Nek2 Is a Novel Regulator of B Cell Development and Immunological Response

The serine/threonine kinase Nek2 is commonly found upregulated in a wide variety of neoplasms including diffuse large B cell lymphoma and multiple myeloma. High expression of Nek2 is implicated in the induction of chromosomal instability, promotion of cell proliferation, and drug resistance in tumor cells as well as a marker for poor clinical outcomes. Despite its well recorded involvement in chromosomal instability and neoplastic growth, little is known about the involvement of Nek2 in B cell development. Here we report the development of a transgenic mouse line with conditional expression of Nek2 in the B cell lineage and the effects it has on the development of B cells. Interestingly, we found that the overexpression of Nek2 does not induce spontaneous tumor formation within the transgenic mice up to 24 months after induction. Instead, overexpression of Nek2 in the B cell lineage affects the development of B cells by increasing the proportion of immature B cells in the bone marrow and decreasing B-1 B cells in peritoneal cavity. Furthermore, Nek2 transgenic mice develop spontaneous germinal centers and exhibit an enhanced T cell dependent immune response. Altogether, our data demonstrates a novel role for Nek2 in regulating B cell development and the immune response.

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bcl-x exhibits regulated expression during B cell development and activation and modulates lymphocyte survival in transgenic mice.

Journal of Experimental Medicine ◽

10.1084/jem.183.2.381 ◽

1996 ◽

Vol 183 (2) ◽

pp. 381-391 ◽

Cited By ~ 140

Author(s):

D A Grillot ◽

R Merino ◽

J C Pena ◽

W C Fanslow ◽

F D Finkelman ◽

...

Keyword(s):

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Cell Lineage ◽

Cell Development ◽

B Cell Development ◽

Immunoglobulin M ◽

Protective Mechanism ◽

And Function ◽

Peripheral B Cells

We have assessed during B cell development, the regulation and function of bcl-x, a member of the bcl-2 family of apoptosis regulatory genes. Here we show that Bcl-xL, a product of bcl-x, is expressed in pre-B cells but downregulated at the immature and mature stages of B cell development. Bcl-xL but not Bcl-2 is rapidly induced in peripheral B cells upon surface immunoglobulin M (IgM) cross-linking, CD40 signaling, or LPS stimulation. Transgenic mice that overexpressed Bcl-xL within the B cell lineage exhibited marked accumulation of peripheral B cells in lymphoid organs and enhanced survival of developing and mature B cells. B cell survival was further increased by simultaneous expression of bcl-xL and bcl-2 transgenes. These studies demonstrate that Bcl-2 and Bcl-xL are regulated differentially during B cell development and activation of mature B cells. Induction of Bcl-xL after signaling through surface IgM and CD40 appears to provide mature B cells with an additional protective mechanism against apoptotic signals associated with antigen-induced activation and proliferation.

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Conditional antibody expression to avoid central B cell deletion in humanized HIV-1 vaccine mouse models

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1921996117 ◽

2020 ◽

Vol 117 (14) ◽

pp. 7929-7940

Author(s):

Ming Tian ◽

Kelly McGovern ◽

Hwei-Ling Cheng ◽

Peyton Waddicor ◽

Lisa Rieble ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Mouse Models ◽

Negative Selection ◽

Cell Development ◽

B Cell Development ◽

Affinity Maturation ◽

Variable Regions ◽

Conditional Expression ◽

Hiv 1

HIV-1 vaccine development aims to elicit broadly neutralizing antibodies (bnAbs) against diverse viral strains. In some HIV-1–infected individuals, bnAbs evolved from precursor antibodies through affinity maturation. To induce bnAbs, a vaccine must mediate a similar antibody maturation process. One way to test a vaccine is to immunize mouse models that express human bnAb precursors and assess whether the vaccine can convert precursor antibodies into bnAbs. A major problem with such mouse models is that bnAb expression often hinders B cell development. Such developmental blocks may be attributed to the unusual properties of bnAb variable regions, such as poly-reactivity and long antigen-binding loops, which are usually under negative selection during primary B cell development. To address this problem, we devised a method to circumvent such B cell developmental blocks by expressing bnAbs conditionally in mature B cells. We validated this method by expressing the unmutated common ancestor (UCA) of the human VRC26 bnAb in transgenic mice. Constitutive expression of the VRC26UCA led to developmental arrest of B cell progenitors in bone marrow; poly-reactivity of the VRC26UCA and poor pairing of the VRC26UCA heavy chain with the mouse surrogate light chain may contribute to this phenotype. The conditional expression strategy bypassed the impediment to VRC26UCA B cell development, enabling the expression of VRC26UCA in mature B cells. This approach should be generally applicable for expressing other bnAbs that are under negative selection during B cell development.

