Overexpression of the Protein Tyrosine Phosphatase Lyp Reduces the Responsiveness of Chronic Lymphocytic Leukemia B-Cells to B-Cell Receptor Ligation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 800-800
Author(s):  
Roberto Negro ◽  
Pablo G Longo ◽  
Michela Tarnani ◽  
Stefania Gobessi ◽  
Luca Laurenti ◽  
...  
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Signaling Molecules ◽  
Negative Regulator ◽  
Nucleotide Sequence Analysis ◽  
Aberrant Expression ◽  
Bcr Signaling

Abstract Abstract 800 CLL B cells display many features that suggest a role for antigen stimulation in the development and progression of the disease. These include the expression of stereotyped B-cell receptors (BCRs), the association between IgVH gene mutation status and prognosis, and the gene-expression profile of antigen-stimulated B cells. In addition, CLL B cells have other BCR-related features that distinguish them from normal B lymphocytes, such as lower levels of surface Ig, less efficient BCR signal transduction and increased basal activity of the proximal BCR signaling molecules Lyn and Syk. We have now investigated whether any of these features are related to aberrant expression or function of the phosphatases SHP-1, SHP-2 and Lyp (PTPN22), which regulate the amplitude and duration of the BCR signal by dephosphorylating various components of the BCR signal transduction unit. These phosphatases are also interesting because mutated or polymorphic variants have been linked to various malignant or autoimmune diseases. We started our study by performing nucleotide sequence analysis of the complete coding region of SHP1, SHP2 and Lyp in 8, 21 and 29 CLL B cell samples, respectively. Overall, only two mutations were identified (an R527C substitution in SHP2 and a Q456E substitution in Lyp, each in a single patient), suggesting that these phosphatases are infrequently mutated in CLL. The previously reported Lyp polymorphisms R620W and R263Q were observed in 2 additional cases. We next investigated expression of these phosphatases in purified CLL and normal B cells by immunoblotting. Expression of SHP1 and SHP2 was relatively uniform in the different CLL B-cells samples (n=42) and was not different from normal B cells (n=4). In contrast, expression of Lyp was markedly higher in most CLL samples, with 35 of the 49 investigated cases exhibiting 2 to more than 10 fold higher levels than normal B cells (n=5) (CLL, mean Lyp levels 4.7, SD +/−3.7; normal B cells, mean Lyp levels 0.9, SD +/−0.1, P=0.022). The mean Lyp levels were somewhat higher in U-CLL than M-CLL (6.0 vs. 3.9) and ZAP-70-positive than ZAP-70-negative cases (5.6 vs. 4.7), but these differences were not statistically significant. Analysis of Lyp expression in various lymphoma B-cell lines (n=9) also did not reveal significant differences with respect to normal B-cells, suggesting that Lyp overexpression is a specific feature of CLL. To determine what are the consequences of Lyp overexpression on BCR signaling, we downregulated Lyp in primary CLL B-cells by RNA interference and investigated activation of BCR signaling molecules following sIgM crosslinking. Downregulation of Lyp resulted in a substantial increase in BCR-induced phosphorylation of Lyn (Y397), Syk (Y352), BLNK (Y84) and ERK (T202/Y204), suggesting that overexpression of this phosphatase may be at least partially responsible for the lower BCR signaling capacity of CLL B-cells. Since Lyp expression can be induced in resting T cells by activation with anti-CD3, we investigated whether BCR stimulation will have a similar effect on CLL B-cells. A two-fold increase in Lyp levels was observed after 24 hours of sustained BCR stimulation with immobilized anti-IgM, whereas transient stimulation with soluble anti-IgM resulted in a 20% decrease in Lyp levels. These effects were specific for Lyp, since no such changes were observed in the expression of SHP1 and SHP2. In summary, this study shows that CLL B-cells specifically overexpress the phosphatase Lyp, and important negative regulator of BCR signaling that has been implicated in the pathogenesis of several common autoimmune diseases. Given the observation that Lyp can be induced by sustained BCR engagement and in view of recent findings that Lyp is also overexpressed in anergic B cells, these data further support the notion that CLL cells are continuously exposed to (auto)antigen in vivo. 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.

