimerization domain is retained (Klein et al., 2006; Iacobucci et al., 2008; Reynaud et al., 2008). Based on a previous study of 12 cases of Ph⁺ ALL, our group described inactivation of the pre–B cell receptor in Ph⁺ ALL based on nonfunctional IGHM gene rearrangements (Klein et al., 2004) and down-regulation of pre–B cell receptor–related signaling molecules (Klein et al., 2004, 2006). Here, we confirm these observations based on 57 cases of human Ph⁺ ALL as compared with normal pre–B cells and 54 cases of Ph⁻ ALL and elucidate the mechanism of pre–B cell receptor–mediated tumor suppression in Ph⁺ ALL.     

A role for the IgH intronic enhancer Eμ in enforcing allelic exclusion

The intronic enhancer (Eμ) of the immunoglobulin heavy chain (IgH) locus is critical for V region gene assembly. To determine Eμ's subsequent functions, we created an Igh allele with assembled V_H gene but with Eμ removed. In mice homozygous for this Eμ-deficient allele, B cell development was normal and indistinguishable from that of mice with the same V_H knockin and Eμ intact. In mice heterozygous for the Eμ-deficient allele, however, allelic exclusion was severely compromised. Surprisingly, this was not a result of reduced suppression of V-DJ assembly on the second allele. Rather, the striking breakdown in allelic exclusion took place at the pre-B to immature B cell transition. These findings reveal both an important role for Eμ in influencing the fate of newly arising B cells and a second checkpoint for allelic exclusion.     

Mutations in the Human λ5/14.1 Gene Result in B Cell Deficiency and Agammaglobulinemia

B cell precursors transiently express a pre–B cell receptor complex consisting of a rearranged mu heavy chain, a surrogate light chain composed of λ5/14.1 and VpreB, and the immunoglobulin (Ig)-associated signal transducing chains, Igα and Igβ. Mutations in the mu heavy chain are associated with a complete failure of B cell development in both humans and mice, whereas mutations in murine λ5 result in a leaky phenotype with detectable humoral responses. In evaluating patients with agammaglobulinemia and markedly reduced numbers of B cells, we identified a boy with mutations on both alleles of the gene for λ5/14.1. The maternal allele carried a premature stop codon in the first exon of λ5/14.1 and the paternal allele demonstrated three basepair substitutions in a 33-basepair sequence in exon 3. The three substitutions correspond to the sequence in the λ5/14.1 pseudogene 16.1 and result in an amino acid substitution at an invariant proline. When expressed in COS cells, the allele carrying the pseudogene sequence resulted in defective folding and secretion of mutant λ5/14.1. These findings indicate that expression of the functional λ5/14.1 is critical for B cell development in the human.     

Regulation of B Cell Development by Variable Gene Complexity in Mice Reconstituted with Human Immunoglobulin Yeast Artificial Chromosomes

The relationship between variable (V) gene complexity and the efficiency of B cell development was studied in strains of mice deficient in mouse antibody production and engineered with yeast artificial chromosomes (YACs) containing different sized fragments of the human heavy (H) chain and κ light (L) chain loci. Each of the two H and the two κ chain fragments encompasses, in germline configuration, the same core variable and constant regions but contains different numbers of unique V_H (5 versus 66) or Vκ genes (3 versus 32). Although each of these YACs was able to substitute for its respective inactivated murine counterpart to induce B cell development and to support production of human immunoglobulins (Igs), major differences in the efficiency of B cell development were detected. Whereas the YACs with great V gene complexity restored efficient development throughout all the different recombination and expression stages, the YACs with limited V gene repertoire exhibited inefficient differentiation with significant blocks at critical stages of B cell development in the bone marrow and peripheral lymphoid tissues. Our analysis identified four key checkpoints regulated by V_H and Vκ gene complexity: (a) production of functional μ chains at the transition from the pre B-I to the pre B-II stage; (b) productive VκJκ recombination at the small pre B-II stage; (c) formation of surface Ig molecules through pairing of μ chains with L chains; and (d) maturation of B cells. These findings demonstrate that V gene complexity is essential not only for production of a diverse repertoire of antigen-specific antibodies but also for efficient development of the B cell lineage.     

Role of Different T Cell Receptors in the Development of Pre–T Cells

The development of pre–T cells with productive TCR-β rearrangements can be mediated by each the pre–T cell receptor (pre-TCR), the TCR-αβ as well as the TCR-γδ, albeit by distinct mechanisms. Although the TCR-γδ affects CD4⁻8⁻ precursor cells irrespective of their rearrangement status by TCR-β mechanisms not involving TCR-β selection, both the preTCR and the TCR-αβ select only cells with productive TCR-β genes for expansion and maturation. The TCR-αβ appears to be much less effective than the pre-TCR because of the paucity of TCR-α proteins in TCR-β–positive precursors since an early expressed transgenic TCR-αβ can largely substitute for the pre-TCR. Thus, the TCR-αβ can assume a role not only in the rescue from programmed cell death of CD4⁺8⁺ but also of CD4⁻8⁻ thymocytes. In evolution this double function of the TCR-αβ may have been responsible for the maturation of αβ T cells before the advent of the pre–TCR-α chain.     

