Counterselection against Dμ Is Mediated through Immunoglobulin (Ig)α-Igβ

The pre-B cell receptor is a key checkpoint regulator in developing B cells. Early events that are controlled by the pre-B cell receptor include positive selection for cells express membrane immunoglobulin heavy chains and negative selection against cells expressing truncated immunoglobulins that lack a complete variable region (Dμ). Positive selection is known to be mediated by membrane immunoglobulin heavy chains through Igα-Igβ, whereas the mechanism for counterselection against Dμ has not been determined. We have examined the role of the Igα-Igβ signal transducers in counterselection against Dμ using mice that lack Igβ. We found that Dμ expression is not selected against in developing B cells in Igβ mutant mice. Thus, the molecular mechanism for counterselection against Dμ in pre-B cells resembles positive selection in that it requires interaction between mDμ and Igα-Igβ.

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Characterisation of B Cell Receptor-Induced NF-KB Activation In Chronic Lymphocytic Leukaemia Cells

Blood ◽

10.1182/blood.v116.21.375.375 ◽

2010 ◽

Vol 116 (21) ◽

pp. 375-375 ◽

Cited By ~ 1

Author(s):

Fatima Talab ◽

Victoria Thompson ◽

John C Allen ◽

Ke Lin ◽

Joseph R Slupsky

Keyword(s):

B Cells ◽

Tyrosine Kinase ◽

Chronic Lymphocytic Leukaemia ◽

B Cell ◽

Cell Cultures ◽

Lymphocytic Leukaemia ◽

Cell Receptor ◽

B Cell Receptor ◽

Nfkb Pathway

Abstract Abstract 375 B cell receptor (BCR) signaling promotes survival of the malignant clone in chronic lymphocytic leukaemia (CLL) through its ability to stimulate NFkB pathway signaling. In lymphoid cells, antigen receptor stimulation of this pathway is achieved by engaging the Carma-1 – Bcl10 – MALT1 (CBM) complex for eventual activation of I-kB kinases (IKKs). In B cells, protein kinase C beta (PKCbeta) is an important mediator of CBM complex activation. However, in CLL cells we found that PKCs do not appear to have a role in BCR-mediated NFkB pathway signaling, despite high expression levels of PKCbeta, because the presence of specific inhibitors of this kinase (LY379196 and bisindolylmaleimide-I) has no effect on the induction of IKK phosphorylation during BCR crosslinking. Examination of CBM complex expression suggests an explanation for this phenomenon; the expression levels of Carma-1 and MALT-1 are largely similar in CLL and normal B cells, but the expression of Bcl10 is much reduced in CLL cells. These findings, taken together with the established role of Bcl10 in the pathway of BCR-induced NFkB activation, suggest that CLL cells may employ a different mechanism to activate this pathway during BCR stimulation. Tyrosine kinases are known to play a role in BCR-induced IKK activation in CLL cells because compounds like dasatinib and PP2 inhibit NFkB pathway activation by BCR. One possible tyrosine kinase is c-Abl because we have shown this protein to be overexpressed in CLL cells, where it plays a role in activation of the NFkB pathway. To investigate the role of c-Abl in BCR-induced IKK activation, we used the inhibitor imatinib and found that the presence of this compound partially inhibited IKK phosphorylation in BCR-stimulated CLL cells. However, imatinib can also inhibit Lck, a T cell-specific src-family tyrosine kinase that is expressed by CLL cells. To differentiate between Lck- and c-Abl-mediated BCR signals we used the specific inhibitor 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolo[3,2d] pyrimidin-7-yl-cyclopentane (Lck-i). We found that the presence of this compound in CLL cell cultures undergoing BCR stimulation almost completely inhibited the induction of IKK activation. Investigation of Lck-i specificity revealed this compound did not inhibit either c-Abl or Lyn at the concentration used to inhibit Lck in CLL cell cultures. Further investigation of the effects of Lck-i showed that this compound was also effective in inhibiting BCR-induced activation of the Akt and ERK signaling pathways. Taken together, these data suggest a major role for Lck in BCR-mediated signaling in CLL cells, and question the existing paradigm on the importance of Lyn. Disclosures: No relevant conflicts of interest to declare.