Overexpression of the Autoimmunity-Associated Tyrosine Phosphatase PTPN22 in CLL Cells Enhances Antiapoptotic B-Cell Receptor Signals Through Selective Activation of the AKT Pathway

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 619-619
Author(s):  
Roberto Negro ◽  
Stefania Gobessi ◽  
Pablo G Longo ◽  
Luca Laurenti ◽  
Dimitar G Efremov
Keyword(s):  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Negative Regulator ◽  
Gain Of Function ◽  
Akt Kinase ◽  
Bcr Signaling ◽  
Increased Activity

Abstract Abstract 619 The phosphatase PTPN22 is an important negative regulator of T cell receptor (TCR) signaling. A gain of function polymorphism in this phosphatase is strongly linked with increased risk for the development of several common autoimmune diseases, including rheumatoid arthritis, insulin-dependent diabetes mellitus and systemic lupus erythematosus. The exact mechanism how the more active PTPN22 variant contributes to the development of these autoimmune diseases is still unclear, but it is has been postulated that increased activity of this enzyme may prevent negative selection of autoreactive T cells by blocking proapoptotic TCR signals induced by autoantigen. We now report that PTPN22 is significantly overexpressed in CLL B cells. Of the 91 investigated CLL samples, in 66 (73%) the levels of PTPN22 were at least 2 fold higher and often exceeded by 10 fold the levels in normal tonsilar B cells (CLL, mean PTPN22 levels 5.7, SD +/−4.3; normal tonsilar B cells, mean PTPN22 levels 0.9, SD +/−0.1). PTPN22 levels were somewhat higher in CD38−positive than CD38−negative cases (P=0.031), whereas no association was observed with IGHV mutation status, ZAP-70 expression and time to treatment. To determine the functional consequences of PTPN22 overexpression, we performed RNA interference experiments in primary CLL B cells with high PTPN22. We evaluated the effects of PTPN22 silencing in unstimulated CLL cells as well as CLL cells stimulated through the B cell receptor (BCR), given the important role of the BCR signaling pathway in the pathophysiology of CLL. Both soluble and immobilized anti-IgM antibodies were used for BCR stimulation, as these two BCR crosslinking agents have opposing effects on leukemic cell survival. Silencing of PTPN22 had no effect on the viability of unstimulated CLL cells (n=11, viable CLL cells: siControl 49%, siPTPN22 48%, P=n.s.), but significantly inhibited the antiapoptotic effect of immobilized anti-IgM (viable CLL cells: siControl 55%, siPTPN22 48%, P=0.001) and enhanced the proapoptotic effect of soluble anti-IgM (viable CLL cells: siControl 44%, siPTPN22 31%, P<0.001). Consistent with these data, overexpression of PTPN22 in the lymphoma B cell line B104 significantly blocked apoptosis induced by soluble anti-IgM (control 22%, PTPN22 35%) without affecting the survival of unstimulated cells (control 52%, PTPN22 51%). The previous experiments suggested that PTPN22 functions as a molecular switch that enhances antiapoptotic and inhibits proapoptotic BCR signals. To establish the molecular mechanisms underlying these different activities, we studied BCR signal transduction in primary CLL cells transfected with control or PTPN22-specific siRNA. Downregulation of PTPN22 increased anti-IgM-induced activation of several BCR signaling molecules, including LYN, SYK, ERK, JNK and the proapoptotic p38MAPK, confirming that PTPN22 is primarily a negative regulator of BCR signaling. However, activation of the antiapoptotic AKT kinase was substantially reduced by silencing of PTPN22, as evidenced by decreased phosphorylation of AKT and its direct targets GSK3b and FOXO3a. Lack of PTPN22 expression was further associated with increased activity of the phosphatase SHIP, a key negative regulator of the AKT kinase. Opposite effects on the activity of these signaling molecules were observed when PTPN22 was overexpressed in B104 cells, thus validating the data obtained by PTPN22 knockdown in primary CLL cells. In summary, this study shows that the phosphatase PTPN22, which has been implicated in the pathogenesis of several common autoimmune diseases, is significantly overexpressed in CLL B-cells. Overexpression of PTPN22 inhibits activation of molecules that propagate the proapoptotic BCR signal, such as p38MAPK, but at the same time enhances the antiapoptotic signal delivered through the AKT kinase. The mechanism how PTPN22 enhances activation of AKT in response to BCR engagement is by preventing Lyn-mediated activation of the negative regulator SHIP. Collectively, these data show that PTPN22 is an important regulatory molecule in CLL and a potential therapeutic target. Inhibitors of this phosphatase, which are currently being developed for the treatment of autoimmune diseases associated with the gain of function PTPN22 variant, would be expected to convert the BCR survival signal into a death signal and may thus provide means for selective targeting of the malignant clone. Disclosures: No relevant conflicts of interest to declare.