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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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Long noncoding RNAs in B-cell development and activation

Blood ◽

10.1182/blood-2015-11-680843 ◽

2016 ◽

Vol 128 (7) ◽

pp. e10-e19 ◽

Cited By ~ 48

Author(s):

Tiago F. Brazão ◽

Jethro S. Johnson ◽

Jennifer Müller ◽

Andreas Heger ◽

Chris P. Ponting ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

De Novo ◽

Lymphoblastic Leukemia ◽

Cell Lineage ◽

Noncoding Rnas ◽

Cell Development ◽

B Cell Development ◽

Long Noncoding Rnas ◽

Human B Cells

AbstractLong noncoding RNAs (lncRNAs) are potentially important regulators of cell differentiation and development, but little is known about their roles in B lymphocytes. Using RNA-seq and de novo transcript assembly, we identified 4516 lncRNAs expressed in 11 stages of B-cell development and activation. Most of these lncRNAs have not been previously detected, even in the closely related T-cell lineage. Comparison with lncRNAs previously described in human B cells identified 185 mouse lncRNAs that have human orthologs. Using chromatin immunoprecipitation-seq, we classified 20% of the lncRNAs as either enhancer-associated (eRNA) or promoter-associated RNAs. We identified 126 eRNAs whose expression closely correlated with the nearest coding gene, thereby indicating the likely location of numerous enhancers active in the B-cell lineage. Furthermore, using this catalog of newly discovered lncRNAs, we show that PAX5, a transcription factor required to specify the B-cell lineage, bound to and regulated the expression of 109 lncRNAs in pro-B and mature B cells and 184 lncRNAs in acute lymphoblastic leukemia.

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Enforced BCL6 Expression Inhibits B Cell Development in Vivo.

Blood ◽

10.1182/blood.v104.11.1535.1535 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1535-1535

Author(s):

Davide F. Robbiani ◽

Kaity Colon ◽

Kruti Naik ◽

Helen Nickerson ◽

Maurizio Affer ◽

...

Keyword(s):

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Light Chain ◽

Germinal Center ◽

Regulatory Elements ◽

Cell Development ◽

B Cell Development ◽

Kappa Light Chain

Abstract The B-Cell Lymphoma 6 (BCL6) gene encodes for a zinc finger motifs containing transcriptional repressor that is frequently dysregulated by chromosomal translocations in germinal center lymphomas. A putative protooncogene, its transforming ability in vivo was reported in I-mu-HA-BCL6 knock-in mice by Cattoretti et al last year. We also tested this assumption in transgenic mice expressing BCL6 in B cells under the control of kappa light chain regulatory elements. We replaced the murine C-kappa locus with the 16kb human BCL6 genomic locus in a construct containing the murine kappa light chain regulatory elements (Vk, EiK, 3′RR). While control transgenics were readily obtained (5/32 founders), only 3/68 founders were positive for the BCL6 transgene, of which only one (bearing a single copy of the transgene) was able to transmit the transgene to its progeny, thus suggesting embryonal toxicity of exogenous BCL6. In the bone marrow, flow cytometry revealed a nearly complete block of B cell development at the pro-B to pre-B transition. This was also the stage at which we first detected expression of EGFP in control reporter mice that were generated in parallel. Spleens of transgenic mice weighed about 50% of control spleens and less than 5% of splenocytes were CD19+ B cells. These were IgM high, IgD intermediate, corresponding to an immature B cell phenotype. Lymph nodes were smaller and B cells barely detected. Peyers’ patches were not visible. Combined, our analysis of 6–8 weeks old VkHABCL6 transgenic mice reveals that enforced expression of BCL6 early in development results in a profound block of B lymphocyte differentiation. How transgenic BCL6 modulates this effect at the transcriptional level remains to be investigated. To test the oncogenic potential of BCL6 in B cells, it will be interesting to precisely turn on this gene in the germinal center.

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Ectopic Expression of HDAC9 in Murine Lymphoid System Leads to Altered Lymphocyte Numbers and Proliferation as Well as Predisposition to Tumorigenesis.

Blood ◽

10.1182/blood.v110.11.376.376 ◽

2007 ◽

Vol 110 (11) ◽

pp. 376-376

Author(s):

Veronica S. Gil ◽

Louise M.C. Howell ◽

Jenny Yeung ◽

Kevin R. Petrie ◽

Adrian Smith ◽

...

