Aberrant Splicing of the SLP65 SH2 Domain Enables Pre-B Cell Transformation and Compromises the Leukemia-Suppressive Function of the Pre-B Cell Receptor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 294-294
Author(s):  
Cihangir Duy ◽  
Lars Klemm ◽  
Rahul Nahar ◽  
Peter van Essen ◽  
Mieke Sprangers ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Full Length ◽  
Aberrant Splicing ◽  
Cell Stage ◽  
Suppressive Function

Abstract The linker molecule SLP65 is critical for pre-B cell receptor-mediated differentiation signals during early B cell development. Germline mutations of the SLP65 gene in mice and humans result in a profound differentiation block at an early pre-B cell stage. Likewise, B cell lineage acute lymphoblast leukemia (ALL) cells are phenotypically arrested at an early pre-B cell stage in the vast majority of cases. In about 2–5% of cases with ALL, mutations and deletions of the SLP65 gene are found (Sprangers et al., 2006; Mullighan et al., 2007). In a comprehensive study of SLP65 function in human ALL cells, we identified numerous aberrant SLP65 splice variants, which all lack one or more exons that encode the C-terminal SLP65 SH2 domain. A splice variant lacking exon 16, which encodes the central part of the SH2 domain was particularly abundant in ALL but not in normal pre-B cells (EMBL/GenBank Accession number AM180337). The SH2 domain may have critical function because it represents the main ligand for the non-ITAM Y204 in the Igα (CD79A) signaling chain of the pre-B cell receptor (Patterson et al., 2006) and thereby connects the pre-B cell receptor to downstream signaling molecules. To examine a potential role of the SLP65 SH2 domain during the process of malignant transformation in ALL, we measured mRNA levels of full-length SLP65 vs SLP65 splice variants, in which the SH2 domain was deleted (SLP65ΔSH2) in normal pre-B cells and primary human ALL cells by quantitative RT-PCR. While normal pre-B cells express full-length SLP65 at high and SLP65ΔSH2 at very low levels, the SLP65/SLP65ΔSH2 ratio was reduced in E2A-PBX1-driven ALL and particularly low in BCR-ABL1-driven ALL. Given that SLP65ΔSH2 is overexpressed in ALL cells at the expense of full-length SLP65, we investigated biochemical properties of SLP65ΔSH2. To this end, we transduced Slp65−/− pre-B cells with either full-length Slp65, SLP65ΔSH2 or a GFP empty vector control. As opposed to full-length SLP65, SLP65ΔSH2 does not interact with Igα and does not confer tyrosine phosphorylation of BTK downstream of the pre-B cell receptor. Also Ca2+-release in response to pre-B cell receptor engagement and induction of differentiation was drastically reduced in Slp65−/− pre-B cells that were reconstituted with SLP65ΔSH2 instead of SLP65. Co-expression experiments with SLP65 showed that SLP65ΔSH2 has a dominant-negative function and impairs pre-B cell receptor signaling. We next transformed Slp65−/− pre-B cells with a retroviral BCR-ABL1 vector and tested whether SLP65 and SLP65ΔSH2 have a different impact on BCR-ABL1-mediated pre-B cell transformation and BCR-ABL1-driven leukemic growth. Reconstitution of SLP65 expression in Slp65−/− BCR-ABL1-transformed pre-B ALL cells resulted in cell death and nine days after transduction more than 90% of the SLP65-transduced cells had undergone apoptosis. Conversely, reconstitution of Slp65−/− BCR-ABL1-transformed pre-B ALL cells with SLP65ΔSH2 had no significant effect on viability of the leukemia cells. In a titration experiment, we reconstituted SLP65−/−BCR-ABL1-driven ALL cells with SLP65 and SLP65ΔSH2 at various ratios and thereby confirmed that SLP65ΔSH2 has a dominant-negative effect and compromises the leukemia-suppressive function of the pre-B cell receptor. We next tested whether aberrant splicing also interferes with the tumor suppressor function of SLP65 in vivo: To this end, we engrafted SLP65−/−BCR-ABL1- driven ALL cells that were transduced with either SLP65-GFP, SLP65ΔSH2 or GFP into sublethally irradiated NOD/SCID mice. After 24 days, mice that were engrafted with ALL cells transduced with SLP65ΔSH2 or GFP became terminally leukemic, whereas mice injected with SLP65-transduced ALL cells showed no signs of disease. At this time, all mice were sacrificed and analyzed. Whereas mice with SLP65ΔSH2- or GFP-transduced ALL cells had enlarged spleens and substantial leukemic infiltration of the bone marrow (79 and 81% leukemia cell content), leukemic infitration was reduced if SLP65-transduced ALL cells were injected (1.8% leukemia infiltration). Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor.