CD22-Targeting Epratuzumab Down-Regulates Multiple BCR Regulators On the Surface of Normal, Lupus, and Malignant B Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1357-1357
Author(s):  
Edmund A Rossi ◽  
Rosana Michel ◽  
Daniel J Wallace ◽  
Chien-Hsing Chang ◽  
David M Goldenberg
Keyword(s):  
B Cells ◽  
Autoimmune Diseases ◽  
B Cell ◽  
Mononuclear Cells ◽  
Substantial Reduction ◽  
Therapeutic Activity ◽  
Down Regulation ◽  
Cell Functions ◽  
Healthy Donors ◽  
Bcr Signaling

Abstract Abstract 1357 Background. The humanized anti-CD22 antibody, epratuzumab, has demonstrated therapeutic activity in clinical trials of lymphoma and autoimmune diseases, treating currently over 1000 cases of non-Hodgkin lymphoma (NHL), leukemias, Waldenström's macroglobulinemia, Sjögren's syndrome, and systemic lupus erythematosus (SLE). Thus, epratuzumab offers a promising option for CD22-targeted immunotherapy, yet its mechanism of action (MOA) remains poorly understood to date. Because epratuzumab reduces on average 35% of circulating B cells in patients, and has minimal antibody–dependent cell-mediated cytotoxicity and negligible complement-dependent cytotoxicity when evaluated in vitro, we reason its therapeutic activity may not result completely from B-cell depletion; instead, ligation of epratuzumab to CD22 could modulate other surface molecules involved in regulating B-cell antigen receptor (BCR) signaling, leading to altered B-cell functions that ultimately mitigate symptoms of the underlying diseases. Here we report for the first time that epratuzumab induces a substantial reduction of CD22 along with CD19, CD21, and CD79b, on the surface of B cells in peripheral blood mononuclear cells (PBMCs) obtained from normal donors or lupus patients, and of cells from three NHL lines (Daudi, Raji, and Ramos) spiked into normal PBMCs. Intriguingly, only CD22, but not others, was appreciably down-regulated by epratuzumab in isolated B cells, implicating additional cell-based factors are required. Methods and Results. PBMCs, from either healthy donors or lupus patients with flares, were incubated with epratuzumab (10 μg/mL) for 2 h or 18 h, and the relative surface expression levels of CD22 and selected BCR regulators, including CD19, CD21, and CD79b, were analyzed by flow cytometry. Treatment of PBMCs from healthy donors with epratuzumab, but not an isotype control mAb, induced a marked down-regulation of CD22 (>80%), CD19 (>50%), CD21 (>50%) and CD79b (>30%) on the surface of B cells, with the stronger effect observed for CD27-negative B cells. The effect of epratuzumab could be observed after 2 h and increased moderately with overnight incubation (18 h). Moreover, substantial reduction of CD22 (>50%), but not others, was achieved with the F(ab')2 of epratuzumab generated from pepsin digestion. Similar results were observed for B cells in PBMCs of lupus patients. In the absence of PBMCs, treatment of NHL cell lines (Daudi, Raji and Ramos) with epratuzumab resulted in more than 80% reduction of CD22, with little, if any, reduction in CD19, CD21, CD79b or surface IgM observed. Inclusion of a crosslinking second antibody with epratuzumab induced only a minimal (<15%) suppression of CD19 and CD21. When these NHL cells (1×105 cells) were mixed with PBMCs (1×106 cells), epratuzumab induced a 40 to 70% reduction of CD19 and CD21, with significant down-regulation of surface IgM and CD79b also. Importantly, in all these experiments, there was no evidence of depletion in B cells, whether malignant or not; nor had epratuzumab induced an increase in apoptotic (Annexin V+) B cells in PBMCs from any of 4 donors, compared to the untreated (22.9 ± 1.8% versus 22.1 ±1.2%; P=0.6864). Conclusions. This study revealed a previously unknown, and potentially important, MOA of epratuzumab. The prominent down-regulation of CD19, CD21, and CD79b by epratuzumab is not only Fc-dependent, but also requires further engagement with certain effector cells present in PBMCs. The findings of reduced levels of CD19 are of particular relevance for the efficacy of epratuzumab in autoimmune diseases, because elevated CD19 has been correlated with susceptibility to SLE in animal models as well as in patients, and down-regulation of CD19 should attenuate activation of B cells by raising the BCR signaling threshold. Disclosures: Rossi: Immunomedics, Inc.: Employment; IBC Pharmaceuticals Inc.: Employment. Michel:Immunomedics, Inc.: Employment. Chang:Immunomedics, Inc.: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.

