Takuya Nojima

Germinal center dysregulation by histone methyltransferase EZH2 promotes lymphomagenesis

Protection against deadly pathogens requires the production of high-affinity antibodies by B cells, which are generated in germinal centers (GCs). Alteration of the GC developmental program is common in many B cell malignancies. Identification of regulators of the GC response is crucial to develop targeted therapies for GC B cell dysfunctions, including lymphomas. The histone H3 lysine 27 methyltransferase enhancer of zeste homolog 2 (EZH2) is highly expressed in GC B cells and is often constitutively activated in GC-derived non-Hodgkin lymphomas (NHLs). The function of EZH2 in GC B cells remains largely unknown. Herein, we show that Ezh2 inactivation in mouse GC B cells caused profound impairment of GC responses, memory B cell formation, and humoral immunity. EZH2 protected GC B cells against activation-induced cytidine deaminase (AID) mutagenesis, facilitated cell cycle progression, and silenced plasma cell determinant and tumor suppressor B-lymphocyte-induced maturation protein 1 (BLIMP1). EZH2 inhibition in NHL cells induced BLIMP1, which impaired tumor growth. In conclusion, EZH2 sustains AID function and prevents terminal differentiation of GC B cells, which allows antibody diversification and affinity maturation. Dysregulation of the GC reaction by constitutively active EZH2 facilitates lymphomagenesis and identifies EZH2 as a possible therapeutic target in NHL and other GC-derived B cell diseases.

In-vitro derived germinal centre B cells differentially generate memory B or plasma cells in vivo

In response to T cell-dependent antigens, B cells proliferate extensively to form germinal centres (GC), and then differentiate into memory B (B(mem)) cells or long-lived plasma cells (LLPCs) by largely unknown mechanisms. Here we show a new culture system in which mouse naïve B cells undergo massive expansion and isotype switching, and generate GC-phenotype B (iGB) cells. The iGB cells expressing IgG1 or IgM/D, but not IgE, differentiate into B(mem) cells in vivo after adoptive transfer and can elicit rapid immune responses with the help of cognate T cells. Secondary culture with IL-21 maintains the proliferation of the iGB cells, while shifting their in vivo developmental fate from B(mem) cells to LLPCs, an outcome that can be reversed by withdrawal of IL-21 in tertiary cultures. Thus, this system enables in vitro manipulation of B-cell fate, into either B(mem) cells or LLPCs, and will facilitate dissection of GC-B cell differentiation programs.

Distinct signaling requirements for Dμ selection, igh allelic exclusion, pre-B cell transition, and tumor suppression in B cell progenitors

The pre-B cell receptor triggers expansion and differentiation of pre-B cells (the pre-B cell transition), as well as inhibition of V(H) to DJ(H) recombination (allelic exclusion). The latter also accounts for counter-selection of pro-B cells expressing Dmu protein (Dmu selection). However, the signaling pathways responsible for these events remain poorly defined. Here we show complete arrest of B cell development at the pre-B cell transition in BASH/CD19 double mutant mice, indicating partial redundancy of the two B cell-specific adaptors. Allelic exclusion remained intact in the double mutant mice, whereas Dmu selection was abolished in BASH mutant mice. Thus, distinct signals are required for these events. In addition, both mutant mice succumbed to pre-B cell leukemia, indicating that BASH and CD19 contribute to tumor suppression.

Impaired Receptor Editing in the Primary B Cell Repertoire of BASH-Deficient Mice

The editing of B cell Ag receptor (BCR) through successive rearrangements of Ig genes has been considered to be a major mechanism for the central B cell tolerance, which precludes appearance of self-reactive B cells, through studies using anti-self-Ig transgenic/knock-in mouse systems. However, contribution of the receptor editing in the development of the normal B cell repertoire remains unclear. In addition, the signaling pathway directing this event is unknown. In this study, we demonstrate that receptor editing in anti-DNA Ig knock-in mice is impaired in the absence of an adaptor protein BASH (BLNK/SLP-65) that is involved in BCR signaling. Remarkably, the supposed hallmarks of receptor editing such as Igλ chain expression, recombination sequence rearrangements at Igκ loci, and presence of in-frame VκJκ joins in the Igκ loci inactivated by the recombination sequence rearrangements, were all diminished in BASH-deficient mice with unmanipulated Ig loci. BCR ligation-induced Igλ gene recombination in vitro was also impaired in BASH-deficient B cells. Furthermore, the BASH-deficient mice showed an excessive Ab response to a DNA carrier immunization, suggesting the presence of unedited DNA-reactive B cells in the periphery. These results not only define a signaling pathway required for receptor editing but indicate that the BCR-signaled receptor editing indeed operates in the development of normal B cell repertoire and contributes to establishing the B cell tolerance.

