Partial block in B lymphocyte development at the transition into the pre-B cell receptor stage in Vpre-B1-deficient mice

Abstract B lymphocyte development proceeds in a stepwise fashion and is tightly linked to the generation of a functional B cell receptor (BCR). At the preB cell stage B lymphocyte progenitors express the precursor B cell receptor (pre-BCR), an immature form of the BCR consisting of two µ heavy chains (µHC) and two surrogate light chains (SLC). Pre-BCR expression marks the proB to preB transition and induces a burst in preB lymphocyte proliferation. In 20% of the cases B cell acute lymphoblastic leukemia (B-ALL) arises from lymphocytes arrested at the pre-BCR positive stage of lymphocyte development (preB-ALL). Due to the essential role of the pre-BCR for preB cell proliferation we hypothesized that pre-BCR signaling also is involved in the maintenance of preB-ALL. Consequently, pharmacological inhibition of Spleen tyrosine kinase (Syk), the main transducer of pre-BCR signaling, may serve as effective treatment for this subtype of B-ALL. We analyzed a panel of six ALL cell lines (SMS-SB, RCH-ACV, Nalm-6, Kasumi-2, 697, KOPN-8) arrested at the pre-BCR+ stage of B lymphocyte development (cytoIgµ+, sIgM-). Assessment of the baseline phosphorylation levels of the pre-BCR associated kinases Lyn, Syk and Btk by immunoblotting and subsequent densitometric analysis allowed us to assign B-ALL cells into groups with either high levels of Lyn, Syk and Btk phosphorylation or with low or absent phosphorylation of these kinases, respectively. Moreover cell lines with highly phosphorylated Lyn, Syk and Btk also exhibited lower surface pre-BCR expression than cell lines with low phosphorylation levels. As pre-BCR activation is followed by its rapid internalization the concomitant presence of low pre-BCR expression and high phosphorylation of pre-BCR associated proteins suggests increased pre-BCR pathway activity. When we investigated the impact of pharmacological inhibition of the pre-BCR associated kinase Syk through the highly specific inhibitor PRT060318, preB-ALL cell lines with highly phosphorylated pre-BCR associated molecules turned out to be more sensitive to Syk inhibition (IC50 < 1.6µM) than preB-ALL cell lines with less phosphorylation (IC50 > 3.9µM). In proliferation assays PRT060318 inhibited preB-ALL proliferation in a dose dependent manner, whereas PRT060318 did not induce apoptosis in concentrations as high as 5µM. This supports the notion that pre-BCR signaling activity may be more relevant for preB-ALL proliferation than for preB-ALL viability. In line with these results the pre-BCR- proB-ALL cell lines REH and RS4;11 were highly resistant to Syk inhibition in all functional assays (IC50 > 10µM), suggesting that pre-BCR expression is a prerequisite for sensitivity to Syk inhibition. To examine the molecular changes following pre-BCR inhibition, ALL cells were treated with increasing concentrations of PRT060318 (100nM-5µM) for two hours and then subjected to immunoblotting. Syk inhibition led to a dose dependent decrease in AKT phosphorylation in all preB-ALL cell lines and subsequently reduced phosphorylation of FOXO transcription factors. In the resistant proB-ALL cell line REH, AKT and FOXO phosphorylation were not affected. Gene expression analysis of the preB-ALL cell lines RCH-ACV and Nalm-6 further suggested that PRT060318 interferes with pre-BCR signaling. Treatment with 1µM PRT060318 for 72h reduced the expression of genes associated with pre-BCR signaling (e.g. BCL6, CD22, PTPN6) and Ingenuity Pathway Analysis identified pre-BCR signaling as the main target of PRT060318 in both cell lines (p<0.05). We are currently validating the GEP analysis by quantitative PCR and immunoblotting. In conclusion, we provide evidence for the efficacy of Syk inhibition in pre-BCR+ ALL. Moreover we were able to correlate the baseline phosphorylation status of pre-BCR associated proteins and pre-BCR expression levels with the sensitivity of preB-ALL to the Syk inhibitor PRT060318. These findings provide a first rationale for the clinical testing of Syk inhibitors in preB-ALL, and suggest that activation status of pre-BCR associated molecules can help in selecting preB-ALL cases that are particularly sensitive to Syk inhibition. Disclosures: Coffey: Portola Pharmaceuticals: Employment.

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Development of CD4+CD8+ thymocytes in RAG-deficient mice through a T cell receptor β chain-independent pathway.