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Essential Role of Phospholipase Cγ2 in Early B-Cell Development and Myc-Mediated Lymphomagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.00839-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9364-9376 ◽

Cited By ~ 22

Author(s):

Renren Wen ◽

Yuhong Chen ◽

Li Bai ◽

Guoping Fu ◽

James Schuman ◽

...

Keyword(s):

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

B Cell Receptor ◽

Wild Type ◽

Interleukin 7

ABSTRACT Phospholipase Cγ2 (PLCγ2) is a critical signaling effector of the B-cell receptor (BCR). Here we show that PLCγ2 deficiency impedes early B-cell development, resulting in an increase of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B cells. PLCγ2 deficiency impairs pre-BCR-mediated functions, leading to enhanced interleukin-7 (IL-7) signaling and elevated levels of RAGs in the selected large pre-B cells. Consequently, PLCγ2 deficiency renders large pre-B cells susceptible to transformation, resulting in dramatic acceleration of Myc-induced lymphomagenesis. PLCγ2 −/− Eμ-Myc transgenic mice mainly develop lymphomas of B220+ CD43+ BP-1+ CD24hi pre-BCR+ large pre-B-cell origin, which are uncommon in wild-type Eμ-Myc transgenics. Furthermore, lymphomas from PLCγ2 −/− Eμ-Myc transgenic mice exhibited a loss of p27Kip1 and often displayed alterations in Arf or p53. Thus, PLCγ2 plays an important role in pre-BCR-mediated early B-cell development, and its deficiency leads to markedly increased pools of the most at-risk large pre-B cells, which display hyperresponsiveness to IL-7 and express high levels of RAGs, making them prone to secondary mutations and Myc-induced malignancy.

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Rapid, Sustained B Cell Receptor Signaling in Lymphoma B Cells Differs from Normal Signaling in Tumor Infiltrating Nonmalignant B Cells.

Blood ◽

10.1182/blood.v106.11.283.283 ◽

2005 ◽

Vol 106 (11) ◽

pp. 283-283

Author(s):

Jonathan M. Irish ◽

Debra K. Czerwinski ◽

Garry P. Nolan ◽

Ronald Levy

Keyword(s):

B Cells ◽

Cell Signaling ◽

B Cell ◽

Peripheral Blood ◽

Cell Receptor ◽

B Cell Receptor ◽

Life And Death ◽

Heavy Chains ◽

Bcr Signaling ◽

Tumor Biopsies

Abstract The B cell receptor (BCR) drives life and death signaling throughout B cell development, and dysregulation of BCR signaling might be expected to play a role in aberrant proliferation of lymphoma B cells. We have previously used flow cytometry based cell signaling profiles to identify patterns of altered signaling in acute myeloid leukemia that were informative of clinical outcome (Irish et al., Cell, 2004). Here we used a similar signaling profiles approach to compare BCR signaling in normal and lymphoma B cells. However, in addition to comparing follicular lymphoma (FL) B cells with peripheral blood B cells from normal donors, we also interrogated signaling within individual non-tumor B cells infiltrating FL tumor biopsies. By staining for CD20 and BCR light chain isotype (κ vs. λ), we could distinguish tumor and normal B cells within each patient biopsy. Following crosslinking of BCR heavy chains (shared by tumor and non-tumor B cells), we measured phosphorylation of Syk and Btk proteins, as markers of early BCR signaling activity, and Erk1/2 and p38, as markers of downstream BCR signaling effector activity. The BCR signaling network in FL tumor B cells was activated more rapidly than infiltrating non-tumor B cells, achieved greater levels of per-cell signaling, and sustained high levels of signaling over a period of hours. In lymphoma B cells, BCR-mediated Btk and Erk1/2 phosphorylation could reach the normal maximum in as little as 4 minutes, which was much more rapid than the 30–60 minutes required for peak signaling in non-tumor B cells. Strikingly, the timing and magnitude of BCR pathway protein phosphorylation we measured in non-tumor B cells within tumor biopsies was the same as that of normal, mature B cells from peripheral blood. These results suggest that the altered BCR signaling we identified in lymphoma is cell-intrinsic and associated with lymphomagenesis, as opposed to being a general change in tumor microenviornment affecting all B cells within a biopsy. FL tumor B cells from different patients were distinguished by the degree and number of changes to BCR signaling, such that variable profiles of lymphoma signaling kinetics distinguished each patient from the consistent signaling of normal B cells. These results identify cell-intrinsic changes to BCR signaling that may contribute to immortalization of lymphoma B cells and suggest that single cell profiles could identify lymphoma specific BCR-mediated signaling responsible for clinical outcomes.

