The Role of the BCR Class Expressed by Eμ-TCL1tg Mice and Human CLL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 182-182
Author(s):  
Marcus Dühren-von Minden ◽  
Thomas Wossning ◽  
Hassan Jumaa ◽  
Hendrik Veelken
Keyword(s):  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Operating Characteristics ◽  
Lymphoma Cells ◽  
Mutational Status ◽  
Significant Difference ◽  
Single Positive

Abstract Abstract 182 The B-cell antigen receptor (BCR) plays a critical role in the development and progression of B-cell lymphomas. In chronic lymphocytic leukemia (CLL), the existence of stereotyped heavy-chain complementarity determining regions (HCDR3) suggested that binding of external antigens might play a role in CLL pathogenesis. In contrast, we recently reported that BCRs derived from both human CLL patients and from Eμ-TCL1tg mice have the unique function to induce antigen-independent signaling. This capacity is mediated by the HCDR3 through binding to a BCR internal motif in adjacent BCRs on the same cell (Dühren-von Minden et al., Nature 2012). Mature B cells as well as CLL B cells co-express IgM and IgD with the same HCDR3. In this study, we address the respective roles of these expressed BCR classes in lymphoma pathogenesis in Eμ-TCL1tg mice and in human CLL. By mating Eμ-TCL1tg-mice with IgM−/− mice, which lacks the μ constant heavy domain (μCH) and instead expresses IgD in all developmental stages, we demonstrate a significantly lower frequency of CD19+CD5+IgM+IgDlow lymphoma cells in heterozygous IgM+/−TCL1 mice compared to conventional IgM+/+TCL1 mice (p=0.007). Furthermore, IgM+/−TCL1 mice show a delayed or slowed disease progression compared to TCL1tg mice carrying both IgM alleles. In both TCL1tg mice strains, lymphoma development was exclusively linked to expression of surface IgM, since no IgD single positive lymphoma was detected in IgM+/−TCL1 mice (n=12). TCL1tg mice that lack both μCH alleles showed an accumulation of CD19+CD5+ cells in the spleen at the age of 6 months. According to the genotype of these mice, this population was indeed IgD single positive. However, no further progression could be observed during follow-up to an age of 8 months, indicating a benign form of lymphoproliferation. In contrast to BCRs derived from Eμ-TCL1 mice, analysis of the signaling properties of BCRs derived from IgM−/−TCL1 mice failed to show any autonomous signaling capacity, even when they were expressed as IgM. To address whether IgD in general is able to mediate autonomous signaling reported for TCL1tg- and CLL-derived BCRs, we tested these receptors for autonomous signaling capacity when expressed as IgD. However, expression as IgD led to a complete loss of autonomous signaling capacity in all cases (n=10). In conclusion, whereas autonomous signaling is a characteristic feature of TCL1tg- and CLL-derived BCRs, the pathogenesis of CLL is dependent on the expression of their BCR as the IgM isotype. Expression of IgD-BCRs leads to loss of autonomous signaling capacity, and mice that lack μCH fail to develop malignant lymphoproliferation. To address the question if differential expression of IgD and IgM also had an impact on the clinical behavior of human CLL, we measured the relative expression levels of surface IgD and IgM on circulating lymphoma cells from 67 CLL patients by flow cytometry with simultaneous staining. According to previous reports (Mockridge et al., Blood 2007), unmutated (UM-CLL) cases (n=22) had a higher level of total surface Ig compared to mutated CLL (M-CLL) cases (n=45). Based on our results that IgM is more potent to drive lymphoproliferation, we calculated the ratio of mean fluorescence intensities for IgD over IgM, further called DvM-Score, for every case. A significant difference in the expression pattern as represented by the DvM-Score was observed for UM-CLL and M-CLL (p=0.003) as well as for ZAP70+ and ZAP70- cases (p=0.0002). Both UM-CLL cases as well as ZAP70+ cases show a higher amount of IgM compared to IgD represented by a DvM-Score of <1, whereas the majority of M-CLL and ZAP70− cases express less IgM than IgD and show a DvM-Score of >1. Based on receiver operating characteristics, an DvM cut-off value of 1.15 was identified to optimally discriminate mutational status (AUC: 0.72) and ZAP70 expression (AUC: 0.78). Preliminary data show that CLL samples with a DvM-Score <1.15 had a more aggressive disease course as indicated by a median time to first treatment (TTFT) of 39 months, whereas cases with a DvM-Score of >1.15 show a median TTFT of 154 months (log rank test, p=0.0014). In summary, our results demonstrate an important role of the BCR class, especially with respect to the pathogenetic role of autonomously active IgM BCRs expressed by CLL B cells, for the outcome of the disease. In addition, the DvM score may represent a convenient and novel prognostic marker for CLL. Disclosures: No relevant conflicts of interest to declare.

