pre-B Cell Receptor Complex Research Articles

Disruption of the preB Cell Receptor Complex Leads to Decreased Bone Mass.

In the bone marrow, preB cells are found adjacent to the bone endosteum where bone synthesizing osteoblast and bone resorbing osteoclasts reside. Although there is evidence of interactions between preB and bone cells, the factors that contribute to such interactions are poorly understood. A critical checkpoint for preB cell development assesses the integrity of the nascent immunoglobulin μ heavy chain (HC) by testing whether it can participate in the formation of a preB cell receptor (preBCR), composed of the μ HC and surrogate light chain (LC). In this work, we tested whether loss of preBCR components can affect bone synthesis. A panel of gene targeted mice with sequential blocks in preBCR formation or function [surrogate light chain component lambda 5 deleted (λ5−/−), transmembrane domain of μHC deleted (IgM-mem−/−), and CD19 preBCR co-receptor deleted (CD19−/−)] were evaluated for effects on postnatal bone synthesis. Postnatal bone mass was analyzed in 6 month old mice using μ-CT, histomorphometry and double calcein labeling. Both cortical and trabecular bone mass were significantly decreased in the femurs of the λ5 and IgM-mem deficient mice. Histomorphometric analysis showed a decrease in the numbers of osteoblasts and osteoclasts in all three mutant strains. Double calcein labeling revealed a significant decrease in dynamic synthesis and mineralization of bone in λ5−/− mice. Our data strongly suggest that interference with preBCR formation or function affects bone homeostasis independent of the presence or absence of mature B cells, and that components of the preBCR play important, and potentially distinct, roles in regulating adult bone mass.

CD79a Is Associated with Central Nervous System Infiltration of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

▪Despite the advances in the treatment of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), infiltration of the central nervous system (CNS) remains a clinical challenge. Certain cytogenetic subtypes such as E2A-PBX1-and BCR-ABL-positive BCP-ALL confer a higher risk for CNS involvement initially and for CNS relapse. Novel strategies to predict CNS and to eradicate leukemic cells from the CNS are subjects of ongoing research.In order to identify targets with diagnostic and therapeutic relevance, comparative RNA-sequencing was performed with patient derived xenograft (PDX) blasts from 5 E2A-PBX1-positive patients, recovered from the bone marrow (BM) and from the CNS of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Differential gene expression analysis revealed the upregulation of various genes of the pre-B cell receptor complex, particularly the signaling component CD79a (Igα) in blasts recovered from the CNS as compared to blasts from the BM. We then investigated the impact of CD79a on CNS infiltration in vivo and in patients. CD79a was downregulated by short-hairpin RNA (shRNA) mediated knockdown in the E2A-PBX1 positive cell line 697. Proliferation rates of 697-shCD79a cells and control-transfected 697 cells (697-shCtrl) in vitro were similar. Furthermore, NSG mice injected with 697-shCD79a cells showed comparable survival times, as well as similar blast infiltration in spleen and BM as animals injected with 697-shCtrl cells. However, downregulation of CD79a led to a significantly lower number of CNS-positive mice (4/15, 26%) as compared to control animals (7/10, 70%) (p=0.0486, Figure A). This indicates that CD79a is not critically involved in proliferation and peripheral engraftment, but in CNS infiltration of E2A-PBX1 positive 697 cells in vivo.To test if CD79a also affects CNS involvement in BCR-ABL-positive leukemia, a murine/murine transplantation model was used. B-cells isolated from CD79a-knockout (CD79a-KO) or wildtype mice (CD79a-Ctrl) were stably transfected with a BCR-ABL fusion gene and cultured independent of cytokines, thereby inducing malignant transformation. Both cell lines were subsequently injected into recipient NSG mice (n=8/group) and leukemic development was followed. The experiment was terminated when all control mice had developed leukemic symptoms and mice were analyzed for leukemic engraftment. A further CD79a-KO group was included for survival analysis. Median spleen volume as a surrogate of leukemic infiltration was significantly lower in mice injected with CD79a-KO as compared to CD79a-Ctrl cells (0.35 cm³ vs. 0.06 cm³; p=0.0001). Median blast percentages in spleens and BM were also markedly reduced (75.3% vs. 5.8%; p=0.0001 and 61.0% vs. 4.5%; p=0.0001, respectively). Importantly, none of the animals in the CD79a-KO group showed blasts in the CNS as assessed by histology whereas blasts were present in all of the animals in the CD79a-Ctrl group. Finally and most importantly, NSG-mice injected with CD79a-KO cells showed a highly significant prolongation in median survival as compared to mice with CD79a-Ctrl cells (29 days vs. 95 days; p=0.0001, Figure B). Altogether, these data suggest that in a model of BCR-ABL-positive leukemia, absence of CD79a impacts the engraftment of blasts in vivo, in the CNS and other leukemic niches.To further validate our findings in patient material, we measured CD79a protein expression in PDX cells from an E2A-PBX1- and a BCR-ABL-positive patient serially transplanted into NSG mice for three passages. For both entities and in all passages, CNS blasts showed a higher CD79a expression than blasts isolated from the bone marrow. In order to assess if CD79a can be used as a marker to predict CNS involvement in patients, CD79a mRNA levels were measured in a selected cohort of 98 pediatric BCP-ALL patients, which contained 26 CNS-positive patients matched to 72 CNS-negative patients. CNS-positive patients showed significantly higher mRNA levels of CD79a than CNS-negative patients (p=0.0225, unpaired t-test, Figure C) suggesting that CD79a may be of value as a potential diagnostic marker for initial CNS involvement in BCP-ALL.Our results indicate a role of CD79a in proliferation and CNS infiltration of BCP-ALL blasts in experimental settings and patients. We intend to prospectively evaluate CD79a as a prognostic marker, which may also be a therapeutic target in CNS-positive BCP-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Physiological Level Production of Antigen-Specific Human Immunoglobulin in Cloned Transchromosomic Cattle

Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

PAX5 Is a Frequently Altered Transcription Factor In Genetically Unclassified Childhood Common/B-Cell Precursor Acute Lymphoblastic Leukemia

Abstract 1458 Introduction:Genetic lesions define treatment and prognosis in childhood common/B cell-precursor Acute Lymphoblastic Leukemia (c/pre-B ALL) (Pui CH et al., J Clin Oncol, 2011). However, up to 40% of childhood pre-B ALL cases, lack the presence of prognostically significant abnormalities; and relapses are common in this group (Mejerink J et al., Semin Hem, 2009). Molecular genetic characterization of genetically unclassified ALL is critical for a better understanding of the biology of this disease, to determine new prognostic markers and to optimize treatment for this group of patients. Several transcription factors, including EBF1, IKZF1 and PAX5, which regulate B-cell development, have recently been shown often to be altered in B-ALL (Mullighan CG et al., Nature, 2007; den Boer ML et al., Lancet, 2009). We decided to investigate the incidence of abnormalities involving B-cell transcription factors, in a cohort of patients with childhood c/pre-B ALL lacking significant prognostic genetic aberrations. Materials and methods:60 patients diagnosed with c/pre-B ALL in Great Ormond Street Hospital (London, UK) from 1994 to 2011 were included in the study. The median age was 3.43 (range: 1.14–12.24), with a median white blood cell count of 14.0 (range: 1.36–213.2). All patients showed an absence of high-hyperploidy/hypodiploidy, BCR-ABL1, MLL, ETV6-RUNX1 and E2A rearrangements, and also i(amp)21 by karyotype, fluorescence in-situ hybridization (FISH) and/or reverse transcriptase-polymerase chain reaction (RT-PCR) investigations. We performed FISH using commercially available probes and BAC clones, in parallel with RT-PCR, to detect deletions/rearrangements of the EBF1, IKZF1 and PAX5 gene regions. Direct sequence analysis was performed for all exons of the PAX5 gene. Real-time PCR was used to determine the level of expression of PAX5 and its transcriptional targets, mostly components of the pre-B cell receptor (pre-BCR) complex including CD19, CD79α, VpreB, BLNK and BLK genes. Western blotting was further performed for the identification of an abnormal PAX5 protein. Results:25/60 patients (42%) showed abnormalities involving PAX5, 2/60 patients (3%) showed deletions of IKZF1. No deletions/rearrangements of EBF1 were detected. Several forms of PAX5 deletions were observed: the entire loss of one PAX5 allele (32%), loss of 5’PAX5 (20%), loss of 3’PAX5 (12%) and loss of 3’PAX5 due to unbalanced rearrangements (8%). Balanced PAX5 rearrangements comprise 12% of the PAX5 alterations. Sequence analysis revealed 9 somatic mutations of PAX5 in 8/60 patients (13%): 6 frameshifts, 2 splice-site and 1 non-stop mutations. The mutations were located in functional exons of PAX5 and resulted in the initiation of premature stop-codons, exon skips, and the formation of abnormal transcripts. Interestingly, 2 patients with an identical splice site mutation, showed the same karyotypic abnormality of an isochromosome of the long arm of chromosome 9 leading to loss of the second PAX5 allele. Moreover, further investigation DNA and mRNA from remission samples demonstrated that in one case the mutation was acquired, and that in the second case the mutation was a germ-line variant. In total, 20% of patients with PAX5 abnormalities had bi-allelic alterations of the PAX 5 gene. PAX5, BLNK and BLK gene-expression levels were lower (p<0.05) in the group of patients with loss of one entire PAX5 allele. There were no significant changes in expression levels of the tested genes in patients with partial PAX5 deletions, rearrangements or mutations. Expression of an abnormal PAX5 protein was confirmed by western blot analysis. Conclusion:The high incidence of PAX5 alterations in childhood c/pre-B ALL, without prognostically significant genetic abnormalities, suggests a pathogenetic role for PAX5 in these acute lymphoblastic leukemia cases. Down-regulation of expression of the BLNK and BLK genes could be one of the mechanisms by which abnormal PAX5 blocks B-cell differentiation and contributes to the initiation of leukemia. Disclosures:No relevant conflicts of interest to declare.