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

Blood ◽

10.1182/blood-2008-08-176057 ◽

2009 ◽

Vol 113 (19) ◽

pp. 4566-4574 ◽

Cited By ~ 57

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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Loss of Precursor B Cell Expansion but Not Allelic Exclusion in VpreB1/VpreB2 Double-Deficient Mice

Journal of Experimental Medicine ◽

10.1084/jem.193.4.435 ◽

2001 ◽

Vol 193 (4) ◽

pp. 435-446 ◽

Cited By ~ 73

Author(s):

Cornelia Mundt ◽

Steve Licence ◽

Takeyuki Shimizu ◽

Fritz Melchers ◽

Inga-Lill Mårtensson

Keyword(s):

B Cells ◽

B Cell ◽

Cell Receptor ◽

Allelic Exclusion ◽

Surrogate Light Chain ◽

Heavy Chains ◽

Immature B Cells ◽

Ig Heavy Chain ◽

Deficient Mice

The pre-B cell receptor consists of immunoglobulin (Ig) μ heavy chains and surrogate light chain, i.e., the VpreB and λ5 proteins. To analyze the role of the two VpreB proteins, mice lacking the VpreB1 and VpreB2 genes were generated. VpreB1−/−VpreB2−/− mice were impaired in their B cell development at the transition from pre-BI to large pre-BII cells. Pre-BII cells did not expand by proliferation, consequently 40-fold less small pre-BII and immature B cells were found in bone marrow, and the generation of immature and mature conventional B cells in spleen appeared reduced. In addition, only low numbers of B-1a cells were detected in the peritoneum. Surprisingly, Ig heavy chain allelic exclusion was still active, apparently ruling out a signaling role of a VpreB1/VpreB2–containing receptor in this process.

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Expression of ZAP-70 is associated with increased B-cell receptor signaling in chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2002-06-1683 ◽

2002 ◽

Vol 100 (13) ◽

pp. 4609-4614 ◽

Cited By ~ 364

Author(s):

Liguang Chen ◽

George Widhopf ◽

Lang Huynh ◽

Laura Rassenti ◽

Kanti R. Rai ◽

...

Keyword(s):

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Tyrosine Phosphorylation ◽

Variable Region ◽

Cell Receptor ◽

B Cell Receptor ◽

Lymphocytic Leukemia ◽

Cytosolic Proteins ◽

V Genes

We examined isolated leukemia B cells of patients with chronic lymphocytic leukemia (CLL) for expression of zeta-associated protein 70 (ZAP-70). CLL B cells that have nonmutated immunoglobulin variable region genes (V genes) expressed levels of ZAP-70 protein that were comparable to those expressed by normal blood T cells. In contrast, CLL B cells that had mutated immunoglobulin variable V genes, or that had low-level expression of CD38, generally did not express detectable amounts of ZAP-70 protein. Leukemia cells from identical twins with CLL were found discordant for expression of ZAP-70, suggesting that B-cell expression of ZAP-70 is not genetically predetermined. Ligation of the B-cell receptor (BCR) complex on CLL cells that expressed ZAP-70 induced significantly greater tyrosine phosphorylation of cytosolic proteins, including p72Syk, than did similar stimulation of CLL cells that did not express ZAP-70. Also, exceptional cases of CLL cells that expressed mutated immunoglobulin V genes and ZAP-70 also experienced higher levels tyrosine phosphorylation of such cytosolic proteins following BCR ligation. Following BCR ligation, ZAP-70 underwent tyrosine phosphorylation and became associated with surface immunoglobulin and CD79b, arguing for the involvement of ZAP-70 in BCR signaling. These data indicate that expression of ZAP-70 is associated with enhanced signal transduction via the BCR complex, which may contribute to the more aggressive clinical course associated with CLL cells that express nonmutated immunoglobulin receptors.