Role Of Lyn Kinase In The Pathogenesis Of Chronic Lymphocytic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 668-668
Author(s):  
Phuong-Hien Nguyen ◽  
Nina Reinart ◽  
Michael Hallek
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Lymphocytic Leukemia ◽  
Negative Regulator ◽  
Lymphoid Tissues ◽  
Malignant Cells ◽  
Deficient Mice

Abstract The Src family kinase Lyn is predominantly expressed in B cells and plays a central role in initiating B cell receptor (BCR) signaling. Lyn is associated with BCR complexes and is renowned for its role in B cell activation and proliferation. Active Lyn contributes to positive regulation of signalling through tyrosine phosphorylation of components of the BCR. Intriguingly, Lyn was also shown as a negative regulator of BCR signal transduction. Lyn plays an essential role in negative regulation of signalling through its unique ability to phosphorylate immunoreceptor tyrosine based inhibition motifs (ITIM) in inhibitory cell surface receptors. ITIM phosphorylation induces the recruitment of inhibitory phosphatases such as SHP-1/2 and SHIP-1, which attenuate BCR signalling. Lyn-deficient mice have reduced number of B cells and increased numbers of myeloid progenitors. It was reported that expression and activity of Lyn in human chronic lymphocytic leukemia (CLL) is elevated compared to healthy B cells. Besides, higher levels of Lyn are associated with a shorter treatment-free survival of CLL patients. This rises up a hypothesis about Lyn’s significant role in B cell tumorigenesis, malignant transformation of B cells, and the balance between myeloid cells and B lymphocytes. We generated Eµ-TCL1 transgenic LYN-deficient mice (TCL1+/wtLYN-/-) and monitored them in order to identify the population of malignant B cells and to characterize the development of malignant cells in these mice in comparison with Eµ-TCL1 transgenic mice (TCL1+/wtLYNwt/wt). In comparison to TCL1+/wtLYNwt/wt mice, TCL1+/wtLYN-/- mice show a significantly reduced number of malignant B cells in the peripheral blood, as well as a reduced leukocyte count. Besides, TCL1+/wtLYN-/- mice have significantly decreased infiltration of malignant B cells in lymphoid tissues such as spleen, liver, lymph node and bone marrow. This result is also resembled in a hepato-splenomegaly in the TCL1+/wtLYNwt/wt mice. These mice develop severe splenomegaly and hepatomegaly due to infiltration of malignant cells, while TCL1+/wtLYN-/- mice do not develop hepatomegaly. The non-transgenic LYN-/- control mice develop splenomegaly due to infiltration of myeloid cells. Although TCL1+/wtLYN-/- mice have hindered development of TCL1-induced CLL, preliminary data suggest it is not only due to LYN-deficiency in B cell compartment of these mice. Indeed, B cell of TCL1+/wtLYN-/- mice show enhanced proliferation and better survival ex vivo compared to TCL1+/wtLYNwt/wt mice. Notably, TCL1+/wtLYN-/- mice developed a skewed microenvironment which might contribute to CLL down regulation. LYN-/- microenvironment, particularly in aged mice, does not support engraftment of TCL1-induced leukemic B cell as well as LYNwt/wt mice in our transplantation model. These results point to a complex regulation of Lyn signalling in CLL involving not only leukemic cells but also cells of the micromillieu, that needs further investigation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Acute Csk inhibition hinders B cell activation by constraining the PI3 kinase pathway