Keyword(s):

Gene Expression ◽

B Cells ◽

Transgenic Mice ◽

B Cell ◽

Ectopic Expression ◽

Cell Development ◽

B Cell Development ◽

Gene Promoters ◽

Marginal Zone B Cells ◽

Lymphoid System

Abstract Reversible acetylation of lysine residues on histone tails is associated with changes to chromatin structure and plays a key role in regulation of gene expression. In this process, histone hypoacetylation is generally associated with gene silencing and pharmacological inhibition of histone deacetylases (HDACs) leads usually to activation of gene expression. Decreased histone acetylation is a hallmark of cancer cells and increased HDAC expression or their mistargetting to specific gene promoters has been associated with a variety of tumors. In the past we have identified and cloned class IIa HDAC9. The HDAC9 gene is located in chromosome 7p21, which is frequently amplified in B-cell tumours such as mantle cell lymphoma (MCL) and in B-cell non-Hodgkin’s lymphoma cell lines. Consistently, initial analysis of patient samples and/or publicly available microarray data highlighted high levels of HDAC9 expression in chronic lymphocytic leukemia, folicullar lymphoma and MCL. Within the normal lymphoid system, HDAC9 is co-expressed with BCL-6 in germinal center B-cells (∼60% of cells). HDAC9 is also expressed in marginal zone B cells and a fraction of CD38 or CD27 positive subepithelial tonsilar cells. In order to examine the role of HDAC9 in the lymphoid development and pathogenesis of lymphoid malignancies we used Ig heavy chain enhancer (Eμ), which drives gene expression from early stages of B-cell development, to ectopically express HDAC9 in transgenic mice. Hemizygous and homozygous mice expressing Flag epitope tagged human HDAC9 (fHDAC9) transgene display throughout their lifespan altered B-cell development. Immunophenotypic analysis of B-cells isolated from bone marrow (BM) revealed an absence of cells expressing the pre-B/immature-B cell markers normally associated with C-E Hardy’s fractions. In vitro functional clonogenic assays for IL-7 responsive BM-derived B-cell progenitors demonstrated an increase (∼50%) in colony numbers in the transgenic BM. Moreover, morphologic and flow cytometric analyses of the transgenic colonies, but not those derived from normal BM, revealed the presence of granulocyte/macrophage colony forming units expressing the HDAC9 transgene, suggesting a lympho-myeloid lineage switch. This correlates with the finding that extramedullary myelopoiesis occurs in a fraction of mice presenting splenomegaly (44%). Furthermore, a subgroup of homozygous Eμ-fHDAC9 mice (n=16) developed tumours (81%) at middle age, and present with enlarged lymph nodes (6%) and abnormal hematopoietic elements in peripheral blood and BM. Taken together these data suggest that HDAC9 plays a role in B-cell maturation and its ectopic expression in early B-cells leads to perturbation of normal B-cell development, possibly predisposing transgenic mice to tumorigenesis.

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Constitutively Activated STAT3 Promotes Cell Proliferation and Survival in the Activated B Cell Subtype of Diffuse Large B Cell Lymphomas.

Blood ◽

10.1182/blood.v110.11.1621.1621 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1621-1621

Author(s):

Bihui Hilda Ye ◽

Beibei Belinda Ding ◽

Jian Jessica Yu ◽

Raymond Y.-L. Yu ◽

Lourdes M. Mendez ◽

...

Keyword(s):

