Immunoglobulin a suppresses B cell receptor-mediated activation of mouse B cells with differential inhibition of signaling molecules

Author(s):  
Kouya Yamaki ◽  
Masato Terashi ◽  
Saori Yamamoto ◽  
Rei Fujiwara ◽  
Jun-ichi Inoue ◽  
...  
Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 147-147
Author(s):  
Cihangir Duy ◽  
Daniel Nowak ◽  
Lars Klemm ◽  
Rahul Nahar ◽  
Carina Ng ◽  
...  

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.


1997 ◽  
Vol 186 (8) ◽  
pp. 1299-1306 ◽  
Author(s):  
James R. Drake ◽  
Paul Webster ◽  
John C. Cambier ◽  
Ira Mellman

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.


Author(s):  
Sarah Wilmore ◽  
Karly-Rai Rogers-Broadway ◽  
Joe Taylor ◽  
Elizabeth Lemm ◽  
Rachel Fell ◽  
...  

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.


2021 ◽  
Author(s):  
Jun Li ◽  
Yurong Pan ◽  
Qingqing Ma ◽  
Long Ma ◽  
Bin Shi ◽  
...  

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.


2021 ◽  
Vol 12 ◽  
Author(s):  
Han Sun ◽  
Hu-Qin Yang ◽  
Kan Zhai ◽  
Zhao-Hui Tong

B cells play vital roles in host defense against Pneumocystis infection. However, the features of the B cell receptor (BCR) repertoire in disease progression remain unclear. Here, we integrated single-cell RNA sequencing and single-cell BCR sequencing of immune cells from mouse lungs in an uninfected state and 1–4 weeks post-infection in order to illustrate the dynamic nature of B cell responses during Pneumocystis infection. We identified continuously increased plasma cells and an elevated ratio of (IgA + IgG) to (IgD + IgM) after infection. Moreover, Pneumocystis infection was associated with an increasing naïve B subset characterized by elevated expression of the transcription factor ATF3. The proportion of clonal expanded cells progressively increased, while BCR diversity decreased. Plasma cells exhibited higher levels of somatic hypermutation than naïve B cells. Biased usage of V(D)J genes was observed, and the usage frequency of IGHV9-3 rose. Overall, these results present a detailed atlas of B cell transcriptional changes and BCR repertoire features in the context of Pneumocystis infection, which provides valuable information for finding diagnostic biomarkers and developing potential immunotherapeutic targets.


2010 ◽  
Vol 185 (12) ◽  
pp. 7405-7412 ◽  
Author(s):  
Tanisha A. Jackson ◽  
Christopher L. Haga ◽  
Götz R. A. Ehrhardt ◽  
Randall S. Davis ◽  
Max D. Cooper
Keyword(s):  
B Cells ◽  
B Cell ◽  

2020 ◽  
Vol 117 (42) ◽  
pp. 26318-26327
Author(s):  
Kamonwan Fish ◽  
Federico Comoglio ◽  
Arthur L. Shaffer ◽  
Yanlong Ji ◽  
Kuan-Ting Pan ◽  
...  

Epstein–Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.


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