TRAF3 Over-Expression Contributes to the Enhanced Alternative NF-κB Activity in Hodgkin's Lymphoma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3665-3665
Author(s):  
Feng Guo ◽  
Peng Zhou ◽  
Liang Ma
Keyword(s):  
B Cells ◽  
B Cell ◽  
Expression Vector ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Activity Assay ◽  
Lymphoma Cells ◽  
Aberrant Expression ◽  
Wide Range ◽  
P100 Processing

Abstract Abstract 3665 Poster Board III-601 Introduction Hodgkin and Reed-Sternberg (H-RS) cells are originated from germinal center B cells. Constitutive nuclear factor κB (NF-κB) activation is one of the molecular characteristic futures of H-RS cells. TNFR-associated factors (TRAFs) participate in a wide range of biological processes, such as adaptive and innate immunity, stress response, and bone metabolism, which are mediated by the induction of cell survival, proliferation, and differentiation. Among those, TRAF3 are reported as a negative regulator of the alternative NF-κB signaling pathway in B cells. How TRAF3 functions in H-RS cells is currently unclear. Methods Electromobility shift assay (EMSA) was performed to examine the NF-κB activity in B cell-derived Hodgkin's cells (L428 and KM-H2). An ELISA-based NF-κB family transcription factor activity assay was performed to quantify NF-κB DNA-binding in nuclear extracts from L428 cells. p100 processing, the expression of other NF-κB family members in the cytoplasm, and TRAF3 expression were detected by Western blot analysis. The effects of TRAF3 in L428 cells were studied by transient expression of TRAF3 expression vector. Results In this study, we found that TRAF3 was minimally detected in B cell-derived Hodgkin's cell lines (L428 and KM-H2) either in mRNA or protein levels. Both the classical (p50-RelA) and the alternative (p52-RelB) NF-kB activity were consistently activated in L428 cells, measured by EMSA and TransAM NF-kB activity assay. The enhanced alternative NF-κB activity, accompanied by increased p100 processing and RelB accumulation in the cytoplasm were detected in L428 cells. Transient transfection of TRAF3-expression vector enforced the expression of TRAF3 and blocked the p100 processing in L428 cells. The alternative NF-kB activity was partially decreased whereas the classical NF-kB activity remained intact. In addition, the increased TRAF3 expression did not affect the anti-apoptotic effects in L428 cells. Conclusions Not only the classical NF-κB activity but also the alternative NF-κB activity characterized by p100 processing and p52-RelB nuclear localization is constitutively activated in B cell-derived lymphoma cells. Lack of TRAF3 expression might be one of the reasons for the aberrant expression of alternative NF-κB activity. TRAF3 is indeed an important molecule regulating the activation of the alternative NF-kB activity but not the classical NF-kB activity in H-RS cells. Disclosures: No relevant conflicts of interest to declare.

Defining The Role Of Microrna-146a In B Cell Lymphomagenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3805-3805
Author(s):  
Jorge Contreras ◽  
Jayanth Kumar Palanichamy ◽  
Tiffany Tran ◽  
Dinesh S. Rao
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Cell Lymphoma ◽  
Expression Patterns ◽  
Significant Proportion ◽  
B Cell Lymphoma ◽  
Gene Expression Patterns