Inactivation of Pre-B Cell Receptor-Mediated Tumor Suppression by Aberrant Splicing in Ph+ Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 579-579
Author(s):  
Cihangir Duy ◽  
Mieke Sprangers ◽  
Lars Klemm ◽  
Rahul R. Nahar ◽  
Daniel Nowak ◽  
...  
Keyword(s):  
B Cells ◽  
Tumor Suppressor ◽  
B Cell ◽  
Splice Site ◽  
Splice Variants ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
B Cell Receptor ◽  
Aberrant Splicing ◽  
Linker Molecules

Abstract Abstract 579 The pre-B cell receptor instructs differentiation of normal pre-B cells and functions as a tumor suppressor in pre-B acute lymphoblastic leukemia (ALL). Its function is impaired in virtually all cases of pre-B ALL carrying the Philadelphia chromosome (Ph), which encodes the BCR-ABL1 kinase. However, genomic deletions in pre-B cell receptor pathway are rare and the mechanism of its inactivation in ALL is not known. Here we report that aberrant splicing of SLP65 (BLNK, BASH) and the pre-B cell receptor-related linker molecules LCP2, SYK and LAT represents a major mechanism of pre-B cell receptor-inactivation, which is consistent with deregulated expression of splice factors in Ph+ ALL. In contrast to normal pre-B cells, BCR-ABL1-transformed pre-B ALL expressed multiple splice variants of the pre-B cell receptor-related linker molecules SLP65, LCP2, SYK and LAT. A detailed sequence analysis of these splice variants revealed that aberrant splicing resulted in functional inactivation of important protein domains in SLP65, LCP2, SYK and LAT transcripts. Of note, the vast majority of SLP65 splice variants lacked a functional SH2 domain, which is required to anchor SLP65 to the Igαa signaling chain of the pre-B cell receptor. We then focused our analysis on the SH2 domain of SLP65 for the following reasons: (1) SLP65 functions as a powerful tumor suppressor downstream of the pre-B cell receptor. (2) Exon 16 which encodes the central part of the SH2 domain was affected in the vast majority of SLP65 splice variants. (3) The SH2 domain of SLP65 is required to link downstream effector molecules (BTK, SYK, PLCγ2) to the Igαa signaling chain of the pre-B cell receptor. We found that deletion of the SH2 domain represents a frequent feature in Ph+ ALL. In reconstitution experiments with pre-B cells from SLP65-/- mice, SLP65 forms lacking the SH2 domain failed to support pre-B cell receptor signaling. By coexpression with full-length SLP65, we demonstrated that SH2-deficient variants have a dominant-negative effect by reducing the Ca2+-release and the leukemia-suppressive function of SLP65. These effects are dose-dependent as demonstrated in a titration experiment, in which SLP65 and SH2-deficient SLP65 were expressed at different ratios. SH2-deficient SLP65 also compromised the leukemia-suppressive function of the pre-B cell receptor in vivo transplantation model: While reconstitution of full-length SLP65 in SLP65-/- BCR-ABL1 ALL cells suppressed leukemia growth and bone marrow infiltration, loss of the SLP65 SH2 domain abolished the tumor suppressor function of the pre-B cell receptor. To address potential mechanisms of aberrant pre-mRNA splicing, we studied expression levels of splicing factors in Ph+ ALL and searched for splice site mutations: Consistent with aberrant splicing of pre-B cell receptor-related linker molecules including SLP65, SYK, LAT and LCP2, we found evidence of splice factor deregulation in Ph+ ALL: Among the 94 pre-mRNA splice factors studied, 24 were differentially expressed between Ph+ ALL (n=15) and normal bone marrow B cell precursors (n=11). Of note, the SRPK1, hnRPC, hnRNP-E2 (PCBP2) and hnRNP-A1 pre-mRNA splice factors that were previously found as BCR-ABL1-regulated, were also identified in our analysis. Sequence analysis of the 3'prime splice site of exon 16 in three Ph+ ALL cell lines and six primary cases of Ph+ ALL revealed one clonal splice site mutation in one primary Ph+ ALL case. In one cell line and one primary Ph+ ALL case, each one additional mutation was found within 2 basepairs distance from the splice site. It is not clear, however, whether and to which extent these mutations affect splice site selection. Interestingly, we found that clones amplified from one primary Ph+ ALL case and one Ph+ ALL cell line harbor mutations encoding premature translation stops close to the end of exon 16. While these mutations do not lead to aberrant splicing of exon 16, they reinforce the notion that Ph+ ALL cells are selected for inactivation of the SLP65 SH2 domain. We conclude that aberrant splicing frequently results in the expression of dominant-negative SH2-deficient SLP65, which is sufficient to compromise the tumor suppressor function of the pre-B cell receptor in Ph+ ALL. Whereas mutations and deletions of the SLP65 gene are rare, we conclude that aberrant splicing of the SLP65 SH2 domain represents a common mechanism in ALL cells to compromise the leukemia-suppressive function of the pre-B cell receptor. Disclosures: Martinelli: Novartis: Research Funding.

