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.

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 Immune Cell Profiling of COVID-19 Patients in the Recovery Stage by Single-Cell Sequencing

2020 ◽  
Author(s):  
Wen Wen ◽  
Wenru Su ◽  
Hao Tang ◽  
Wenqing Le ◽  
Xiaopeng Zhang ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Hospital Discharge ◽  
Immune Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Early Recovery ◽  
Recovery Stage

AbstractCOVID-19, caused by SARS-CoV-2, has recently affected over 300,000 people and killed more than 10,000. The manner in which the key immune cell subsets change and their states during the course of COVID-19 remain unclear. Here, we applied single-cell technology to comprehensively characterize transcriptional changes in peripheral blood mononuclear cells during the recovery stage of COVID-19. Compared with healthy controls, in patients in the early recovery stage (ERS) of COVID-19, T cells decreased remarkably, whereas monocytes increased. A detailed analysis of the monocytes revealed that there was an increased ratio of classical CD14++ monocytes with high inflammatory gene expression as well as a greater abundance of CD14++IL1B+ monocytes in the ERS. CD4+ and CD8+ T cells decreased significantly and expressed high levels of inflammatory genes in the ERS. Among the B cells, the plasma cells increased remarkably, whereas the naïve B cells decreased. Our study identified several novel B cell-receptor (BCR) changes, such as IGHV3-23 and IGHV3-7, and confirmed isotypes (IGHV3-15, IGHV3-30, and IGKV3-11) previously used for virus vaccine development. The strongest pairing frequencies, IGHV3-23-IGHJ4, indicated a monoclonal state associated with SARS-CoV-2 specificity. Furthermore, integrated analysis predicted that IL-1β and M-CSF may be novel candidate target genes for inflammatory storm and that TNFSF13, IL-18, IL-2 and IL-4 may be beneficial for the recovery of COVID-19 patients. Our study provides the first evidence of an inflammatory immune signature in the ERS, suggesting that COVID-19 patients are still vulnerable after hospital discharge. Our identification of novel BCR signaling may lead to the development of vaccines and antibodies for the treatment of COVID-19.Highlights-The immune response was sustained for more than 7 days in the early recovery stage of COVID-19, suggesting that COVID-19 patients are still vulnerable after hospital discharge.-Single-cell analysis revealed a predominant subset of CD14++ IL1β+ monocytes in patients in the ERS of COVID-19.-Newly identified virus-specific B cell-receptor changes, such as IGHV3-23, IGHV3-7, IGHV3-15, IGHV3-30, and IGKV3-11, could be helpful in the development of vaccines and antibodies against SARS-CoV-2.-IL-1β and M-CSF were discovered as novel mediators of inflammatory cytokine storm, and TNFSF13, IL-2, IL-4, and IL-18 may be beneficial for recovery.

CD8+ T Cells Engineered to Express a ROR1-Specific Chimeric Antigen Receptor Specifically Recognize ROR1 Positive B Cell Tumors.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 930-930
Author(s):  
Michael Hudecek ◽  
Thomas M Schmitt ◽  
Sivasubramanian Baskar ◽  
Wen-Chung Chang ◽  
David G Maloney ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Tyrosine Kinase ◽  
B Cell ◽  
Cell Line ◽  
Chimeric Antigen Receptor ◽  
Cd8 T Cells ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
Surface Expression