Tolerance Induction of IgG+ Memory B Cells by T Cell-Independent Type II Antigens

Memory B cells generated during a T cell-dependent immune response rapidly respond to a secondary immunization by producing abundant IgG Abs that bind cognate Ag with high affinity. It is currently unclear whether this heightened recall response by memory B cells is due to augmented IgG-BCR signaling, which has only been demonstrated in the context of naive transgenic B cells. To address this question, we examined whether memory B cells can respond in vivo to Ags that stimulate only through BCR, namely T cell-independent type II (TI-II) Ags. In this study, we show that the TI-II Ag (4-hydroxy-3-nitrophenyl) acetyl (NP)-Ficoll cannot elicit the recall response in mice first immunized with the T cell-dependent Ag NP-chicken γ-globulin. Moreover, the NP-Ficoll challenge in vivo as well as in vitro significantly inhibits a subsequent recall response to NP-chicken γ-globulin in a B cell-intrinsic manner. This NP-Ficoll–mediated tolerance is caused by the preferential elimination of IgG+ memory B cells binding to NP with high affinity. These data indicate that BCR cross-linking with a TI-II Ag does not activate IgG+ memory B cells, but rather tolerizes them, identifying a terminal checkpoint of memory B cell differentiation that may prevent autoimmunity.

JNK Regulatory Molecule G5PR Induces IgG Autoantibody–Producing Plasmablasts from Peritoneal B1a Cells

Peritoneal B1a cells expressing CD5 and CD11b generate autoantibody-producing precursors in autoimmune-prone mice. Previous studies show reduced JNK signaling in peritoneal B1a cells of female New Zealand Black mice and an abnormal increase of protein phosphatase 2A subunit G5PR that regulates BCR-mediated JNK signaling as a cause of autoimmunity. To investigate the mechanism regulating B1a differentiation into autoantibody-secreting plasmablasts (PBs), we applied an in vitro culture system that supports long-term growth of germinal center (GC) B cells (iGB) with IL-4, CD40L, and BAFF. Compared with spleen B2 cells, B1a cells differentiated into GC-like B cells, but more markedly into PBs, and underwent class switching toward IgG1. During iGB culture, B1a cells expressed GC-associated aicda, g5pr, and bcl6, and markedly PB-associated prdm1, irf4, and xbp1. B1a-derived iGB cells from New Zealand Black × New Zealand White F1 mice highly differentiated into autoantibody-secreting PBs in vitro and localized to the GC area in vivo. In iGB culture, JNK inhibitor SP600125 augmented the differentiation of C57BL/6 B1a cells into PBs. Furthermore, B1a cells from G5PR transgenic mice markedly differentiated into IgM and IgG autoantibody–secreting PBs. In conclusion, JNK regulation is critical to suppress autoantibody-secreting PBs from peritoneal B1a cells.

gp49B-Mediated Negative Regulation of Antibody Production by Memory and Marginal Zone B Cells

The rapid Ab responses observed after primary and secondary immunizations are mainly derived from marginal zone (MZ) and memory B cells, respectively, but it is largely unknown how these responses are negatively regulated. Several inhibitory receptors have been identified and their roles have been studied, but mainly on follicular B cells and much less so on MZ B, and never on memory B cells. gp49B is an Ig superfamily member that contains two ITIMs in its cytoplasmic tail, and it has been shown to negatively regulate mast cell, macrophage, and NK cell responses. In this study, we demonstrate that gp49B is preferentially expressed on memory and MZ B cells. We show that gp49B−/− mice produce more IgM after a primary immunization and more IgM and IgG1 after a secondary immunization than gp49B+/+ mice in T cell–dependent immune responses. Memory and MZ B cells from gp49B−/− mice also produce more Abs upon in vitro stimulation with CD40 than those from gp49B+/+ mice. The in vitro IgM production by MZ B cells from gp49B+/+, but not gp49B−/−, mice is suppressed by interaction with a putative gp49B ligand, the integrin αvβ3 heterodimer. In addition, gp49B−/− mice exhibited exaggerated IgE production in the memory recall response. These results suggest that plasma cell development from memory and MZ B cells, as well as subsequent Ab production, are suppressed via gp49B. In memory B cells, this suppression also prevents excessive IgE production, thus curtailing allergic diseases.