The Normal Counterpart of IgD Myeloma Cells in Germinal Center Displays Extensively Mutated IgVH Gene, Cμ–Cδ Switch, and λ Light Chain Expression

Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre–B cells, germinal center (GC) B cells, circulating memory cells, or plasma blasts. This situation reflects what has been a general problem in cancer research for years: how to compare a tumor with its normal counterpart. Although several studies have demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a biased λ light chain expression and a Cμ–Cδ isotype switch. Using surface markers, we have previously isolated a population of surface IgM⁻IgD⁺CD38⁺ GC B cells that carry the most impressive somatic mutation in their IgV genes. Here we show that this population of GC B cells displays the two molecular features of IgD-secreting myeloma cells: a biased λ light chain expression and a Cμ–Cδ isotype switch. The demonstration of these peculiar GC B cells to differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro suggests that IgD-secreting plasma and myeloma cells are derived from GCs.     

Abnormal Development of Intestinal Intraepithelial Lymphocytes and Peripheral Natural Killer Cells in Mice Lacking the IL-2 Receptor β Chain

The interleukin-2 receptor β chain (IL-2Rβ) is expressed on a variety of hematopoietic cell types, including natural killer (NK) cells and nonconventional T lymphocyte subsets such as intestinal intraepithelial lymphocytes (IEL). However, the importance of IL-2Rβ-mediated signaling in the growth and development of these cells has yet to be clearly established. We have investigated IEL and NK cells in mice deficient for IL-2Rβ and describe here striking defects in the development of these cells. IL-2Rβ^−/− mice exhibited an abnormal IEL cell population, characterized by a dramatic reduction in T cell receptor αβ CD8αα and T cell receptor γδ lymphocytes. This selective decrease indicates that IEL can be classified into those whose development and/or differentiation is dependent on IL-2Rβ function and those for which IL-2Rβ–mediated signaling is not essential. NK cell development was also found to be disrupted in IL-2Rβ–deficient mice, characterized by a reduction in NK1.1⁺CD3⁻ cells in the peripheral circulation and an absence of NK cytotoxic activity in vitro. The dependence of NK cells and certain subclasses of IEL cells on IL-2Rβ expression points to an essential role for signaling through this receptor, presumably by IL-2 and/or IL-15, in the development of lymphocyte subsets of extrathymic origin.The cytokine interleukin-2 (IL-2) and its receptor (IL2R) have long been known to play a role of prime importance in the activation and proliferation of T lymphocytes (reviewed in reference 1). However, it is unclear whether signaling via the IL-2R is required for the normal development and differentiation of other lymphoid or myeloid cells. The IL-2R is expressed on T lymphocytes (1), B cells (2), NK cells (3), neutrophils (4), and monocytes (5). The high affinity IL-2R is composed of three subunits, the α, β, and γ chains, whereas the intermediate affinity form contains only the β and γ chains (6, 7). Components of the IL-2R have been identified in other cytokine receptors: the IL-2Rγ chain (γ_c, or common γ chain) is common to the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors, whereas the IL-2Rβ chain is shared between the IL-2 and IL-15 receptors (8).The importance of individual IL-2R chains in signal transduction has been clarified recently by the engineered deletion of the gene product for each of the three known receptor subunits. Given the diverse nature of the cytokine receptors that contain the β and γ chains, and the wide range of cell types that express components of the IL-2R, the disruption of multiple immunomodulatory roles might be expected in mice deficient for any one of the three subunits. Mice deficient in IL-2Rα exhibit polyclonal T and B cell expansion (9), which correlates with a defect in activation-induced cell death in T cells, and the development of autoimmune disorders and inflammatory bowel disease. Similarly, in IL-2Rβ–deficient mice, T cells are spontaneously activated, resulting in plasma cell accumulation and high levels of autoantibodies (10). IL-2Rγ–deficient mice also show a defect in mature T and B cell development, but in contrast with the lymphoproliferation seen in IL-2Rα– and IL-2Rβ–deficient mice, a 10-fold reduction in absolute lymphocyte numbers is observed (11). In addition, NK cells are completely absent in IL-2Rγ–deficient mice, and the number of intestinal intraepithelial cells (IEL)¹ cells is severely diminished (11). In humans, loss of the γ_c function leads to X-linked severe combined immunodeficiency (SCID) (12), a disease characterized by multiple defects in T, NK, and B cell development, as might be expected from the impairment of several cytokine receptors (13). Thus, signaling via components of the IL-2R plays an essential regulatory role in homeostasis and autoimmunity.The importance of IL-2 and IL-15 in NK cell activation and proliferation has been well documented (1, 8, 14). Because both cytokines signal via receptors containing the β and γ chains, and because IL-2Rγ–deficient mice exhibit profound defects in IEL and NK cell development, it was of interest to examine the role of IL-2Rβ in these lymphocyte populations. We report here that expression of IL-2Rβ is crucial for the normal development of both TCRγδ and TCRαβ CD8αα IEL and for NK cells. These results suggest that certain cell populations that undergo extrathymic development and differentiation have an important developmental requirement for either IL-15 and/or IL-2.     