Abstract Diffuse large B cell lymphoma (DLBCL) is one of the most common Non-Hodgkin lymphomas among adults. It is a heterogeneous disease characterized by multiple mutations and translocations. Gene expression profiling studies have revealed several characteristic gene expression patterns, with two main patterns emerging, namely Germinal Center(GC) type, and Activated B Cell (ABC) type. ABC-type DLBCL shows gene expression patterns that resemble activated B-cells, with increased expression of anti-apoptotic, and pro-proliferative genes. Critically, upregulation of the NF-κB the pathway is a hallmark of ABC-type DLBCL and has been shown to be necessary for survival, and is caused by several different mutations at different levels within the pathway. Recent work has revealed the critical importance of a new class of small RNA molecules, namely microRNAs, in gene regulation. Of these, microRNA-146a (miR-146a) was discovered as an NF-κB induced microRNA that plays a role as a negative feedback regulator of this pathway by targeting adaptor proteins. To further characterize miR-146a, mice deficient for this miRNA were created, and were found to develop lymphadenopathy, splenomegaly, and myeloid proliferation. As expected, immune cells in these mice have an upregulated NF-κB pathway and many of the phenotypes can be ameliorated by inhibition of the NF-κB pathway. Importantly, a significant proportion of the animals develop B-cell lymphoma at older ages. In this study, we examined the role of miR-146a in the development of malignancy in B-cells. To accelerate the role of miR-146a in tumor formation we overlaid the miR-146a deficient allele onto the Eμ-Myc like mouse model. Eμ-Myc mice develop tumors on average by 14weeks of age. The transgenic status of animals was verified by genotyping, RNA and protein expression analyses. miR-146a sufficient and deficient animals on the Eμ-Myc background were followed for tumor latency by peripheral blood analysis and careful physical examination. Based on approved humane criteria for animal discomfort, animals were sacrificed and hematopoietic tissue was harvested for analysis. Mice deficient for miR-146a had a statistically reduced survival in comparison with miR-146a sufficient animals with a p-value of .0098 (Kaplan Meir survival analysis). Complete Blood Count of animals at time of death revealed an increase leukemia presentation in the miR-146a deficient background. FACS analysis of tumor tissue from both groups revealed an increase in the number of IgM positive tumors in the miR-146a-deficient background indicating skewing towards more mature B cell neoplasms when miR-146a is lacking. Lineage analysis of tumors verified them to be of B cell origin although a subset of miR-146a sufficient tumors had higher numbers of infiltrating myeloid cells compared to deficient animals. Furthermore, histologic analysis of hematopoietic organs showed that while infiltration remained similar in kidneys and liver, more spleens in the miR-146a deficient background tended to be less involved. Our extensive histopathologic and immunophenotypic analyses indicate that miR-146a deficiency drives a more aggressive malignant phenotype in the B-cell lineage. In keeping with this, our profiling studies of human DLBCL suggest that a subset of DLBCL show decreased expression of miR-146a. We are currently examining the status of NF-κB in the murine tumors and using high throughput sequencing approaches to delineate gene expression differences between miR-146a sufficient and deficient tumors. We anticipate the discovery of novel gene targets of miR-146a and expect that these studies will lead to improved diagnostic and therapeutic options for patients of B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

Clinical Monoclonal B Lymphocytosis (cMBL), Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL): Diagnostic Criteria, Features At Diagnosis and Natural History

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5273-5273
Author(s):  
Rodrigo Santacruz ◽  
Julio Delgado ◽  
Tycho Baumann ◽  
Maria Rozman ◽  
Martha Aymerich ◽  
...  
Keyword(s):  
Overall Survival ◽  
Multivariate Analysis ◽  
B Cells ◽  
B Cell ◽  
Cell Count ◽  
Diagnostic Criteria ◽  
Conflicts Of Interest ◽  
Consistent Difference ◽  
Transformation Rate ◽  
Mutational Status

Abstract Introduction CLL, SLL and cMBL are considered to be part of the same spectrum of clonal expansions of CD5+ B cells. Clinically, the transition from one to another of these forms over time is a well recognized event. Diagnostic criteria to separate these disorders have been proposed (IWCLL, 2008; WHO, 2008). Aim To compare presenting and evolving features of three groups of patients with cMBL, Rai 0 CLL or SLL and to ascertain the usefulness of current diagnostic criteria for these disorders. Patients and Methods Retrospective study of clinical, biologic and evolving characteristics of patients diagnosed with cMBL, Rai 0 CLL or SLL according to current criteria (CLL: ≥5x109 clonal B cells/L in peripheral blood; SLL <5x109/L clonal B cells with lymphadenopathy, organomegaly, cytopenia or disease-related symptoms; cMBL <5x109 clonal B cells with no signs or symptoms). Results Baseline characteristics of the patients are shown in the Table. Median age was 68 years (range, 24-94) and 57% of patients were males. Out of 1,093 patients, 79 had cMBL (7.2%), 522 Rai 0 CLL (48%) and 94 SLL (8.6%). Overall, adverse biomarkers such as high LDH (p<0.001), high B2M (p<0.001), increased ZAP70 (p<0.001), high CD38 (p<0.001), unmutated IGHV status (p=0.002), +12 (p=0.02) and 11q- (p=0.01) were significantly more frequent in SLL. In subgroup analyses, the only difference between cMBL and Rai 0 CLL was a higher proportion of cases with mutated IGHV in cMBL (p=0.008). Furthermore, when SLL was compared to Rai I to IV CLL no differences were observed (data not shown). The actuarial risk for transformation from cMBL or SLL to CLL was 4.2% and 4.4 % per year (p=0.5), respectively. Median TTFT was significantly shorter in the SLL group (12 m.) than in Rai 0 CLL (174 m.) or cMBL (244 m.) (p<0.001). Median overall survival was also significantly shorter for SLL (94 m.) compared with Rai 0 CLL and cMBL (153 and 157 m., respectively) (p=0.028). Multivariate analysis of 695 patients (cMBL/Rai 0-CLL/SLL) revealed four variables independently correlated with shorter TTFT: diagnosis of SLL vs. Rai 0 CLL vs. cMBL (HR 2.28; p=0.008), high ZAP70 (HR 4.08; p<0.001), high CD38 (HR 4.68; p=0.001) and increased serum B2M levels (HR 1.54; p = 0.031). Importantly, however, when the multivariate analysis was restricted to patients with cMBL and Rai 0 CLL, variables correlated with TTFT were the clonal B-cell count (HR 3.76; p=0.01), ZAP70 (HR 3.31; p<0.001), and CD38 (HR 4.61; p=0.02). FISH cytogenetics, IGHV mutational status,NOTCH1 or SF3B1 mutations were not included in the analysis because of missing data. Conclusions Disparities in biologic and clinical features of cMBL, CLL and SLL mainly reflect the different tumor burden in this spectrum of CD5+ monoclonal B cell disorders. In this study, the transformation rate from either cMBL or SLL to CLL was around 4% per year. As a result of differences in tumor mass, the need for therapy was shorter in SLL than in Rai 0 CLL and cMBL, and the overall survival poorer. Biologically, the only consistent difference between cMBL and Rai O CLL was a higher proportion of mutated IGHV in cMBL. Clinically, however, no differences in median survival were observed. Moreover, the clonal B cell count was the most reliable predictor of disease outcome in both cMBL and Rai 0 CLL. Therefore, the distinction between cMBL and Rai 0 CLL seems hardly justified. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Circadian Clock Protein CRY Controls B-Cell Intrinsic Tolerance