Abstract Abstract 930 The orphan tyrosine kinase receptor ROR1 was previously identified as a highly expressed gene by expression profiling of B cell chronic lymphocytic leukemia (B-CLL), [Klein et al. J Exp Med 2001], and has subsequently been shown to be expressed on mantle cell lymphoma (MCL) and a subset of B cell acute lymphoblastic leukemias (B-ALL). ROR1 encodes a 105 kDa protein that contains Ig-like, cysteine rich, kringle, tyrosine kinase and proline rich domains and is expressed during embryonic development but is absent on normal adult tissues including non-malignant B cells. The function of ROR1 in normal and malignant cells is not known, although secreted Wnt proteins have been proposed as candidate ligands. Analysis of ROR1 protein expression using specific polyclonal antibodies revealed uniform, stable, and restricted cell surface expression on B-CLL, suggesting this molecule is a candidate for targeted immunotherapy of B cell malignancies [Baskar et al. Clin Cancer Res 2008]. We constructed a lentiviral vector that encodes a chimeric antigen receptor (CAR) consisting of single chain variable (scFV) fragments of the heavy and light chains of a murine monoclonal antibody specific for ROR1, linked to an IgG4 Fc domain, the T cell receptor CD3 zeta chain and a CD28 costimulatory domain. The specificity and function of the ROR1 CAR was compared with a similarly designed CAR specific for the CD20 molecule, which is expressed on both malignant and normal B cells, and is being targeted with gene-modified T cells in clinical trials. Primary human CD8+ T cells were transduced with the ROR1 and CD20-specific CARs respectively, and T cells expressing high levels of the receptors were sort-purified using an anti-Fc antibody. T cells that expressed either the ROR1-specific CAR or the CD20-specific CAR efficiently lysed primary B-CLL samples (5/5) obtained from patients with advanced disease, and also lysed a MCL cell line (JeKo-1), and a ROR1+ B-ALL cell line (BALL-1). ROR1-specific T cells did not recognize the myeloid leukemia cell line K562, but efficiently lysed K562 cells that had been transfected to express ROR1, confirming the specific recognition of ROR1 on target cells. Consistent with the expression pattern of the target molecules, T cells that expressed the CD20-specific CAR also efficiently lysed normal primary and EBV-transformed B cells, but T cells that expressed the ROR1-specific CAR did not recognize nonmalignant or EBV-transformed B cells. Activation of normal B cells by engagement of the B cell receptor or activation through CD40 induced B cell proliferation and upregulation of the CD80 and CD86 costimulatory molecules, but did not result in ROR1 surface expression by flow cytometry or recognition by T cells that expressed the ROR1-specific CAR. These results suggest that targeting ROR1 with gene-modified T cells may have advantages over targeting B cell-lineage restricted molecules such as CD19 and CD20 that are expressed on normal mature B cells. Studies to determine whether ROR1 is expressed during a stage of normal B cell development are in progress. ROR1 is highly conserved in non-human primates and this model may be suitable to determine potential toxicities of adoptive immunotherapy with ROR1-specific CAR expressing T cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals B cell receptor triggering sensitizes human B cells to TRAIL-induced apoptosis

2010 ◽  
Vol 88 (5) ◽  
pp. 937-945 ◽  
Author(s):  
Andre Ortlieb Guerreiro-Cacais ◽  
Jelena Levitskaya ◽  
Victor Levitsky
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Human B Cells ◽  
Induced Apoptosis

SYK and LYN Mediate B-cell Receptor-independent Calcium-induced Apoptosis in DT-40 Lymphoma B-cells

2002 ◽  
Vol 43 (11) ◽  
pp. 2165-2170 ◽  
Author(s):  
De-Min Zhu ◽  
Heather E. Tibbles ◽  
Alexei O. Vassilev ◽  
Fatih M. Uckun
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Induced Apoptosis

scholarly journals Machine Learning Analysis of Naïve B-Cell Receptor Repertoires Stratifies Celiac Disease Patients and Controls

2021 ◽  
Vol 12 ◽  
Author(s):  
Or Shemesh ◽  
Pazit Polak ◽  
Knut E. A. Lundin ◽  
Ludvig M. Sollid ◽  
Gur Yaari
Keyword(s):  
Machine Learning ◽  
T Cells ◽  
Celiac Disease ◽  
B Cells ◽  
B Cell ◽  
Cd4 T Cells ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Machine Learning Classification ◽  
Gluten Peptides

Celiac disease (CeD) is a common autoimmune disorder caused by an abnormal immune response to dietary gluten proteins. The disease has high heritability. HLA is the major susceptibility factor, and the HLA effect is mediated via presentation of deamidated gluten peptides by disease-associated HLA-DQ variants to CD4+ T cells. In addition to gluten-specific CD4+ T cells the patients have antibodies to transglutaminase 2 (autoantigen) and deamidated gluten peptides. These disease-specific antibodies recognize defined epitopes and they display common usage of specific heavy and light chains across patients. Interactions between T cells and B cells are likely central in the pathogenesis, but how the repertoires of naïve T and B cells relate to the pathogenic effector cells is unexplored. To this end, we applied machine learning classification models to naïve B cell receptor (BCR) repertoires from CeD patients and healthy controls. Strikingly, we obtained a promising classification performance with an F1 score of 85%. Clusters of heavy and light chain sequences were inferred and used as features for the model, and signatures associated with the disease were then characterized. These signatures included amino acid (AA) 3-mers with distinct bio-physiochemical characteristics and enriched V and J genes. We found that CeD-associated clusters can be identified and that common motifs can be characterized from naïve BCR repertoires. The results may indicate a genetic influence by BCR encoding genes in CeD. Analysis of naïve BCRs as presented here may become an important part of assessing the risk of individuals to develop CeD. Our model demonstrates the potential of using BCR repertoires and in particular, naïve BCR repertoires, as disease susceptibility markers.