Mechanisms of Pre-B Cell Receptor-Inactivation In Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 147-147
Author(s):  
Cihangir Duy ◽  
Daniel Nowak ◽  
Lars Klemm ◽  
Rahul Nahar ◽  
Carina Ng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Signaling Molecules ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Leukemia Cells ◽  
Immunoglobulin Gene ◽  
Receptor Inactivation

Abstract Abstract 147 Background: We recently established that the pre-B cell receptor functions as a tumor suppressor in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The pre-B cell receptor promotes differentiation of normal pre-B cells and couples the immunoglobulin μ -chain to activating tyrosine kinases (e.g. SYK) via linker molecules (e.g. BLNK). In virtually all cases of Ph+ ALL, pre-B cell receptor function is compromised and its reconstitution induces rapid cell cycle arrest. However, genomic deletions in pre-B cell receptor pathway are rare and the mechanisms of inactivation are not known. Here we report that pre-B cell receptor inactivation occurs at multiple levels and involves at least four different mechanisms, namely (1) deleterious immunoglobulin gene rearrangement, (2) defective splicing of pre-B cell receptor signaling molecules, (3) expression of dominant-negative PAX5 fusion genes and (4) overexpression of inhibitory signaling molecules. Result: (1) Studying progressive transformation of pre-B cells in BCR-ABL1-transgenic mice, we observed that surface expression of the immunoglobulin μ -chain was downregulated after 60 days of age, which was a prerequisite for the onset of full-blown leukemia. While the repertoire of immunoglobulin gene rearrangements was polyclonal in wildtype pre-B cells, BCR-ABL1-transgenic pre-B cells show clonal expansions, which are derived from one ancestral productive immunoglobulin gene rearrangement in the transformed pre-B cell. However, the ancestral immunoglobulin gene rearrangements were rendered non-functional through deleterious secondary rearrangements. Likewise, in 47 of 57 cases of primary human Ph+ ALL, we detected traces of pre-B cell receptor-inactivation through secondary deleterious recombination events at the immunoglobulin μ -chain locus. (2) We studied pre-B cell receptor signaling molecules in primary human pre-B cells and 10 patient-derived Ph+ ALL samples by Western blotting and RT-PCR. As opposed to normal bone marrow pre-B cells, in all 10 cases of Ph+ ALL defective splice variants of the SYK tyrosine kinase and its linker molecule BLNK were found. Sequence analysis revealed a frequent 4 bp slippage during SYK pre-mRNA splicing which resulted in a truncated protein lacking the kinase domain, as confirmed by Western blot. To study the functional significance of defective Syk expression in Ph+ ALL cells, we transformed pre-B cells from Syk-fl/fl mice with BCR-ABL1 and deleted the Syk kinase using tamoxifen-inducible Cre. As opposed to Syk-fl/fl leukemia cells, inducible ablation of Syk rendered the leukemia cells insensitive to forced expression of the pre-B cell receptor. Multiple defective transcript variants of BLNK were found that all lacked exon 16 encoding the central part of the BLNK SH2 domain. In the absence of exon 16, BLNK splice variants were detached from the pre-B cell receptor and function in a dominant-negative way as they reduce Ca2+-mobilization in response to pre-B cell receptor stimulation. In a titration experiment, BLNK−/− leukemia cells were reconstituted with full-length and exon 16-deficient BLNK. Dominant-negative BLNK interfered with pre-B cell receptor-mediated tumor suppression at a ratio of 0.1 relative to full-length BLNK. Of note, we found somatic mutations within the splice site of exon 16 in 2 of 6 primary Ph+ ALL cases. (3) Ph+ ALL cells often carry chromosomal translocations leading to the expression of dominant-negative PAX5-fusion molecules. In a systematic gene expression analysis, we observed that ectopic expression of the dominant-negative PAX5-C20orf112 fusion led to downregulation of immunoglobulin μ -chain and the signaling molecules including SYK and BLNK. As a consequence, Ca2+-mobilization in response to pre-B cell receptor stimulation was significantly diminished. (4) Correction of defective immunoglobulin-μ chain and BLNK expression results in compensatory overexpression of a broad array of inhibitory signaling molecules. These molecules share an ITIM signaling motif, which attenuates pre-B cell receptor signal transduction through recruitment of inhibitory phosphatases. Conclusion: Even though loss of pre-B cell receptor function represents the uniform outcome of a diverse spectrum of lesions, individual Ph+ ALL subclones exhibit a complex pattern of shared and distinct defects involving one or more of these 4 mechanisms. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RasGRP1 Sensitizes an Immature B Cell Line to Antigen Receptor-induced Apoptosis