Pro-B cells sense productive immunoglobulin heavy chain rearrangement irrespective of polypeptide production

B-lymphocyte development is dictated by the protein products of functionally rearranged Ig heavy (H) and light (L) chain genes. Ig rearrangement begins in pro-B cells at the IgH locus. If pro-B cells generate a productive allele, they assemble a pre-B cell receptor complex, which signals their differentiation into pre-B cells and their clonal expansion. Pre-B cell receptor signals are also thought to contribute to allelic exclusion by preventing further IgH rearrangements. Here we show in two independent mouse models that the accumulation of a stabilized μH mRNA that does not encode μH chain protein specifically impairs pro-B cell differentiation and reduces the frequency of rearranged IgH genes in a dose-dependent manner. Because noncoding IgH mRNA is usually rapidly degraded by the nonsense-mediated mRNA decay machinery, we propose that the difference in mRNA stability allows pro-B cells to distinguish between productive and nonproductive Ig gene rearrangements and that μH mRNA may thus contribute to efficient H chain allelic exclusion.

Genetic analysis of patients with defects in early B‐cell development

Approximately 85% of patients with defects in early B-cell development have X-linked agammaglobulinemia (XLA), a disorder caused by mutations in the cytoplasmic Bruton's tyrosine kinase (Btk). Although Btk is activated by cross-linking of a variety of cell-surface receptors, the most critical signal transduction pathway is the one initiated by the pre-B cell and B-cell antigen receptor complex. Mutations in Btk are highly diverse, and no single mutation accounts for more than 3% of patients. Although there is no strong genotype/phenotype correlation in XLA, the specific mutation in Btk is one of the factors that influences the severity of disease. Mutations in the components of the pre-B cell and B-cell antigen receptor complex account for an additional 5-7% of patients with defects in early B-cell development. Patients with defects in these proteins are clinically indistinguishable from those with XLA. However, they tend to be younger at the time of diagnosis, and whereas most patients with XLA have a small number of B cells in the peripheral circulation, these cells are not found in patients with defects in micro heavy chain or Igalpha. Polymorphic variants in the components of the pre-B cell and B-cell receptor complex, particularly micro heavy chain and lambda5, may contribute to the severity of XLA.

Immunoglobulin mu heavy chains do not mediate tyrosine phosphorylation of Ig alpha from the ER-cis-Golgi.