2021 ◽  
Vol 118 (43) ◽  
pp. e2108957118
Author(s):  
Wen Lu ◽  
Katarzyna M. Skrzypczynska ◽  
Arthur Weiss
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Cell Activation ◽  
Antigen Receptor ◽  
Negative Regulator ◽  
B Cell Activation ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Bcr Signaling

T cell antigen receptor (TCR) and B cell antigen receptor (BCR) signaling are initiated and tightly regulated by Src-family kinases (SFKs). SFKs positively regulate TCR signaling in naïve T cells but have both positive and negative regulatory roles in BCR signaling in naïve B cells. The proper regulation of their activities depends on the opposing actions of receptor tyrosine phosphatases CD45 and CD148 and the cytoplasmic tyrosine kinase C-terminal Src kinase Csk. Csk is a major negative regulator of SFKs. Using a PP1-analog-sensitive Csk (CskAS) system, we have previously shown that inhibition of CskAS increases SFK activity, leading to augmentation of responses to weak TCR stimuli in T cells. However, the effects of Csk inhibition in B cells were not known. In this study, we surprisingly found that inhibition of CskAS led to marked inhibition of BCR-stimulated cytoplasmic free calcium increase and Erk activation despite increased SFK activation in B cells, contrasting the effects observed in T cells. Further investigation revealed that acute CskAS inhibition suppressed BCR-mediated phosphatidylinositol 3,4,5-trisphosphate (PIP3) production in B cells. Restoring PIP3 levels in B cells by CD19 cross-linking or SHIP1 deficiency eliminated the negative regulatory effect of CskAS inhibition. This reveals the critical role of Csk in maintaining an appropriate level of SFK activity and regulating PIP3 amounts as a means of compensating for SFK fluctuations to prevent inappropriate B cell activation. This regulatory mechanism controlling PIP3 amounts may also contribute to B cell anergy and self-tolerance.

scholarly journals Myc and the Warburg Effect

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. SCI-12-SCI-12
Author(s):  
Stefano Casola ◽  
Gabriele Varano ◽  
Laura Perucho ◽  
Marianna Ossorio ◽  
Federica Zanardi ◽  
...  
Keyword(s):  
B Cells ◽  
Mouse Model ◽  
B Cell ◽  
Metabolic Pathways ◽  
Gene Networks ◽  
Conflicts Of Interest ◽  
Lymphoproliferative Disorders ◽  
B Cell Malignancies ◽  
Lymphoma Cells ◽  
Bcr Signaling