Germinal center entry not selection of B cells is controlled by peptide-MHCII complex density

B cells expressing high affinity antigen receptors are advantaged in germinal centers (GC), perhaps by increased acquisition of antigen for presentation to follicular helper T cells and improved T-cell help. In this model for affinity-dependent selection, the density of peptide/MHCII (pMHCII) complexes on GC B cells is the primary determinant of selection. Here we show in chimeric mice populated by B cells differing only in their capacity to express MHCII (MHCII+/+ and MHCII+/−) that GC selection is insensitive to halving pMHCII density. Alone, both B cell types generate identical humoral responses; in competition, MHCII+/+ B cells are preferentially recruited to early GCs but this advantage does not persist once GCs are established. During GC responses, competing MHCII+/+ and MHCII+/− GC B cells comparably accumulate mutations and have indistinguishable rates of affinity maturation. We conclude that B-cell selection by pMHCII density is stringent in the establishment of GCs, but relaxed during GC responses.It has been thought that germinal center (GC) B cells with the highest density of peptide-MHCII molecules receive the most T cell help. Here the authors use heterozygous MHCII inactivation to instead show that after recruitment into the GCs, BCR affinity increases over time independent of the MHC density.

A Novel and Effective Cancer Immunotherapy Mouse Model Using Antigen-Specific B Cells Selected In Vitro

Immunotherapies such as adoptive transfer of T cells or natural killer cells, or monoclonal antibody (MoAb) treatment have recently been recognized as effective means to treat cancer patients. However, adoptive transfer of B cells or plasma cells producing tumor-specific antibodies has not been applied as a therapy because long-term culture and selective expansion of antigen-specific B cells has been technically very difficult. Here, we describe a novel cancer immunotherapy that uses B-cell adoptive transfer. We demonstrate that germinal-center-like B cells (iGB cells) induced in vitro from mouse naïve B cells become plasma cells and produce IgG antibodies for more than a month in the bone marrow of non-irradiated recipient mice. When transferred into mice, iGB cells producing antibody against a surrogate tumor antigen suppressed lung metastasis and growth of mouse melanoma cells expressing the same antigen and prolonged survival of the recipients. In addition, we have developed a novel culture system called FAIS to selectively expand antigen-specific iGB cells utilizing the fact that iGB cells are sensitive to Fas-induced cell death unless their antigen receptors are ligated by membrane-bound antigens. The selected iGB cells efficiently suppressed lung metastasis of melanoma cells in the adoptive immunotherapy model. As human blood B cells can be propagated as iGB cells using culture conditions similar to the mouse iGB cell cultures, our data suggest that it will be possible to treat cancer-bearing patients by the adoptive transfer of cancer-antigen-specific iGB cells selected in vitro. This new adoptive immunotherapy should be an alternative to the laborious development of MoAb drugs against cancers for which no effective treatments currently exist.

Differing Requirements for MALT1 Function in Peripheral B Cell Survival and Differentiation

During a T cell-dependent immune response, formation of the germinal center (GC) is essential for the generation of high-affinity plasma cells and memory B cells. The canonical NF-κB pathway has been implicated in the initiation of GC reaction, and defects in this pathway have been linked to immune deficiencies. The paracaspase MALT1 plays an important role in regulating NF-κB activation upon triggering of Ag receptors. Although previous studies have reported that MALT1 deficiency abrogates the GC response, the relative contribution of B cells and T cells to the defective phenotype remains unclear. We used chimeric mouse models to demonstrate that MALT1 function is required in B cells for GC formation. This role is restricted to BCR signaling where MALT1 is critical for B cell proliferation and survival. Moreover, the proapoptotic signal transmitted in the absence of MALT1 is dominant to the prosurvival effects of T cell-derived stimuli. In addition to GC B cell differentiation, MALT1 is required for plasma cell differentiation, but not mitogenic responses. Lastly, we show that ectopic expression of Bcl-2 can partially rescue the GC phenotype in MALT1-deficient animals by prolonging the lifespan of BCR-activated B cells, but plasma cell differentiation and Ab production remain defective. Thus, our data uncover previously unappreciated aspects of MALT1 function in B cells and highlight its importance in humoral immunity.