Journal of Experimental Medicine ◽

10.1084/jem.181.3.1187 ◽

1995 ◽

Vol 181 (3) ◽

pp. 1187-1195 ◽

Cited By ~ 51

Author(s):

C J Guidos ◽

C J Williams ◽

G E Wu ◽

C J Paige ◽

J S Danska

Keyword(s):

T Cell ◽

T Cell Receptor ◽

B Lymphocyte ◽

Cell Receptor ◽

Sublethal Dose ◽

Lymphocyte Development ◽

Site Specific ◽

B Lymphocyte Development ◽

Rag 1 ◽

Deficient Mice

Antigen-binding diversity is generated by site-specific V(D)J recombination of the T cell receptor (TCR) and immunoglobulin loci in lymphocyte precursors. Coordinate expression of two structurally distinct recombinase activating genes, RAG-1 and RAG-2, is necessary for activation of site-specific V(D)J recombination. In mice bearing targeted disruptions of either the RAG-1 or RAG-2 genes, T and B lymphocyte development is arrested at the CD4-8- double negative (DN) thymocyte or B220+/CD43+ pro-B cell stage. Development of CD4+CD8+ double positive (DP) thymocytes is restored by expression of a functionally rearranged TCR beta transgene, suggesting that TCR beta expression is critical for this developmental transition. We have found that treatment of adult or newborn RAG-deficient mice with a single sublethal dose of gamma-irradiation rescues the DN to DP transition in early thymocytes, and this is accompanied by a dramatic increase in thymus cellularity. In contrast to the observed induction of thymocyte maturation, there was no phenotypic or functional evidence of coincident B lymphocyte development in irradiated RAG-deficient mice. Interestingly, maturation of DP thymocytes occurred without expression of TCR beta protein in the cytoplasm or on the cell surface. These results suggest an in vivo pathway for DP thymocyte development which is TCR beta chain independent.

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Faculty Opinions recommendation of Ablation of CD22 in ligand-deficient mice restores B cell receptor signaling.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029979.351430 ◽

2005 ◽

Author(s):

Marcus Clark

Keyword(s):

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Receptor Signaling ◽

B Cell Receptor Signaling ◽

Deficient Mice ◽

Cell Receptor Signaling

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ERP, a new member of the ets transcription factor/oncoprotein family: cloning, characterization, and differential expression during B-lymphocyte development

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3292-3309.1994 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3292-3309

Author(s):

M Lopez ◽

P Oettgen ◽

Y Akbarali ◽

U Dendorfer ◽

T A Libermann

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Gene Family ◽

B Cell ◽

Binding Sites ◽

B Lymphocyte ◽

Striking Similarity ◽

Lymphocyte Development ◽

B Lymphocyte Development ◽

Ets Transcription Factor

The ets gene family encodes a group of proteins which function as transcription factors under physiological conditions and, if aberrantly expressed, can cause cellular transformation. We have recently identified two regulatory elements in the murine immunoglobulin heavy-chain (IgH) enhancer, pi and microB, which exhibit striking similarity to binding sites for ets-related proteins. To identify ets-related transcriptional regulators expressed in pre-B lymphocytes that may interact with either the pi or the microB site, we have used a PCR approach with degenerate oligonucleotides encoding conserved sequences in all members of the ets family. We have cloned the gene for a new ets-related transcription factor, ERP (ets-related protein), from the murine pre-B cell line BASC 6C2 and from mouse lung tissue. The ERP protein contains a region of high homology with the ETS DNA-binding domain common to all members of the ets transcription factor/oncoprotein family. Three additional smaller regions show homology to the ELK-1 and SAP-1 genes, a subgroup of the ets gene family that interacts with the serum response factor. Full-length ERP expresses only negligible DNA-binding activity by itself. Removal of the carboxy terminus enables ERP to interact with a variety of ets-binding sites including the E74 site, the IgH enhancer pi site, and the lck promoter ets site, suggesting a carboxy-terminal negative regulatory domain. At least three ERP-related transcripts are expressed in a variety of tissues. However, within the B-cell lineage, ERP is highly expressed primarily at early stages of B-lymphocyte development, and expression declines drastically upon B-cell maturation, correlating with the enhancer activity of the IgH pi site. These data suggest that ERP might play a role in B-cell development and in IgH gene regulation.

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Antigens Varying in Affinity for the B Cell Receptor Induce Differential B Lymphocyte Responses

Journal of Experimental Medicine ◽

10.1084/jem.188.8.1453 ◽

1998 ◽

Vol 188 (8) ◽

pp. 1453-1464 ◽

Cited By ~ 109

Author(s):

Valerie Kouskoff ◽

Sara Famiglietti ◽

Georges Lacaud ◽

Paul Lang ◽

James E. Rider ◽

...

Keyword(s):

Cell Proliferation ◽

B Cell ◽

Cell Activation ◽

B Lymphocyte ◽

Receptor Occupancy ◽

Cell Receptor ◽

Interferon Γ ◽

B Cell Receptor ◽

Multiple Parameters ◽

Biological Responses

The B cell receptor (BCR) triggers a variety of biological responses that differ depending upon the properties of the antigen. A panel of M13 phage-displayed peptide ligands with varying affinity for the 3-83 antibody was generated to explore the role of antigen-BCR affinity in cell activation studies using primary 3-83 transgenic mouse B cells. Multiple parameters of activation were measured. T cell–independent B cell proliferation, antibody secretion, induction of germline immunoglobulin γ1 transcripts, and B cell production of interleukin (IL) 2 and interferon γ responses were better correlated with antigen-BCR affinity than with receptor occupancy. In contrast, other responses, such as upregulation of major histocompatibility complex class II and B7.2 (CD86), secretion of IL-6, and B cell proliferation in the context of CD40 signaling were only weakly dependent on antigen affinity. Biochemical analysis revealed that at saturating ligand concentrations the ability of phage to stimulate some early signaling responses, such as Ca++ mobilization and tyrosine phosphorylation of syk or Igα, was highly affinity dependent, whereas the ability to stimulate Lyn phosphorylation was less so. These data suggest that the BCR is capable of differential signaling. The possibility that differential BCR signaling by antigen determines whether an antibody response will be T independent or dependent is discussed.