Abstract Mature B cells recognize and respond in a highly-specific fashion to a multitude of environmental antigens through membrane-bound immunoglobulins forming together with the Igα and Igβ proteins a functional unit called the B cell antigen receptor (BCR). Through a complex network of effector molecules, the BCR transforms environmental signals into biochemical reactions which are responsible for highly codified cellular responses affecting survival, proliferation, migration and terminal differentiation of B cells. Surface BCR expression is conserved in most types of B cell malignancies arising from mature B cells. This observation, together with genetic and biochemical evidence pointing to sustained BCR signaling in different types of B cell neoplasms represents the rationale for the current use of pharmacological inhibitors of BCR signaling to treat several forms of B lymphoproliferative disorders. Nevertheless, our understanding of how the BCR influences malignant B cell behavior remains poorly understood. In an attempt to fill this knowledge gap, we engineered a mouse model to monitor the effects of acute ablation of the BCR in highly-aggressive MYC-driven lymphomas. Inducible BCR ablation did not, per se, prevent the outgrowth of receptor-less MYC lymphoma cells both in vitro and in vivo. Instead, BCR loss weakened the fitness of the malignant B cells leading to the rapid elimination of BCR-less tumor cells in the presence of their BCR-expressing counterparts (Varano et al., 2017). Through the integration of data generated from genomics, metabolomics and bulk/single cell transcriptomics analyses, comparing BCR-deficient lymphoma cells to their proficient counterparts, we have started to elucidate the gene networks and metabolic pathways influenced by BCR expression that sustain competitive fitness of MYC-transformed lymphoma B cells. Data from CRISPR/Cas9-mediated disruption of candidate fitness genes in primary malignant B cells will be presented. In support of the findings in the mouse model, we will provide evidence that BCR-less malignant B cells are spontaneously generated during tumor progression in several forms of human B cell lymphoproliferative disorders, establishing a possible Achilles heel of anti-BCR therapies. Finally, we will report possible strategies enabling the clearance of BCR-less lymphoma cells, taking advantage of their acquired addiction to specific signaling and metabolic pathways. Our results shed light on the coordinated regulation of signaling and metabolism imposed on malignant B cells by BCR expression/signaling and provide indications for improved treatment options to fight several forms of mature B cell malignancies. Reference: Varano G, Raffel S, Sormani M, Zanardi F, Lonardi S, Zasada C, Perucho L, Petrocelli V, Haake A, Lee AK, Bugatti M, Paul U, Van Anken E, Pasqualucci L, Rabadan R, Siebert R, Kempa S, Ponzoni M, Facchetti F, Rajewsky K, Casola S. Nature. 2017; 546:302-306. Disclosures No relevant conflicts of interest to declare.

scholarly journals Functional analysis of Csk in signal transduction through the B-cell antigen receptor

1994 ◽  
Vol 14 (11) ◽  
pp. 7306-7313
Author(s):  
A Hata ◽  
H Sabe ◽  
T Kurosaki ◽  
M Takata ◽  
H Hanafusa
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
B Cell ◽  
Tyrosine Phosphorylation ◽  
Kinase Activity ◽  
B Cell Antigen Receptor ◽  
Wild Type ◽  
Inactive State ◽  
Cell Clones ◽  
Bcr Signaling

In B cells, two classes of protein tyrosine kinases (PTKs), the Src family of PTKs (Lyn, Fyn, Lck, and Blk) and non-Src family of PTKs (Syk), are known to be involved in signal transduction induced by the stimulation of the B-cell antigen receptor (BCR). Previous studies using Lyn-negative chicken B-cell clones revealed that Lyn is necessary for transduction of signals through the BCR. The kinase activity of the Src family of PTKs is negatively regulated by phosphorylation at the C-terminal tyrosine residue, and the PTK Csk has been demonstrated to phosphorylate this C-terminal residue of the Src family of PTKs. To investigate the role of Csk in BCR signaling, Csk-negative chicken B-cell clones were generated. In these Csk-negative cells, Lyn became constitutively active and highly phosphorylated at the autophosphorylation site, indicating that Csk is necessary to sustain Lyn in an inactive state. Since the C-terminal tyrosine phosphorylation of Lyn is barely detectable in the unstimulated, wild-type B cells, our data suggest that the activities of Csk and a certain protein tyrosine phosphatase(s) are balanced to maintain Lyn at a hypophosphorylated and inactive state. Moreover, we show that the kinase activity of Syk was also constitutively activated in Csk-negative cells. The degree of activation of both the Lyn and Syk kinases in Csk-negative cells was comparable to that observed in wild-type cells after BCR stimulation. However, BCR stimulation was still necessary in Csk-negative cells to elicit tyrosine phosphorylation of cellular proteins, as well as calcium mobilization and inositol 1,4,5-trisphosphate generation. These results suggest that not only activation of the Lyn and Syk kinases but also additional signals induced by the cross-linking of the BCR are required for full transduction of BCR signaling.