2004 ◽  
Vol 279 (19) ◽  
pp. 19523-19530 ◽  
Author(s):  
Benoit Guilbault ◽  
Robert J. Kay
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wehi 231 ◽  
Induced Apoptosis

RasGRP1 is a guanine nucleotide exchange factor that activates Ras GTPases and is activated downstream of antigen receptors on both T and B lymphocytes. Ras-GRP1 provides signals to immature T cells that confer survival and proliferation, but RasGRP1 also promotes T cell receptor-mediated deletion of mature T cells. We used the WEHI-231 cell line as an experimental system to determine whether RasGRP1 can serve as a quantitative modifier of B cell receptor-induced deletion of immature B cells. A 2-fold elevation in RasGRP1 expression markedly increased apoptosis of WEHI-231 cells following B cell receptor ligation, whereas a dominant negative mutant of RasGRP1 suppressed B cell receptor-induced apoptosis. Activation of ERK1 or ERK2 kinases was not required for RasGRP1-mediated apoptosis. Instead, elevated RasGRP1 expression caused down-regulation of NF-κB and Bcl-xL, which provide survival signals counter-acting apoptosis induction by B cell receptor. Inhibition of NF-κB was sufficient to enhance B cell receptor-induced apoptosis of WEHI-231 cells, and ligation of co-stimulatory receptors that activate NF-κB suppressed the ability of RasGRP1 to promote B cell receptor-induced apoptosis. These experiments define a novel apoptosis-promoting pathway leading from B cell receptor to the inhibition of NF-κB and demonstrate that differential expression of RasGRP1 has the potential to modulate the sensitivities of B cells to negative selection following antigen encounter.

scholarly journals Igβ tyrosine residues contribute to the control of B cell receptor signaling by regulating receptor internalization

2006 ◽  
Vol 203 (7) ◽  
pp. 1785-1794 ◽  
Author(s):  
Anna Gazumyan ◽  
Amy Reichlin ◽  
Michel C. Nussenzweig
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
B Cell Receptor ◽  
Receptor Internalization ◽  
Bcr Signaling ◽  
B Cell Receptor Signaling ◽  
B1 Cells

Immunoglobulin (Ig)α and Igβ initiate B cell receptor (BCR) signaling through immune receptor tyrosine activation motifs (ITAMs) that are targets of SH2 domain–containing kinases. To examine the function of Igβ ITAM tyrosine resides in mature B cells in vivo, we exchanged these residues for alanine by gene targeting (IgβAA). Mutant mice showed normal development of all B cell subtypes with the exception of B1 cells that were reduced by fivefold. However, primary B cells purified from IgβAA mice showed significantly decreased steady-state and ligand-mediated BCR internalization and higher levels of cell surface IgM and IgD. BCR cross-linking resulted in decreased Src and Syk activation but paradoxically enhanced and prolonged BCR signaling, as measured by cellular tyrosine phosphorylation, Ca++ flux, AKT, and ERK activation. In addition, B cells with the ITAM mutant receptor showed an enhanced response to a T-independent antigen. Thus, Igβ ITAM tyrosines help set BCR signaling threshold by regulating receptor internalization.