Sustained Signaling through the B-Cell Receptor Induces Mcl-1 and Promotes Survival of Chronic Lymphocytic Leukemia B-Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 178-178
Author(s):  
Dimitar G. Efremov ◽  
Aleksandar Petlickovski ◽  
Luca Laurenti ◽  
Xiaoping Li ◽  
Sara Marietti ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Spontaneous Apoptosis ◽  
B Cell Survival ◽  
Bcr Signaling ◽  
Induced Apoptosis

Abstract The clinical course of chronic lymphocytic leukemia (CLL) differs significantly between patients with mutated (M-CLL) and unmutated (U-CLL) immunoglobulin V genes, implying a role for B-cell receptor (BCR) signaling in the pathogenesis of this disease. BCR stimulation in normal B-cells triggers several crucial signaling pathways, including PI3K/Akt, IKK/NF- κB and the mitogen-activated protein kinases Erk, JNK and p38 MAPK, which can induce proliferation, survival, differentiation or apoptosis, depending on the nature and context of the antigenic stimulation. We have now investigated activation of these downstream signaling pathways, as well as induction of anti-apoptotic proteins and survival of CLL B-cells stimulated with soluble (sol-IgM) and immobilized (imm-IgM) anti-IgM antibodies, which were used to mimic stimulation with soluble and particulate/membrane-bound antigen, respectively. Stimulation with sol-IgM revealed similar activation patterns in the 10 U-CLL and 12 M-CLL cases that partially resembled the pattern described for tolerant B-cells. The response in the U-CLL cases was characterized by transient (<45 minutes) phosphorylation of Akt and Erk, no activation of JNK and p38 MAPK, and activation of IKKβ in 50% of the cases. Most M-CLL cases showed similar activation of Akt and Erk, but lacked activation of IKKβ, whereas three M-CLL cases were completely non-responsive. To investigate the effects on CLL B-cell survival, 14 U-CLL and 19 M-CLL cases were analyzed by Annexin V/PI staining after 48 hours stimulation with sol-IgM. A 10–40% increase in apoptotic cells was observed in the majority of cases from both CLL subsets (p<0.001 with respect to spontaneous apoptosis). Induction of apoptosis was confirmed by analyzing cleavage of the Caspase 3 substrate PARP, and was accompanied by an approximately 50% reduction in the levels of Mcl-1, an antiapoptotic protein implicated in CLL B-cell survival and resistance to chemotherapy. A markedly different response was induced by imm-IgM, which was characterized by activation of IKKβ in all cases and sustained Akt and Erk phosphorylation that persisted over 24 hours. This response resulted in a 2.5 fold mean increase in the levels of Mcl-1, whereas no changes were observed in the levels of Bcl-2 and Bcl-xL. Imm-IgM slightly reduced the percentage of cells undergoing spontaneous apoptosis after 48 hours, but significantly protected from fludarabine- and methylprednisolone-induced apoptosis. To investigate which of the three imm-IgM activated pathways is responsible for induction of Mcl-1 and protection from chemotherapy-induced apoptosis, we incubated CLL B-cells with LY294002, U0126 and BAY-11 (inhibitors of PI3K, ERK and NF- κB, respectively) prior to stimulation with imm-IgM and addition of fludarabine. Induction of Mcl-1 and inhibition of fludarabine-induced PARP cleavage were significantly abrogated only by LY294002, indicating that the PI3K/Akt pathway is the major link between the BCR and apoptosis resistance of CLL B-cells. In conclusion, this study shows that the response of CLL B-cells to BCR stimulation primarily depends on the nature of the antigenic stimulus. Moreover, it shows that only sustained BCR signaling can promote survival of CLL B-cells, and raises the possibility that the distinct clinical and biological behavior of U-CLL and M-CLL is determined by the availability of such stimulation.

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