B Cells at the Interface of Innate and Adoptive Immunity: Identification of IRF8 Target Genes in Germinal Center B Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4914-4914
Author(s):  
Dong-Mi Shin ◽  
Chang-Hoon Leeand Herbert Morse
Keyword(s):  
Signal Transduction ◽  
Innate Immunity ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Activation ◽  
Target Genes ◽  
Negative Regulator ◽  
B Cell Differentiation ◽  
And Function

Abstract IRF8 is a transcription factor widely expressed in hematopoietic cells and best known for its effects on myeloid and dendritic cell (DC) differentiation and function. Recent studies showed that IRF8 is expressed at low levels in naïve B cells, at significantly increased levels in germinal center (GC) B cells but little if at all in plasma cells. In GC, IRF8 was shown to contribute to the regulation of AID and BCL6. To better understand IRF8 contributions to the biology of mature B cells and possibly to transformation, we studied three mouse and three human lymphoma cell lines of GC origin by ChIP-Chip (C-C) analyses for IRF8 and POLII targets. We identified 87 IRF8 targets that were common to mouse and human B cells and were also positive for POLII. 17/18 of these putative IRF8 targets tested by PCR ChIP analyses were validated. IRF8 is transcriptionally activated by IFNg, and many of the newly identified targets were previously known to be IFN-responsive. Analyses of the targets identified 14 that contribute to various aspects of innate immunity at the cell surface (CD37, HSP90B1, Ly86), via signal transduction (AZI2, NFKBIZ, STAT2, IRF9), or with direct anti-viral activity (ADAR, PML, S100, ZC3HAV1 and others). Another 19 are active contributors to acquired immune functions of B cells with many involved in peptide processing and antigen presentation by both MHC class I and class II molecules (B2M, CD74, TAP1, TAP2, CTSS and 20S proteosomal components) and some in signal transduction (BLK, BLNK). Still others are active in B cell differentiation and function (MS4A1 [CD20], ELF1 and GNA13). CYLD plays in both arenas as a negative regulator of RIG-I-mediated antiviral responses and by regulating proper B cell activation after BCR ligation. The regulatory contributions of IRF8 to innate immunity in DC, macrophages, granulocytes and now B cells as well as elements of acquired immunity in B cells suggest that it contributes to an evolutionary bridge that positions a single cell type to integrate critical defense mechanisms from the innate and acquired immune repertoires.

scholarly journals Distinct Roles of Phosphoinositide-3 Kinase and Phospholipase Cγ2 in B-Cell Receptor-Mediated Signal Transduction

2006 ◽  
Vol 26 (1) ◽  
pp. 88-99 ◽  
Author(s):  
Xuezhi Dai ◽  
Yuhong Chen ◽  
James Schuman ◽  
Zichun Hua ◽  
John W. Adamson ◽  
...  
Keyword(s):  
Signal Transduction ◽  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Cell Development ◽  
B Cell Development ◽  
B Cell Receptor ◽  
Bcr Signaling ◽  
Phosphoinositide 3 Kinase ◽  
Cell Transition

ABSTRACT During B-cell receptor (BCR) signaling, phosphoinositide-3 kinase (PI3K) is thought to function upstream of phospholipase Cγ2 (PLCγ2). PLCγ2 deficiency specifically impedes transitional type 2 (T2) to follicular (FO) mature B-cell transition. Here, we demonstrate that PI3K deficiency specifically impaired T2-to-FO mature B-cell transition and marginal zone B-cell development. Furthermore, we investigated the functional relationship between PI3K and PLCγ2 using PI3K−/−, PLCγ2−/−, and PI3K−/− PLCγ2−/− B cells. Interestingly, PLCγ2 deficiency had no effect on BCR-mediated PI3K activation, whereas PI3K deficiency only partially blocked activation of PLCγ2. Moreover, whereas PI3K−/− PLCγ2−/− double deficiency did not affect hematopoiesis, it resulted in embryonic lethality. PI3K−/− PLCγ2−/− fetal liver cells transplanted into B-cell null JAK3−/− mice failed to restore development of peripheral B cells and failed to progress through early B-cell development at the pro-B- to pre-B-cell transition, a more severe phenotype than was observed with either PI3K or PLCγ2 single-deficiency B cells. Consistent with this finding, BCR signaling was more severely impaired in the absence of both PI3K and PLCγ2 genes than in the absence of either one alone. Taken together, these results demonstrate that whereas PI3K functions upstream of PLCγ2, activation of PLCγ2 can occur independently of PI3K and that PI3K and PLCγ2 also have distinct functions in BCR signal transduction.