Grb2 Directly Interacts with HGAL and Ameliorates Its Effects on B-Cell Receptor Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 702-702
Author(s):  
Xiaoyu Jiang ◽  
Xiaoqing Lu ◽  
Brett J Schuchardt ◽  
David C Mikles ◽  
Amjad Farooq ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Germinal Center ◽  
Kinase Activity ◽  
Cell Receptor ◽  
Sh2 Domain ◽  
B Cell Receptor ◽  
Syk Kinase ◽  
Bcr Signaling

Abstract Human Germinal center Associated Lymphoma (HGAL) is specifically expressed in germinal center (GC) B-cells and GC-derived lymphomas. High expression of HGAL is an independent predictor of prolonged survival of Diffuse Large B-Cell (DLBCL) and classical Hodgkin (cHL) lymphoma patients. HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. HGAL increases BCR signaling by binding to and enhancing Syk kinase activity. However, our previous studies also suggested that other proteins may be involved in HGAL-mediated regulation of BCR signaling. In vitro kinase assays demonstrated that both Syk and Lyn can phosphorylate HGAL. Mass spectrometry (μ LC/MS/MS) demonstrated that these kinases can phosphorylate HGAL's tyrosines Y80, Y86, Y106Y107, Y128 and Y148. The HGAL Y106Y107 comprise a YYENV motif (aa 106-110) similar to the phosphopeptide motif pYXNX frequently used as a binding site to the SH2 domain of Growth Factor Receptor bound protein 2 (Grb2). Grb2 signaling in B cells controls lymphoid follicle organization and the GC reaction. Specifically, Grb2 is an integral component of the BCR signalosome and decreases BCR-induced Ca2+influx. The presence of the phosphorylated YYENV motif in HGAL raised the hypothesis that HGAL-Grb2 interactions may play a role in HGAL -mediated regulation of BCR signaling. To address this possibility, we performed reciprocal coimmunoprecipitations (Co-IPs) of endogenous HGAL and Grb2 in Raji and VAL lymphoma cell lines. These studies demonstrated that HGAL Co-IPs with Grb2. The interaction between these two proteins is dependent on the presence and phosphorylation of tyrosines in the YYENV motif, since an HGAL mutant in which these tyrosines were mutated to phenylalanine (FFENV) failed to Co-IP with Grb2. Isothermal titration calorimetry confirmed that phosphorylated (pYEN) but not unphosphorylated (YEN) HGAL-derived 12-mer peptides bind to the SH2 domain of Grb2 with an affinity of 5µM. GST-Grb2 pull down assays with recombinant Trx-HGAL(FFENV) and Trx-HGAL proteins confirmed that the HGAL-Grb2 interaction is direct and occurs only if the HGAL tyrosines are phosphorylated. Concordantly, addition of phosphatase to cellular lysates decreased the HGAL-Grb2 interaction. Furthermore, CO-IP studies demonstrated that HGAL's interaction with Grb2 increases following BCR stimulation-induced HGAL phosphorylation. Concordantly, confocal microscopy studies demonstrated HGAL-Grb2 colocalization in the cell membrane following BCR signaling activation. We next examined the functional significance of the HGAL-Grb2 interaction on BCR activation as measured by intracellular and transmembrane Ca2+ mobilization and phosphorylation of proximal BCR effectors (Syk (Y352), BLNK (Y84), BTK (Y551) and PLCγ2 (Y753) in several lymphoma cell lines (U2942, TMD8 and Mino) stablly transfected to express HGAL protein. HGAL expression markedly increased Ca2+ influx and phosphorylation of these proteins, while Grb2 knockdown only slightly increased transmembrane Ca2+ mobilization. Of note, concomitant HGAL expression and Grb2 knockdown further increased intracellular and transmembrane Ca2+ influx and phosphorylation of BCR effectors in comparison to HGAL expression alone. Expression of the HGAL (FFENV) mutant also enhanced Ca2+ influx and phosphorylation of BCR effectors in comparison to wild type HGAL. Concordantly, expression of the dominant negative Grb2 (W193K) mutant also enhanced HGAL's effects on BCR signaling. These observations suggest that Grb2's interaction with HGAL ameliorates HGAL's effects on BCR signaling. We previously showed that HGAL interacts with Syk and enhances Syk kinase activity. We now demonstrate that Grb2 Co-IPs with both Syk and HGAL and thus may potentially interfere with HGAL-Syk interaction. Indeed, knockdown of Grb2 increased HGAL Co-IP with the Syk kinase and this was associated with increased BCR signaling. These findings indicate that Grb2 ameliorates HGAL-mediated enhancement of BCR signaling by decreasing HGAL binding to Syk. In summary, out data demonstrates that Grb2 directly interacts with HGAL and ameliorates HGAL-enhanced BCR signaling. These interactions may play an important function in regulating the magnitude of BCR signaling during the GC reaction. Disclosures No relevant conflicts of interest to declare.