A vanillin-based copper(ii) metal complex with a DNA-mediated apoptotic activity

Vanillin (vanH) is the major component of vanilla and one of the most widely used flavoring agents. In this work the complex [Cu(phen)(van)₂] was prepared and characterized by structural (X-ray), spectroscopic (IR, UV-Vis, EPR) and electrochemical techniques. This compound showed an octahedral geometry with an unusual arrangement of the vanillin ligands, where the methoxy groups of the vanillinate ions are coordinated opposite to each other. The compound promoted DNA cleavage in the presence of glutathione (GSH) and H₂O₂. At 40 μmol L⁻¹ of complex with GSH (10 mmol L⁻¹), there is a complete cleavage of DNA to nicked form II, while only at 10 μmol L⁻¹ of this complex with H₂O₂ (1 mmol L⁻¹) an extensive cleavage leading to form III took place. Additionally, we have evidences of superoxide generation upon reaction with GSH. Therefore, DNA fragmentation occurs likely through an oxidative pathway. MTT assays indicated that the complex is highly cytotoxic against three distinct cell lines: B16–F10 (IC₅₀ = 3.39 ± 0.61 μmol L⁻¹), HUH-7 (IC₅₀ = 4.22 ± 0.31 μmol L⁻¹) and 786-0 (IC₅₀ = 10.38 ± 0.91 μmol L⁻¹). Flow cytometry studies conducted with 786-0 cell line indicated cell death might occur by apoptosis. Cell cycle progression evaluated at 5 and 10 μmol L⁻¹ resulted in a clear increase of 786-0 cells at G1 phase and depletion of G2/M, while higher doses showed an expressive increase of sub-G1 phase. Altogether, these results pointed out to a promising biological activity and potential as an anti-cancer agent.

Proposed catalytic cycle for ROS production in the vicinity of DNA after reduction of [Cu(phen)(van)₂] by glutathion.     

Enforced Bcl-2 Expression Inhibits Antigen-mediated Clonal Elimination of Peripheral B Cells in an Antigen Dose–dependent Manner and Promotes Receptor Editing in Autoreactive, Immature B Cells

The mechanisms that establish immune tolerance in immature and mature B cells appear to be distinct. Membrane-bound autoantigen is thought to induce developmental arrest and receptor editing in immature B cells, whereas mature B cells have shortened lifespans when exposed to the same stimulus. In this study, we used Eμ–bcl-2-22 transgenic (Tg) mice to test the prediction that enforced expression of the Bcl-2 apoptotic inhibitor in B cells would rescue mature, but not immature, B cells from tolerance induction. To monitor tolerance to the natural membrane autoantigen H-2K^b, we bred 3–83μδ (anti-K^k,b) Ig Tg mice to H-2^b mice or to mice expressing transgene-driven K^b in the periphery. In 3–83μδ/bcl-2 Tg mice, deletion of autoreactive B cells induced by peripheral K^b antigen expression in the liver (MT-K^b Tg) or epithelia (KerIV-K^b Tg), was partly or completely inhibited, respectively. Furthermore, Bcl-2 protected peritoneal B-2 B cells from deletion mediated by acute antigen exposure, but this protection could be overcome by higher antigen dose. In contrast to its ability to block peripheral self-tolerance, Bcl-2 overexpression failed to inhibit central tolerance induced by bone marrow antigen expression, but instead, enhanced the receptor editing process. These studies indicate that apoptosis plays distinct roles in central and peripheral B cell tolerance.     

B-1 Cell Development: Evidence for an Uncommitted Immunoglobulin (Ig)M+ B Cell Precursor in B-1 Cell Differentiation

Murine phosphatidyl choline (PtC)–specific B cells in normal mice belong exclusively to the B-1 subset. Analysis of anti-PtC (V_H12 and V_H12/Vκ4) transgenic (Tg) mice indicates that exclusion from B-0 (also known as B-2) occurs after immunoglobulin gene rearrangement. This predicts that PtC-specific B-0 cells are generated, but subsequently eliminated by either apoptosis or differentiation to B-1. To investigate the mechanism of exclusion, PtC-specific B cell differentiation was examined in mice expressing the X-linked immunodeficiency (xid) mutation. xid mice lack functional Bruton's tyrosine kinase (Btk), a component of the B cell receptor signal transduction pathway, and are deficient in B-1 cell development. We find in C57BL/ 6.xid mice that V_H12 pre-BII cell selection is normal and that PtC-specific B cells undergo modest clonal expansion. However, the majority of splenic PtC-specific B cells in anti-PtC Tg/xid mice are B-0, rather than B-1 as in their non-xid counterparts. These data indicate that PtC-specific B-0 cell generation precedes segregation as predicted, and that Btk function is required for efficient segregation to B-1. Since xid mice exhibit defective B cell differentiation, not programmed cell death, these data are most consistent with an inability of PtC-specific B-0 cells to convert to B-1 and a single B cell lineage.