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1029-1029 ◽  
Author(s):  
Qi Cao ◽  
Xuan Zhao ◽  
Sigal Gery ◽  
Zhengshan Chen ◽  
Ruishu Deng ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Signaling Pathway ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Adaptive Immune System ◽  
Cell Tolerance ◽  
Double Knockout ◽  
B Cell Tolerance ◽  
Physiological Processes

Abstract The circadian system regulates numerous physiological processes including adaptive immune system. Here we show that mice deficient for the circadian genes Cry1 and Cry2, (Cry double knockout [DKO]) display an autoimmune phenotype including higher serum IgG concentration than wild type (WT) mice, presence of serum anti-nuclear antibodies, precipitation of IgG, IgM and complement 3 (C3) in glomeruli, and massive infiltrations of leukocytes into the lung and kidney. A large panel of autoantigens demonstrated that the sera of the Cry DKO mice but not the WT mice, had autoantibodies covering most of the specificities reported to be present in patients with SLE, rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome and other autoimmune disorders. Taken together, lost of the CRY circadian protein leds to severe autoimmunity. Furthermore, flow cytometry analysis of lymphoid organs showed lower pre-B cell numbers and higher mature recirculating B cells in the bone marrow as well as increased number of B2 B cells in the peritoneal cavity of Cry DKO mice. The BCR-proximal signaling pathway plays a critical role in peripheral B cell tolerance and activation. Activation of splenic B cells from the Cry DKO mice elicited markedly enhanced and prolonged tyrosine phosphorylation of cellular proteins compared to WT mice, suggesting that a very active BCR signaling pathway may contribute to impaired B cell tolerance in the Cry DKO mice. In summary, our results suggest that B cell development, as well as the intrinsic checkpoints of immune tolerance, are under direct circadian control. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting BCL6 and Germinal Centers (GCs) in Chronic Graft-Versus-Host Disease (cGVHD) Using Direct and Epigenomic Therapies

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 535-535 ◽  
Author(s):  
Ryan P Flynn ◽  
Katelyn Goodman ◽  
Jun Qi ◽  
Du Jing ◽  
Angela Panoskaltsis-Mortari ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Pulmonary Function Tests ◽  
Critical Role ◽  
Fold Increase ◽  
Affinity Maturation ◽  
Lung Dysfunction ◽  
Direct Acting