Signals delivered by Ig receptors guide the development of functional B lymphocytes. For example, clonal expansion of early mu heavy chain ( mu HC)-positive pre-B cells requires the assembly of a signal-competent pre-B cell receptor complex (pre-BCR) consisting of a mu HC, a surrogate L chain, and the signal dimer Ig alpha beta. However, only a small fraction of the pre-BCR is transported to the cell surface, suggesting that pre-BCR signaling initiates already from an intracellular compartment, e.g., the endoplasmic reticulum (ER). The finding that differentiation of pre-B cells and allelic exclusion at the IgH locus take place in surrogate L chain-deficient mice further supports the presence of a mu HC-mediated intracellular signal pathway. To determine whether a signal-competent Ig complex can already be assembled in the ER, we analyzed the consequence of pervanadate on tyrosine phosphorylation of Ig alpha in J558L plasmacytoma and 38B9 pre-B cells transfected with either a transport-competent IgL chain-pairing or an ER-retained nonpairing micro HC. Flow cytometry, combined Western blot-immunoprecipitation-kinase assays, and confocal microscopy revealed that both the nonpairing and pairing mu HC assembled with the Ig alpha beta dimer; however, in contrast to a pairing mu HC, the nonpairing mu HC was retained in the ER-cis-Golgi compartment, and neither colocalized with the src kinase lyn nor induced tyrosine phosphorylation of Ig alpha after pervanadate treatment of cells. On the basis of these findings, we propose that a signal-competent Ig complex consisting of mu HC, Ig alpha beta, and associated kinases is assembled in a post-ER compartment, thereby supporting the idea that a pre-BCR must be transported to the cell surface to initiate pre-BCR signaling.

Role of the BCR complex in B cell development, activation, and leukemic transformation.

A primary focus of signal transduction in B cells, from the pre-B cell to the mature B cell, is the B cell receptor complex. Here we describe work demonstrating the importance of signaling via the pre-B cell receptor complex (pre-BCR) to the pre-B cell transition, the central checkpoint in B-cell development. We have shown tht pre-BCR complex components Igalpha and Igbeta are critical to allowing the pre-B cell to move through this transition, but may not be required for allelic exclusion. Pre-BCR expression also directly affects the response of leukemic cells to steroid treatment, suggesting that signals initiated by the pre-BCR complex may present therapeutic targets in acute leukemia. Additionally, interleukin-7 may also modulate the response of leukemic cells arising from early B-cell stages to treatment. This observation has lead directly to proposals to test drugs which may antagonize early B-cell growth signals, such as rapamycin, in acute lymphoid leukemia.

Recent progress in the diagnosis and treatment of patients with defects in early B-cell development.

Mutation detection for X-linked agammaglobulinemia (XLA) has revealed the heterogeneity of the clinical phenotype of patients with defects in Bruton's tyrosine kinase (Btk), the gene that is abnormal in XLA. Over 50% of patients with mutations in Btk have no family history of the disease because their cases are the first manifestation of a new mutation in their family. In 10% to 20% of patients, the serum immunoglobulins are higher than expected or the onset of disease is delayed; however, a marked reduction in B-cell numbers is consistent in all patients. Mutation detection has also shown that not all patients with presumed XLA have mutations in Btk. Mutations in mu heavy chain, and other components of the pre-B cell receptor complex, including lambda 5/14.1, cause a disorder that is clinically identical to XLA. Although new strategies for therapy are not yet available, the groundwork is being laid for cell or gene therapy.

Novel mechanisms control the folding and assembly of lambda5/14.1 and VpreB to produce an intact surrogate light chain.

Surrogate light chain, which escorts the mu heavy chain to the cell surface, is a critical component of the pre-B cell receptor complex. The two proteins that comprise the surrogate light chain, VpreB and lambda5/14.1, contain both unique regions and Ig-like domains. The unique regions have been postulated to function in the assembly of the surrogate light chain. However, by using transient transfection of COS7 cells, we show that deletion of the unique regions of both proteins did not inhibit the assembly of surrogate light chain. Instead, in vivo folding studies showed that the unique region of lambda5/14.1 acts as an intramolecular chaperone by preventing the folding of this protein when it is expressed in the absence of its partner, VpreB. The Ig domains of both lambda5/14.1 and VpreB are atypical. The one in VpreB lacks one of the canonical beta strands whereas the one in lambda5/14.1 has an extra beta strand. Deletion of the extra beta strand in lambda5/14.1 completely abrogated the formation of the surrogate light chain, demonstrating that complementation of the incomplete Ig domain in VpreB by the extra beta strand in lambda5/14.1 was necessary and sufficient for the folding and assembly of these proteins. Our studies reveal two novel mechanisms for regulating surrogate light chain formation: (i) the presence of an intramolecular chaperone that prevents folding of the unassembled subunit but that remains part of the mature assembled protein, and (ii) splitting an Ig domain between two proteins to control their folding and assembly.