Cell Proliferation ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

B Cell Lymphoma ◽

Cell Development ◽

B Cell Development ◽

Reciprocal Relationship ◽

B Cell Lymphomas ◽

Large B Cell

Abstract During B cell development, cell proliferation and survival are regulated by stage-specific transcription factors. Accordingly, distinct oncogenic pathways are employed by B cell lymphomas representing different stages of B cell development. Diffuse large B cell lymphoma (DLBCL) contains at least two main phenotypic subtypes, i.e. the germinal center B cell-like (GCB-DLBCL) and the activated B cell-like (ABC-DLBCL) groups. It has been shown that GCB-DLBCL responds favorably to chemotherapy and expresses high levels of BCL6, a transcription repressor known to play a causative role in lymphomagenesis. In comparison, ABC-DLBCL has lower levels of BCL6, constitutively activated NF-kappaB and tends to be refractory to chemotherapy. In this study, we investigated the relationship between BCL6 and STAT3 expression/activation in DLBCL and normal GC B cells. Our results demonstrate that BCL6 directly inhibits transcription of the STAT3 gene by binding to two BCL6 sites in its 5′ regulatory region. As a result, high level STAT3 expression and activation are preferentially detected in ABC-DLBCL and BCL6-negative normal germinal center B cells. Specifically, in tonsillar GCs, STAT3 expression and activation is restricted to a previously uncharacterized subset of BCL6−Blimp-1− B cells in the apical light zone. The location and phenotype of these cells suggest that they are in the process of exiting the BCL6-directed GC program and transitioning to a plasma cell differentiation process governed by Blimp-1. The reciprocal relationship between BCL6 and STAT3 is also conserved in DLBCL such that STAT3 expression and activation is preferentially associated with the BCL6-low, ABC subtype. Most importantly, inactivating STAT3 by either AG490 or small interference RNA in ABC-DLBCL cells inhibits cell proliferation and triggers apoptosis. These phenotypes are accompanied by decreased expression of several known STAT3 target genes, including c-Myc, JunB and Mcl-1, and increased expression of the cell cycle inhibitor p27. In addition to identifying STAT3 as a novel BCL6 target gene, our results define STAT3 activation as a second oncogenic pathway operating in ABC-DLBCL and suggest that blocking STAT3 may be potentially therapeutic in treatment of these aggressive lymphomas.

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Harnessing lymphoma epigenetics to improve therapies

Blood ◽

10.1182/blood.2020006908 ◽

2020 ◽

Vol 136 (21) ◽

pp. 2386-2391

Author(s):

Haopeng Yang ◽

Michael R. Green

Keyword(s):

Transcription Factors ◽

B Cells ◽

B Cell ◽

Germinal Center ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Genetic Alterations ◽

Cell Development ◽

B Cell Development ◽

Affinity Maturation

Abstract Affinity maturation and terminal differentiation of B cells via the germinal center reaction is a complex multistep process controlled by transcription factors that induce or suppress large dynamic transcriptional programs. This occurs via the recruitment of coactivator or corepressor complexes that epigenetically regulate gene expression by post-translationally modifying histones and/or remodeling chromatin structure. B-cell–intrinsic developmental programs both regulate and respond to interactions with other cells in the germinal center that provide survival and differentiation signals, such as T-follicular helper cells and follicular dendritic cells. Epigenetic and transcriptional programs that naturally occur during B-cell development are hijacked in B-cell lymphoma by genetic alterations that directly or indirectly change the function of transcription factors and/or chromatin-modifying genes. These in turn skew differentiation toward the tumor cell of origin and alter interactions between lymphoma B cells and other cells within the microenvironment. Understanding the mechanisms by which genetic alterations perturb epigenetic and transcriptional programs regulating B-cell development and immune interactions may identify opportunities to target these programs using epigenetic-modifying agents. Here, we discuss recently published studies centered on follicular lymphoma and diffuse large B-cell lymphoma within the context of prior knowledge, and we highlight how these insights have informed potential avenues for rational therapeutic interventions.

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Transcription factor Zfx controls BCR-induced proliferation and survival of B lymphocytes

Blood ◽

10.1182/blood-2008-11-188888 ◽

2009 ◽

Vol 113 (23) ◽

pp. 5857-5867 ◽

Cited By ~ 15

Author(s):

Teresita L. Arenzana ◽

Matthew R. Smith-Raska ◽

Boris Reizis

Keyword(s):

Transcription Factor ◽

B Cells ◽

B Cell ◽

B Lymphocytes ◽

Cell Lineage ◽

Cell Development ◽

B Cell Development ◽

Stem Cell Maintenance ◽

And Function ◽

Cell Maintenance

Abstract The development, homeostasis, and function of B lymphocytes involve multiple rounds of B-cell receptor (BCR)–controlled proliferation and prolonged maintenance. We analyzed the role of transcription factor Zfx, a recently identified regulator of hematopoietic stem cell maintenance, in B-cell development and homeostasis. Panhematopoietic or B cell–specific deletion of Zfx in the bone marrow blocked B-cell development at the pre-BCR selection checkpoint. Zfx deficiency in peripheral B cells caused accelerated B-cell turnover, depletion of mature recirculating B cells, and delayed T-dependent antibody responses. In addition, the numbers and function of B-1 cell lineage were reduced. Zfx-deficient B cells showed normal proximal BCR signaling, but impaired BCR-induced proliferation and survival in vitro. This was accompanied by aberrantly enhanced and prolonged integrated stress response and by delayed induction of cyclin D2 and Bcl-xL proteins. Thus, Zfx restrains the stress response and couples antigen receptor signaling to cell expansion and maintenance during B-cell development and peripheral homeostasis. These results identify a novel transcriptional regulator of the B-cell lineage and highlight the common genetic control of stem cell maintenance and lymphocyte homeostasis.

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