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B-Cell Receptor- and Phorbol Ester-Induced NF-κB and c-Jun N-Terminal Kinase Activation in B Cells Requires Novel Protein Kinase C's

Molecular and Cellular Biology ◽

10.1128/mcb.21.19.6640-6650.2001 ◽

2001 ◽

Vol 21 (19) ◽

pp. 6640-6650 ◽

Cited By ~ 63

Author(s):

Daniel Krappmann ◽

Alina Patke ◽

Vigo Heissmeyer ◽

Claus Scheidereit

Keyword(s):

B Cells ◽

Protein Kinase ◽

B Cell ◽

Phorbol Ester ◽

Critical Role ◽

Ectopic Expression ◽

Cell Receptor ◽

B Cell Receptor ◽

Novel Protein

ABSTRACT Antigen receptor signaling is known to activate NF-κB in lymphocytes. While T-cell-receptor-induced NF-κB activation critically depends on novel protein kinase C θ (PKCθ), the role of novel PKCs in B-cell stimulation has not been elucidated. In primary murine splenic B cells, we found high expression of the novel PKCs δ and ɛ but only weak expression of the θ isoform. Rottlerin blocks phorbol ester (phorbol myristate acetate [PMA])- or B-cell receptor (BCR)-mediated NF-κB and c-Jun N-terminal kinase (JNK) activation in primary B and T cells to a similar extent, suggesting that novel PKCs are positive regulators of signaling in hematopoietic cells. Mouse 70Z/3 pre-B cells have been widely used as a model for NF-κB activation in B cells. Similar to the situation in splenic B cells, rottlerin inhibits BCR and PMA stimulation of NF-κB in 70Z/3 cells. A derivative of 70Z/3 cells, 1.3E2 cells, are defective in NF-κB activation due to the lack of the IκB kinase (IKKγ) protein. Ectopic expression of IKKγ can rescue NF-κB activation in response to lipopolysaccharides (LPS) and interleukin-1β (IL-1β), but not to PMA. In addition, PMA-induced activation of the mitogen-activated protein kinase JNK is blocked in 1.3E2 cells, suggesting that an upstream component common to both pathways is either missing or mutated. Analysis of various PKC isoforms revealed that exclusively PKCθ was absent in 1.3E2 cells while it was expressed in 70Z/3 cells. Stable expression of either novel PKCθ or -δ but not classical PKCβII in 1.3E2 IKKγ-expressing cells rescues PMA activation of NF-κB and JNK signaling, demonstrating a critical role of novel PKCs for B-cell activation.

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Biological Significance of B Cell Receptor Mediated Regulation of Autophagy in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v126.23.4130.4130 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4130-4130

Author(s):

Lindsay Smith ◽

Elizabeth Hogg ◽

Angus Haynes ◽

Jonathan C Strefford ◽

Francesco Forconi ◽

...

Keyword(s):