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B Lymphocyte Receptor (B cell receptor, BCR)

Encyclopedia of Genetics, Genomics, Proteomics and Informatics ◽

10.1007/978-1-4020-6754-9_1469 ◽

2008 ◽

pp. 178-178

Keyword(s):

B Cell ◽

B Lymphocyte ◽

Cell Receptor ◽

B Cell Receptor

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IVIg modulates BCR signaling through CD22 and promotes apoptosis in mature human B lymphocytes

Blood ◽

10.1182/blood-2009-12-261461 ◽

2010 ◽

Vol 116 (10) ◽

pp. 1698-1704 ◽

Cited By ~ 99

Author(s):

Jean-François Séïté ◽

Divi Cornec ◽

Yves Renaudineau ◽

Pierre Youinou ◽

Rizgar A. Mageed ◽

...

Keyword(s):

B Cell ◽

B Lymphocytes ◽

B Lymphocyte ◽

Cell Receptor ◽

B Cell Receptor ◽

Time Dependent ◽

G1 Phase ◽

Dependent Manner ◽

Bcr Signaling ◽

Sustained Activation

Abstract Among various mechanisms for interactions with B cells, intravenous immunoglobulin (IVIg) may operate through the insertion of its Fc part into the Fc-γ receptor, or the binding of its sialic acid (SA)–bearing glycans to the negatively regulating CD22 lectin. It appeared that IVIg reduces B lymphocyte viability in a dose- and time-dependent manner. Furthermore, we show by confocal microscopy that SA-positive IgG, but not SA-negative IgG bind to CD22. This interaction reduces the strength of B-cell receptor–mediated signaling trough down-regulating tyrosine phosphorylation of Lyn and the B-cell linker proteins, and up-regulating phospholipase Cγ2 activation. This cascade resulted in a sustained activation of Erk 1/2 and arrest of the cell cycle at the G1 phase. These changes may be accounted for the efficacy of IVIg in autoimmune diseases.

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B-Lymphocyte Receptor (B-Cell Receptor, BCR)

Encyclopedic Dictionary of Genetics, Genomics and Proteomics ◽

10.1002/0471684228.egp01485 ◽

2004 ◽

Keyword(s):

B Cell ◽

B Lymphocyte ◽

Cell Receptor ◽

B Cell Receptor

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B Lymphocyte Chemotaxis Regulated in Association with Microanatomic Localization, Differentiation State, and B Cell Receptor Engagement

Journal of Experimental Medicine ◽

10.1084/jem.187.5.753 ◽

1998 ◽

Vol 187 (5) ◽

pp. 753-762 ◽

Cited By ~ 196

Author(s):

Conrad C. Bleul ◽

Joachim L. Schultze ◽

Timothy A. Springer

Keyword(s):

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Messenger Rna ◽

B Lymphocyte ◽

Somatic Hypermutation ◽

Cell Receptor ◽

B Cell Receptor ◽

Cxc Chemokine

Migration of mature B lymphocytes within secondary lymphoid organs and recirculation between these sites are thought to allow B cells to obtain T cell help, to undergo somatic hypermutation, to differentiate into effector cells, and to home to sites of antibody production. The mechanisms that direct migration of B lymphocytes are unknown, but there is evidence that G protein–coupled receptors, and possibly chemokine receptors, may be involved. Stromal cell– derived factor (SDF)-1α is a CXC chemokine previously characterized as an efficacious chemoattractant for T lymphocytes and monocytes in peripheral blood. Here we show with purified tonsillar B cells that SDF-1α also attracts naive and memory, but not germinal center (GC) B lymphocytes. Furthermore, GC B cells could be converted to respond to SDF-1α by in vitro differentiation into memory B lymphocytes. Conversely, the migratory response in naive and memory B cells was significantly reduced after B cell receptor engagement and CD40 signaling. The receptor for SDF-1, CXC chemokine receptor 4 (CXCR4), was found to be expressed on responsive as well as unresponsive B cell subsets, but was more rapidly downregulated on responsive cells by ligand. Finally, messenger RNA for SDF-1 was detected by in situ hybridization in a layer of cells surrounding the GC. These findings show that responsiveness to the chemoattractant SDF-1α is regulated during B lymphocyte activation, and correlates with positioning of B lymphocytes within a secondary lymphoid organ.

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