Abstract To study transcriptional pathways and develop targeted therapeutics for cGVHD, we studied the effects of emerging therapeutic agents targeting GCs. BCL6 is a master regulatory transcription factor, which facilitates GC development in a cooperative manner with emerging chromatin-associated factors, namely the EZH2 lysine methyltransferase and the BRD4 epigenetic reader protein. Using a murine multi-organ system cGVHD model induced by low T cell numbers, we have shown that cGVHD is dependent on pathogenic antibodies and the GC reaction. To determine whether strategies that disrupt GC integrity could be used to treat cGVHD we targeted Bcl6 using a direct-acting ligand (79-6), EZH2 using three structurally-distinct inhibitors (JQ-E, UNC1999 and DZNep), and a first-in-class BRD4 inhibitor (JQ1). In T cells, Bcl6 is selectively expressed in T follicular helper and regulatory cells. Using donor T cells from a Bcl6-mCherry reporter, we observed a 10-fold increase in % of CD4+/mCherry+ splenic T cells in cGVHD vs bone marrow (BM) only controls on d56. The small-molecule Bcl6 inhibitor 79-6 was synthesized and used to treat mice with active cGVHD from d28-56. Treatment reversed cGVHD-induced bronchiolitis obliterans (BO) measured by d56 pulmonary function tests (PFTs). This correlated with a 2-fold decrease in GC B cells and decrease in lung collagen (Fig 1 C). EZH2 catalyzes the methylation of lysine 27 on histone 3 (H3K4me3) silencing genes to a transcriptionally repressive state. EZH2 is massively upregulated during the GC reaction, and prevents GC B cell terminal differentiation, allowing affinity maturation to occur. Furthermore, naive T cells express low EZH2 levels, EZH2 is rapidly upregulated upon allostimulation.To determine whether EZH2 expression in donor BM or splenic T cells was critical for cGVHD, recipients were given wild type (WT) vs EZH2 floxed, Cg1-Cre (for B cell specific EZH2 deletion) BM + WT T cells, WT BM + EZH2 floxed, CD4-Cre vs WT T cells or WT BM + WT T cells. EZH2 deletion in either BM-derived B cells or splenic T cells completely prevented cGVHD (Fig 1 A) with a decrease in d56 splenic GC frequency (Fig 1 B). We compared 3 drugs that reduce H3K4me3: two pyridinone inhibitors, which inhibit SAM-dependent methylation (JQ-E and UNC1999) and DZNep, which destabilizes EZH2 complexes. At established doses given d28-56, DZNep and UNC1999 were ineffective or toxic, respectively. In contrast, JQ-E fully reversed cGVHD lung dysfunction and fibrosis around the bronchioles was significantly decreased (Fig 1 C). JQ1 is a first in class epigenetic reader that recognizes histone modifications and has been shown to reduce B cell lymphomas via inhibiting super-enhancer-associated transcripts and to treat cardiac failure-induced fibrosis. JQ1 given to cGVHD mice d28-56 significantly inhibited BO as assessed by PFTs and collagen deposition (Fig 1 D). Taken together, these data demonstrate for the first time the critical role of Bcl6 and targeting of histones that affect the transcriptional repressive states via EZH2 and a BET bromodomain epigenetic reader. These data provide a strong foundation for clinical trials of inhibitors that directly or epigenetic modifiers and readers that indirectly target Bcl6. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

scholarly journals Ridd Is Required for the Prevention of Chronic Gvhd By Targeting IRE-1a/Xbp-1s Signaling

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1681-1681
Author(s):  
Hee-Jin Choi ◽  
Chih-Hang Anthony Tang ◽  
Linlu Tian ◽  
Yongxia Wu ◽  
Mohammed Hanief Sofi ◽  
...  
Keyword(s):  
T Cells ◽  
Stress Response ◽  
B Cells ◽  
Er Stress ◽  
B Cell ◽  
Critical Role ◽  
Secretory Cells ◽  
Hematopoietic Stem ◽  
Er Stress Response

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an effective therapeutic procedure to treat hematological malignancies. However, the benefit of allo-HCT is limited by a major complication, chronic graft-versus-host disease (cGVHD). Since transmembrane and secretory proteins are generated and modified in the endoplasmic reticulum (ER), the ER stress response is of great importance to secretory cells including B cells. By using conditional knock-out (KO) of XBP-1, IRE-1α or both specifically on B cells, we demonstrated that the IRE-1α/XBP-1s pathway, one of the major ER stress response mediators, plays a critical role in B cell pathogenicity on the induction of cGVHD in murine models of allo-HCT. Endoribonuclease activity of IRE-1α not only activates XBP-1s transcription factor by converting unspliced XBP-1 (XBP-1u) mRNA into spliced XBP-1 (XBP-1s) mRNA but also cleaves other ER-associated mRNAs through regulated IRE-1α-dependent decay (RIDD). Besides, it is known that ablation of XBP-1s production leads to unleashed activation of RIDD. Therefore, we hypothesized that RIDD plays an important role in B cells during cGVHD development. In this study, we found that B cells deficient for XBP-1s reduced ability to induce cGVHD, which however was reversed by inactivation of IRE-1α, highlighting the role of RIDD in controlling cGVHD (Fig. A). Activation of RIDD targets IgM mRNA of (Fig. B), a contributor to organ damage and fibrosis in cGVHD, which correlated with dysregulated expression of MHC II and costimulatory molecules such as CD86, CD40, and ICOSL in B cells (Fig. C). Alloreactive T cells need to be primed by APCs to initiate GVHD, and specifically, CD86 and CD40 mediated-costimulation from APCs has been demonstrated to play an essential role in eliciting cGVHD. We demonstrated that alloreactivity of T cells, especially CD4 T cells, can be recovered by suppressing RIDD in XBP-1s-deficient B cells (Fig. D). Since IRE-1α carrying a S729A mutation shows ablated RIDD activity without effect on splicing XBP-1 mRNA, we investigated the contribution of B cells from S729A knock-in mice to confirm the role of RIDD in B cells. We found that B cells from S729A mice increased GVHD severity (Fig. E). S729A B cells showed significant increases in IgM secretion (Fig. F), GC cell differentiation (Fig. G), and the expression levels of MHCII and co-stimulatory factors (Fig. H). In conclusion, these results provide a novel insight on how ER stress response regulates B cell activity after allo-HCT and suggest RIDD is an important mediator for reducing cGVHD pathogenesis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The Role of Microtubule Regulatory Protein Stathmin (STMN1) in Megakaryopoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4591-4591
Author(s):  
Camelia Iancu-Rubin ◽  
Joseph Tripodi ◽  
Vesna Najfeld ◽  
George F. Atweh
Keyword(s):  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Critical Role ◽  
Regulatory Protein ◽  
Active Form ◽  
Fish Analysis ◽  
Ploidy Levels ◽  
Significant Difference