B Cells ◽

Protein Expression ◽

B Cell ◽

Cell Receptor ◽

B Cell Receptor ◽

Autophagic Flux ◽

Normal Donor ◽

Dependent Manner ◽

Resistance To Therapy

Abstract Chronic lymphocytic leukaemia (CLL) is characterised by an accumulation of B cells which is broadly split into two groups representing a progressive IGHV unmutated (U-CLL) and a more indolent IGHV mutated (M-CLL) disease. Activation of the B cell receptor (BCR) by antigen/autoantigen engagement is crucial for CLL cell survival, disease progression and resistance to therapy, however further research is required to better understand how BCR signalling impacts on CLL biology. Autophagy is known to play a role in tumorigenesis and resistance to therapy in solid tumors, however whether autophagy has a role in CLL biology and how it is regulated has not been fully investigated. Autophagy is important for normal B cell development and is known to be regulated by various drug treatments in vitro in CLL samples. A previous study showed that activation of the BCR on murine splenic B cells with soluble or bead immobilised (BI) anti-IgM induced autophagy and subsequent apoptosis, however, the role of BCR-induced autophagy has not been explored in B cell malignancies and particularly CLL. Firstly, we assessed basal protein expression of key autophagy markers LC3BII, and ATG3 in CLL samples and age-matched normal donor B cells (NDB). CLL cells expressed significantly more LC3BII (p=.014, n=57) and ATG3 (p=.04, n=58) compared with NDB (n=8), with a greater LC3BII protein expression in U-CLL compared to M-CLL (p=.039, n=57), indicating more autophagy occurs in U-CLL. Furthermore basal increases in autophagy markers GABARAPL2 (LC3B family member) (p=.0004, n=34) and ATG4A (p=.04, n=20) at the RNA level were significantly associated with the ability of CLL cells to flux calcium (>10%) in response to anti-IgM. This indicated a possible role of the BCR in the regulation of autophagy in CLL samples and a possible association with progressive disease. Activation of the CLL BCR with BI anti-IgM significantly induced expression of autophagy markers ATG3 (p=.002, n=22), LC3BII (p<.0001, n=23) and p62 (p=.0011, n=9) at the protein level and ATG3 (p=.04, n=8) and GABARAPL2 (p=.03, n=6) at the RNA level in a time dependent manner. Next, CLL samples were treated with BI anti-IgM in the presence or absence of the autophagy inhibitor hydroxychloroquine (HCQ) to confirm the increase in autophagic flux indicated at the RNA level. Addition of HCQ in combination with BI anti-IgM significantly increased accumulation of LC3BII protein expression compared with HCQ or anti-IgM alone (p=.01 and p=.02 respectively, n=5) indicating an increase in autophagic flux. Treatment with ibrutinib or R406 prevented BI anti-IgM dependent increases in LC3BII (p=.02 and p=.009 respectively, n=9) and p62 expression (p=.02 and p=.006 respectively, n=9). Confirming the role of the BCR in the regulation of autophagy in CLL samples and indicating that inhibition of autophagy may contribute to the clinical responses seen with these BCR kinase inhibitors. Finally we showed that BI anti-IgM increased CLL cell viability compared to BI isotype control (p=.002, n=7), whilst the addition of HCQ significantly reversed this effect (p=.001, n=7). More importantly treatment with BI anti-IgM protected CLL cells against fludarabine induced cell death (p=.0004, n=3) which again was reversed by the addition of HCQ (p=.05, n=3). These data emphasise the importance of BCR signalling in the regulation of autophagy and its impact on therapy resistance and suggest a possible role for autophagy inhibitors in the treatment of CLL. Disclosures Strefford: Roche: Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.

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IgG1 B cell receptor signaling is inhibited by CD22 and promotes the development of B cells whose survival is less dependent on Igα/β

Journal of Experimental Medicine ◽

10.1084/jem.20062024 ◽

2007 ◽

Vol 204 (4) ◽

pp. 747-758 ◽

Cited By ~ 87

Author(s):

Ari Waisman ◽

Manfred Kraus ◽

Jane Seagal ◽

Snigdha Ghosh ◽

Doron Melamed ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Receptor ◽

Cell Development ◽

B Cell Development ◽

B Cell Receptor ◽

Cell Physiology ◽

Cell Compartment ◽

Heavy Chains

We describe a mouse strain in which B cell development relies either on the expression of membrane-bound immunoglobulin (Ig) γ1 or μ heavy chains. Progenitor cells expressing γ1 chains from the beginning generate a peripheral B cell compartment of normal size with all subsets, but a partial block is seen at the pro– to pre–B cell transition. Accordingly, γ1-driven B cell development is disfavored in competition with developing B cells expressing a wild-type (WT) IgH locus. However, the mutant B cells display a long half-life and accumulate in the mature B cell compartment, and even though partial truncation of the Igα cytoplasmic tail compromises their development, it does not affect their maintenance, as it does in WT cells. IgG1-expressing B cells showed an enhanced Ca2+ response upon B cell receptor cross-linking, which was not due to a lack of inhibition by CD22. The enhanced Ca2+ response was also observed in mature B cells that had been switched from IgM to IgG1 expression in vivo. Collectively, these results suggest that the γ1 chain can exert a unique signaling function that can partially replace that of the Igα/β heterodimer in B cell maintenance and may contribute to memory B cell physiology.

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