#630 Role of the pre-B cell receptor complex in B cell development

#630 Role of the pre-B cell receptor complex in B cell development

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.

Secretion of soluble pre-B cell receptors by pre-B cells.

Pre-B cells can express secretory mu (mu(s))- as well as membrane mu (mu(m))-chains. We evaluated the ability of mu(s)-chains to associate with surrogate light chains and assemble into a pre-B cell receptor (BCR) complex in pre-B cells, and explored whether mu(s)-chains could be exploited to generate a secreted soluble pre-BCR. We demonstrate that mu(s)-chains can associate with SLC internally. The mu(s)-containing complexes form higher order polymeric structures, but these are never assembled into completed covalent structures. Instead, the complexes are efficiently retained and rapidly degraded. Alteration of the intracellular redox state by incubation with 2-ME resulted in the secretion of mu(s)-chains, suggesting that they are retained by a thiol-mediated retention mechanism. To identify the sequences on mu(s)-chains responsible for their retention, we generated stable transfectants of a mu-negative pre-B cell line expressing either wild-type or mutant mu(s) constructs. Mutation of a single cysteine (Cys575) in the mu(s) tailpiece resulted in the release and secretion of the mu(s) H chains. These were associated with the surrogate light chain proteins lambda5 and VpreB, and thus appear to constitute an authentic secreted soluble pre-BCR. The soluble pre-BCR has a specificity distinct from Ab consisting of the same heavy chain V region paired with conventional light chains.

Assembled Pre-B Cell Receptor Complexes Are Retained in the Endoplasmic Reticulum by a Mechanism That Is Not Selective for the Pseudo-light Chain

The pre-B cell receptor (BCR) complex, consisting of micro heavy chain, a pseudo-light chain, and the Mb-1/B29 heterodimer, directs the transition to the mature B cell stage. Plasma membrane expression of the pre-BCR is extremely low, despite its presumed signaling function. We have compared assembly and intracellular transport of the pre-BCR complex with that of the BCR complex in mature B cells. Synthesis and assembly rate of pre-BCR and BCR components are comparable. However, the pre-BCR is subject to a highly efficient retention mechanism, which only allows exit of a few percent of the complexes from the endoplasmic reticulum (ER). This small transported pool of pre-BCR complexes is significantly enriched for protein-tyrosine kinase activity, as compared with the ER-localized receptor pool. Accordingly, the Src-related tyrosine kinase Lyn was found in the transported glycoprotein fraction but not in association with ER-localized glycoproteins. Upon introduction of a conventional light chain into pre-B cells, plasma membrane receptor levels increased, but the efficiency of intracellular transport of the receptor complex was not restored to that in mature B cells. This indicates that the ER retention mechanism is not selective for the pseudo-light chain and may be inherent to pre-B cells. We propose that this retention mechanism contributes to the regulation of pre-BCR-mediated signal transduction.

Immunoglobulin heavy and light chain genes rearrange independently at early stages of B cell development

The compartment of mouse B cell progenitors can be resolved into five developmentally related fractions by multicolor flow cytometry. Using this system and employing mutant mice in which the membrane exon of the μ chain, the λ5 gene, or the J H locus was inactivated by gene targeting, we found that expression of the pre-B cell receptor complex is necessary for the transition from the large CD43 + to the small CD43 − pre-B cell stage. We report the occurrence of immunoglobulin heavy and light chain gene rearrangement at the stage of large B cell precursors. We show that neither the pre-B cell receptor complex nor any gene rearrangement in the heavy chain locus is required for the induction of κ light chain gene rearrangement in early B cell progenitors.