Abstract Abstract 4591 Megakaryopoiesis is a complex process in which hematopoietic progenitor cells proliferate and acquire megakaryocyte (MK)-specific markers then undergo polyploidization (i.e. acquisition of DNA content >2n) and cytoplasmic maturation, and start producing platelets. Polyploidization and platelet formation are highly dependent on microtubule (MT) function. To become polyploid, MK undergo abortive mitosis that is mediated by a mitotic spindle that consists of MT. Mature polyploid MK extend cytoplasmic extensions (i.e. proplatelets) into the vascular space and release platelets into the circulation. MT provide the structural scaffold for the proplatelets and mediate the transport of organelles and specific granules into nascent platelets. Despite the critical role of MT in MK biology, the regulation of MT in MK is poorly defined. Stathmin (STMN1) is a cytosolic phosphoprotein whose major function is to regulate MT function by promoting their depolymerization. We had previously shown that STMN1 is expressed at high levels early during megakaryopoiesis and is downregulated later during MK maturation. We also showed that inhibition of STMN1 expression increased ploidy while its overexpression decreased ploidy of MK-like cell lines. Thus, we hypothesized that the dynamic regulation of STMN1 expression may be necessary for megakaryopoiesis and that perturbing its expression may impair MK polyploidization and platelet production. To test this hypothesis, we developed feline immunodeficiency virus (FIV)-based lentiviruses that express STMN1 to investigate the effects of overexpression in primary MK. Since the depolymerizing activity of STMN1 can be inactivated by a variety of cellular kinases, we generated a STMN1 vectors that expresses wild-type (WT) and another that expresses a contitutively active phosphorylation-deficient mutant of STMN1 (MT). We also developed a vector that expresses GFP as a negative control. Human MK generated ex vivo in liquid culture from CD34+ cells were infected with these different lentiviruses. After ectopic STMN1 expression by RT-PCR and flow cytometry was confirmed, MK differentiation was assessed in the presence or absence of STMN1 overexpression. Uninfected MK and MK infected with GFP lentiviruses differentiated and matured into large, easily recognizable cells with typical nuclear morphology and expressed similar levels of CD41 and CD42b by flow cytometry. The numbers of MK generated in the presence of WT-STMN1 expressing lentiviruses was similar to that generated in the cultures infected with control lentiviruses, while the number of MK generated in the presence of phosphorylation-deficient MT-STMN1 was drasticaly reduced. Similarly, the numbers of CD41+ and CD42b+ MK generated in the presence of MT-STMN1 was reduced two and three times, respectively, suggesting that overexpression of a contitutively active form of STMN1 prevents MK differentiation and maturation. We then evaluated the effects of STMN1 overexpression on MK polyploidization by determining the number X and Y chromosomes by FISH analysis. While a normal diploid cell has one copy of each chromosome, cells with ploidy levels of 4N, 8N and 16N will have 2, 4 and 8 copies, respectively. There was no significant difference between the fraction of polyploid MK infected with control-GFP and those infected with WT-STMN1 lentiviruses. In contrast, the fraction of polyploid MK infected with MT-STMN1 lentiviruses was reduced by approximately 50%, suggesting that STMN1 overexpression impairs the ability of MK to become polyploid. In conclusion, we demonstrated that perturbing the normal downregulation of STMN1 in primary human MK impairs differentiation and polyploidization. Since STMN1 is expressesd at extremely high levels in a variety of human leukemias, we have started assessing STMN1 expression expression in patients with hematological malignancies characterized by striking abnormalities in their MK lineage. Such studies might validate the role of MT regulation in MK biology in vivo and support the development of potential therapeutic strategies to target MT and/or STMN1 function in MK and platelet disorders. Disclosures: No relevant conflicts of interest to declare.