scholarly journals Delivery of B Cell Receptor–internalized Antigen to Endosomes and Class II Vesicles

1997 ◽  
Vol 186 (8) ◽  
pp. 1299-1306 ◽  
Author(s):  
James R. Drake ◽  
Paul Webster ◽  
John C. Cambier ◽  
Ira Mellman
Keyword(s):  
B Cells ◽  
B Cell ◽  
Antigen Processing ◽  
Specific Antigen ◽  
Cell Receptor ◽  
Class Ii ◽  
Time Frame ◽  
B Cell Receptor ◽  
Subcellular Compartment ◽  
Endocytic Vesicles

B cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II–restricted presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II– containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide–class II complexes occurs. Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative. The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing. Additionally, we have identified a putative CIIV-marker protein, immunologically related to the Igα subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.

scholarly journals Targeted inhibition of eIF4A suppresses B-cell receptor-induced translation and expression of MYC and MCL1 in chronic lymphocytic leukemia cells

2021 ◽  
Author(s):  
Sarah Wilmore ◽  
Karly-Rai Rogers-Broadway ◽  
Joe Taylor ◽  
Elizabeth Lemm ◽  
Rachel Fell ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Mrna Translation ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Mrna Levels ◽  
Mrna Stabilization

AbstractSignaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

scholarly journals Characterization of B-cell Receptor Heavy Chain Complementarity-determining Region 3 Repertoire Based on the Differences of Symbiotic Microorganisms in the Gut of Mice

2021 ◽  
Author(s):  
Jun Li ◽  
Yurong Pan ◽  
Qingqing Ma ◽  
Long Ma ◽  
Bin Shi ◽  
...  
Keyword(s):  
Small Intestine ◽  
B Cells ◽  
B Cell ◽  
Heavy Chain ◽  
Intestinal Microflora ◽  
Cell Receptor ◽  
Memory B Cells ◽  
B Cell Receptor ◽  
Positive Effects ◽  
Significant Difference

Abstract Background Colonization of gut microorganism is related to maturation of B cells in peripheral immune organs. This study aims to investigate the effect of intestinal microflora in Germ-free (GF), Specific Pathogen-free (SPF) and Clean (CL) BALB/C mice to small intestine total B-cell and memory B-cell receptor (BCR) complementary-determining region 3 (CDR3) repertoire. Results The composition and characteristics of intestinal microflora were analyzed by 16S rDNA sequencing. Genomic DNA extracted from small intestine tissue and memory B-cells of GF, SPF and CL mice were conducted via high-throughput DNA sequencing methods. As expected, significant differences of gut microflora diversity were observed in the three mice groups. CL group showed the most diversity, followed by SPF group, and GF group had the lowest diversity. Moreover, anormogenesis of intestinal lymphoid tissue were observed in GF mice. Diversity of the BCR heavy chain CDR3 repertoire in memory B cells were significant difference among three groups, but not in total B cells. The nucleotide polymorphism, usage frequency of gene segments (V, D, J, V–J gene segments) and amino acid of total B cells and memory B cells CDR3 were comparable among three mice groups, and there was significant difference between CL and GF mice groups. Conclusions The results of this study advocate that the colonization of intestinal microorganisms affect the diversity of B cells CDR3 repertoire. Elucidating mechanism of microbiome participated in the function of intestinal mucosal immune system may have positive effects on human health, and it requires further investigation.

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