DC-SIGN Binds Preferentially Highly Glycosylated IgM to Trigger Classical BCR Signaling in Follicular Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2968-2968 ◽  
Author(s):  
Rada Amin ◽  
Frédéric Mourcin ◽  
Fabrice Uhel ◽  
Marion Guirriec ◽  
Thierry Lamy ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Conflicts Of Interest ◽  
Genetic Alterations ◽  
Variable Regions ◽  
Bcr Signaling

Abstract BCR is supposed to deliver key stimulatory signals to malignant B cells in various mature hematological malignancies. In follicular lymphoma (FL), unlike in aggressive lymphomas, very few genetic alterations in BCR signaling pathway have been described, and BCR activation has been reported to rely on the dynamic interaction of mannosylated Ig with C-type lectins, such as Mannose receptor (MR) and DC-SIGN. In fact, the variable regions of FL Ig genes are characterized by the specific accumulation of N-glycan motifs introduced by the somatic hypermutation process and positively selected during lymphomagenesis. BCR activation is thus induced by an antigen-independent functional bridge between FL B cells and lectin-expressing non-malignant cells within tumor cell niche. In agreement, FL cells are well known to be strongly dependent on their microenvironment, including in particular stromal cells, CD4pos T cells, and macrophages that collectively support malignant cell growth and suppress antitumor immune response. In particular, a high number of infiltrating tumor associated macrophages (TAM) is associated with poor prognosis in patients treated by conventional therapy. Since DC-SIGN and MR are expressed by myeloid cells, we explored the possibility that they could trigger FL BCR activation in vitro and in situ. We first demonstrated that primary FL B cells of IgM isotype exhibited a higher response to anti-Ig triggering than normal germinal center B cells, as highlighted by phosphorylation of CD79a, Syk, BLNK, and Erk. Interestingly, IgG-expressing FL B cells showed reduced BCR activation compared to IgM-expressing malignant B cells, an observation that shed new light on the potential role of allelic paradox in FL, where malignant B cells essentially express membrane IgM despite active class-switch recombination on the translocated allele. Furthermore, only IgM-expressing FL cells were able to bind recombinant DC-SIGN with variable affinity, correlated to the level of glycosylation of Ig. Crosslinking of FL IgM by DC-SIGN induces an immune complex where CD19 was detected and led to a sustained phosphorylation of Syk, Akt, and Erk, whereas crosslinking by anti-IgM antibodies triggered a quick and transient activation. Co-culture of FL cells expressing highly glycosylated IgM with DC-SIGNhi M2 macrophages, unlike with DC-SIGNlo M1 macrophages, led to relocalization of DC-SIGN at the M2/B-cell interface and activation of Syk, Akt, and Erk in malignant B cells. Interestingly, M2-dependent activation of Erk was abrogated by BCR inhibitors such as imatinib. Finally, IL-4, which is overexpressed by infiltrating CD4pos follicular helper T cells in FL, upregulated DC-SIGN expression on macrophages, thus favoring such BCR-dependent crosstalk. Overall, our results support the hypothesis that glycosylated BCR contributes to DC-SIGN-dependent activation in FL and reveal the role of IgM isotype in this process. Such study paves the way for a better understanding of TAM/B cell crosstalk, which could constitute an important therapeutic target in this still fatal malignancy. Disclosures No relevant conflicts of interest to declare.

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