B Cell Stimulation through BCR and CD40 Modulates the Response of Chronic Lymphocytic Leukemia Cells to BAFF and APRIL.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1361-1361
Author(s):  
Gerardo Ferrer ◽  
Kate E Hodgson ◽  
Victor Ciria ◽  
Gael Roue ◽  
Dolors Colomer ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Activation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Cd40 Ligation ◽  
Stimulation Of

Abstract Abstract 1361 The two TNF family proteins (B-cell activating factor [BAFF] and a proliferation-inducing ligand [APRIL]) and their three receptors (transmembrane activator and CAML interactor [TACI], B-cell maturation antigen [BCMA], and BAFF receptor [BAFF-R]) play a critical role in the process of differentiation, maturation and survival of normal B cells. Additionally, recent studies indicate that activation or inhibitory signals can modulate the sensitivity of normal B cells to BAFF and APRIL through the regulation of their receptors. In chronic lymphocytic leukemia (CLL), BAFF and APRIL have been shown to increase survival of neoplastic B cells in vitro. We investigated whether stimulation of CLL cells through the B cell receptor (BCR) or CD40 ligation could regulate the expression of BAFF-R, TACI and BCMA and enhance BAFF and APRIL sensitivity. Purified B cells were obtained from 23 CLL patients and nine healthy controls. Receptor expression was measured by flow cytometry at baseline and at 48 hours after stimulation with F(ab’)2 antihuman IgM (10 μg/ml) and CD40L (500ng/ml) plus IL-4 (20ng/ml). Cell activation and viability, as assessed by labeling CD69 and Annexin V/TO-PRO-3, were evaluated at 48, and at 72 hours after co-stimulation with either soluble BAFF (100ng/ml) or APRIL (500ng/ml). Baseline analyses showed that BAFF-R was the most highly expressed receptor in CLL cells and normal B cells (Mean fluorescence intensity (MFI) ratios, 213.5 and 185.8, respectively). TACI and BCMA were also expressed in all CLL cells and normal B cells (MFI ratios TACI: 2.5 and 1.9; BCMA: 14.8 and 6.6, respectively), but at a significantly lower level than BAFF-R (p<0.001). Furthermore, BCMA MFI ratio was significantly higher in CLL than in normal B cells (p=0.015). After 48h of culture, an increase of all three receptors was observed in normal B cells in response to either BCR stimulation or CD40 ligation. In contrast, in CLL cells BCR stimulation induced almost no variation in the receptors expression in all cases. This was accompanied by a failure of cell activation and a significant decreased viability of CLL cells (from 36% to 24% p=0.013). By contrast, CD40 ligation in CLL cells induced a significant upregulation of TACI expression (p=0.007) and a significant reduction of BCMA (p=0.007), which correlated with an increase of CLL cell activation and viability (p<0.001). BAFF-R levels did not change. The addition of exogenous soluble BAFF or APRIL showed increase in the viability of normal B cells at 72 hours independently of whether cells were unstimulated or stimulated through the BCR or by CD40 ligation. In CLL cells, however, the viability was significantly increased in CD40-stimulated cells whereas in either unstimulated or BCR-stimulated CLL cells, the addition of BAFF and APRIL had a modest effect on viability (Table). These findings indicate that stimulation of CLL cells through the BCR and CD40 modifies the sensitivity of CLL cells to respond to BAFF and APRIL which reflects the regulation of BCMA, TACI and BAFF-R. In contrast to normal B cells, CD40-ligation in CLL cells upregulated only TACI expression. The fact that the addition of CD40L plus IL-4 and BAFF increased viability in CLL cells while BAFF alone had almost no effect may be related to the ability of CD40 ligation to increase TACI expression. Although BCR stimulation failed to increase the expression of the receptors, co-stimulation by BAFF plus BCR increased viability in CLL cells. Disclosures: No relevant conflicts of interest to declare.

B Cell Deficiency and CD21low B Cells As Markers For Measuring Cgvhd Severity and Activity In a Large Patient Cohort

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4623-4623
Author(s):  
Zoya Kuzmina ◽  
Jeremy Rose ◽  
Najibah Rehman ◽  
Edward W. Cowen ◽  
Kristin Baird ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Antigen Presentation ◽  
Conflicts Of Interest ◽  
Receptor Expression ◽  
Hla Dr ◽  
Cellular Marker ◽  
Anti Cd20 ◽  
B Cell Deficiency

Background In chronic graft versus host disease (cGVHD) a role for B cells in disease pathogenesis is supported by the presence of autoantibodies and the therapeutic efficacy of B cell depletion. Active cGVHD is also associated with altered B cell homeostasis. Elevated levels of B cell activation factor (BAFF) are coupled to a high incidence of lymphopenia and an elevated frequency of an atypical population of CD21- B cells. In order to assess the utility of altered B cell populations as markers of cGVHD, we examined the frequency and clinical features of B lymphopenia and characterized atypical CD21- B cells in a large cohort of patients. Patients and methods Between October 2004 and May 2013, B cells (CD19+) were assessed in 260 patients consecutively enrolled in a cross-sectional natural history study of cGVHD (NCT00092235). We assessed B cell transitional, naïve, memory and plasmablast populations and the expression of markers, of antigen presentation (CD86, HLA-DR), tissue homing (CXCR3, CXCR4, CXCR5) and activation (CD95, CD11c, CD38, HLA-DR). Non-parametric Mann-Whitney comparisons of populations were used with Bonferroni correction for multiple comparisons. Results Of 260 patients examined, 44% (n=114) were severely B-lymphopenic (fewer than 100 B cells/µl; median 22). Compared to cGVHD patients with normal to elevated B cell numbers (median 563), these patients had reduced levels of immunoglobulin (IgM, IgG, IgA), decreased T subset and NK cells and significantly higher levels of BAFF (4.7 vs. 2.9 ng/ml, p=.003). B-lymphopenic patients had more active cGVHD (34% vs. 12%, p=.001), and had received more lines of systemic therapy (4.4 vs. 3.3, p<.0001), including prior anti-CD20 therapy for cGVHD (39% vs. 10%, p<.0001). These patients had shorter durations of cGVHD (2.5 years vs. 3.7, p=.007) and time from transplant (3.6 vs. 4.8 years, p=.007) Clinically they had a higher frequency of skin involvement (54% vs. 27%, p<.001), mainly erythematous (p<.001) and a lower Karnofsky score (p=.005). Anti-CD20 can result in prolonged B cell deficiency. Excluding such patients from our analysis, we identified a significant expansion (>10%) of an atypical population of CD19+CD21low B cells in 40% of the patients. Consistent with previous reports, these high %CD21low patients were receiving more immunosuppression (p=.008), had lower Karnofsky scores (p=.036), and increased mortality (p=.018). To further characterize the potential role of CD21low cells in cGVHD, in 26 patients we then studied expression of markers associated with B cell maturation, activation, trafficking and capacity for antigen presentation. Comparing patients with more than 10% CD21low B cells (12) with those with less, the high %CD21low group had a lower frequency of naïve (IgD+CD27-) B cells (p=.006), but a higher frequency of IgD-CD27- B cells. The CD21low B cells, and in particular those that were IgD- and CD27+, showed evidence of prior activation: increased size (forward scatter), expression of CD95 and of the Toll receptor-inducible integrin CD11c. Chemokine receptor expression on CD21low B cells reflected an increased capacity to home to inflammatory sites. CXCR3 (receptor responding to interferon-induced chemokines) was increased, while CXCR4 and CXCR5 (supporting trafficking of naïve B cells to lymph nodes) were reduced. Finally, expression of CD86, a critical molecule for antigen presentation by B cells, was increased. All of these changes were more pronounced in the patient subset with a higher frequency of CD21lowCD27- cells: higher expression of CXCR3 (p=.027), CD11c (p=.001), CD95 (p=.001) and CD86 (p=.008), but decreased CXCR4+CXCR5+ (p=.035). Conclusion B-lymphopenia is common in cGVHD population and is associated with severe and active disease, thus posing important and frequently underappreciated limitations to studying B cells. Patients with severe cGVHD and circulating B cells show accumulation of CD21low B cells. Disease activity and “chronicity” can be related to B cells activation (CD95, CD11c), altered migration (downregulation of CXCR4/5, increased CXCR3) and an increased capacity to function as APC (CD86+). Alterations in B cell subpopulations may explain the role of B cells in cGVHD pathogenesis as well as provide noninvasive cellular marker for objective diagnosis and monitoring of cGVHD activity. Disclosures: No relevant conflicts of interest to declare.

scholarly journals A critical role of TAK1 in B-cell receptor–mediated nuclear factor κB activation

Blood ◽  
2009 ◽  
Vol 113 (19) ◽  
pp. 4566-4574 ◽  
Author(s):  
James Schuman ◽  
Yuhong Chen ◽  
Andrew Podd ◽  
Mei Yu ◽  
Hong-Hsing Liu ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Critical Role ◽  
Cell Receptor ◽  
Nuclear Factor Κb ◽  
Cell Development ◽  
B Cell Receptor ◽  
Nuclear Factor

Abstract The kinase TAK1 is essential for T-cell receptor (TCR)–mediated nuclear factor κB (NF-κB) activation and T-cell development. However, the role of TAK1 in B-cell receptor (BCR)–mediated NF-κB activation and B-cell development is not clear. Here we show that B-cell–specific deletion of TAK1 impaired the transition from transitional type 2 to mature follicular (FO) B cells and caused a marked decrease of marginal zone (MZ) B cells. TAK1-deficient B cells exhibited an increase of BCR-induced apoptosis and impaired proliferation in response to BCR ligation. Importantly, TAK1-deficient B cells failed to activate NF-κB after BCR stimulation. Thus, TAK1 is critical for B-cell maturation and BCR-induced NF-κB activation.

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