Complex Antigens Drive Permissive Clonal Selection in Germinal Centers.

Germinal center (GC) B cells evolve toward increased affinity by a Darwinian process that has been studied primarily in genetically restricted, hapten-specific responses. We explored the population dynamics of genetically diverse GC responses to two complex antigens-Bacillus anthracis protective antigen and influenza hemagglutinin-in which B cells competed both intra- and interclonally for distinct epitopes. Preferred VH rearrangements among antigen-binding, naive B cells were similarly abundant in early GCs but, unlike responses to haptens, clonal diversity increased in GC B cells as early "winners" were replaced by rarer, high-affinity clones. Despite affinity maturation, inter- and intraclonal avidities varied greatly, and half of GC B cells did not bind the immunogen but nonetheless exhibited biased VH use, V(D)J mutation, and clonal expansion comparable to antigen-binding cells. GC reactions to complex antigens permit a range of specificities and affinities, with potential advantages for broad protection.

Tracing Self-Reactive B Cells in Normal Mice.

BCR transgenic mice dominate studies of B cell tolerance; consequently, tolerance in normal mice expressing diverse sets of autoreactive B cells is poorly characterized. We have used single B cell cultures to trace self-reactivity in BCR repertoires across the first and second tolerance checkpoints and in tolerized B cell compartments of normal mice. This approach reveals affinity "setpoints" that define each checkpoint and a subset of tolerized, autoreactive B cells that is long-lived. In normal mice, the numbers of B cells avidly specific for DNA fall significantly as small pre-B become immature and transitional-1 B cells, revealing the first tolerance checkpoint. By contrast, DNA reactivity does not significantly change when immature and transitional-1 B cells become mature follicular B cells, showing that the second checkpoint does not reduce DNA reactivity. In the spleen, autoreactivity was high in transitional-3 (T3) B cells, CD93+IgM-/loIgDhi anergic B cells, and a CD93- anergic subset. Whereas splenic T3 and CD93+ anergic B cells are short-lived, CD93-IgM-/loIgDhi B cells have half-lives comparable to mature follicular B cells. B cell-specific deletion of proapoptotic genes, Bak and Bax, resulted in increased CD93-IgM-/loIgDhi B cell numbers but not T3 B cell numbers, suggesting that apoptosis regulates differently persistent and ephemeral autoreactive B cells. The self-reactivity and longevity of CD93-IgM-/loIgDhi B cells and their capacity to proliferate and differentiate into plasmacytes in response to CD40 activation in vitro lead us to propose that this persistent, self-reactive compartment may be the origin of systemic autoimmunity and a potential target for vaccines to elicit protective Abs cross-reactive with self-antigens.

Cross-Reactivity to Kynureninase Tolerizes B Cells That Express the HIV-1 Broadly Neutralizing Antibody 2F5.

2F5 is an HIV-1 broadly neutralizing Ab that also binds the autoantigens kynureninase (KYNU) and anionic lipids. Generation of 2F5-like Abs is proscribed by immune tolerance, but it is unclear which autospecificity is responsible. We sampled the BCR repertoire of 2F5 knock-in mice before and after the first and second tolerance checkpoints. Nearly all small pre-B (precheckpoint) and 35-70% of anergic peripheral B cells (postcheckpoint) expressed the 2F5 BCR and maintained KYNU, lipid, and HIV-1 gp41 reactivity. In contrast, all postcheckpoint mature follicular (MF) B cells had undergone L chain editing that purged KYNU and gp41 binding but left lipid reactivity largely intact. We conclude that specificity for KYNU is the primary driver of tolerization of 2F5-expressing B cells. The MF and anergic B cell populations favored distinct collections of editor L chains; surprisingly, however, MF and anergic B cells also frequently expressed identical BCRs. These results imply that BCR autoreactivity is the primary determinant of whether a developing B cell enters the MF or anergic compartments, with a secondary role for stochastic factors that slightly mix the two pools. Our study provides mechanistic insights into how immunological tolerance impairs humoral responses to HIV-1 and supports activation of anergic B cells as a potential method for HIV-1 vaccination.

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.

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 αvß3 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.

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.

Deformation Matching: Force Computation Based on Deformation Optimization.

The tactile information to be presented to a user during interaction with a virtual object is calculated by simulating the contact between the object model and user model. In the simulation, a distributed force is applied to the contact area on the skin tissue of users' hands and results in deformation of the skin tissue. The skin deformation caused by the distributed force is the target contact state that should be presented by the device. However, most multipoint haptic displays do not have sufficient degrees of freedom (DoF) to represent the target contact state. This paper presents the concept and formulation of "deformation matching," whereby the output force is calculated to minimize the error between the target skin deformation and skin deformation that can be realized by the limited DoF device's output force. For comparison, the conventional concept of "force matching" was also formulated. The difference in human perception between these two concepts in the expression of friction was investigated through experiments using a pin-array tactile display capable of stimulating 128 points. It was demonstrated that the perception of the friction coefficient was more sensitive and the perception of the friction direction was more accurate in deformation matching than in force matching.

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.

In Vitro-Induced Germinal Center B Cell Culture System.

In germinal centers (GCs), B cells undergo repeated cycles of proliferation and affinity-based selection, and differentiate into memory B cells or long-lived plasma cells. It has been difficult to elucidate regulatory mechanisms for the dynamic GC B cell maturation and differentiation, partly because experimental manipulation of GC B cells has been limited. Here we describe a culture system in which we can induce massive expansion of naive B cells that exhibit GC B cell-like phenotype and acquire abilities to differentiate into memory B cells or bone marrow plasma cells depending on cytokine conditions. This system will allow us to elucidate the molecular mechanisms of GC B cell differentiation.

BCR and Endosomal TLR Signals Synergize to Increase AID Expression and Establish Central B Cell Tolerance.

Activation-induced cytidine deaminase (AID) is required to purge autoreactive immature and transitional-1 (immature/T1) B cells at the first tolerance checkpoint, but how AID selectively removes self-reactive B cells is unclear. We now show that B cell antigen receptor (BCR) and endosomal Toll-like receptor (TLR) signals synergize to elicit high levels of AID expression in immature/T1 B cells. This synergy is restricted to ligands for endocytic TLR and requires phospholipase-D activation, endosomal acidification, and MyD88. The first checkpoint is significantly impaired in AID- or MyD88-deficient mice and in mice doubly heterozygous for AID and MyD88, suggesting interaction of these factors in central B cell tolerance. Moreover, administration of chloroquine, an inhibitor of endosomal acidification, results in a failure to remove autoreactive immature/T1 B cells in mice. We propose that a BCR/TLR pathway coordinately establishes central tolerance by hyper-activating AID in immature/T1 B cells that bind ligands for endosomal TLRs.

The AID-Cre-ERT2 Model: A Tool for Monitoring B Cell Immune Responses and Generating Selective Hybridomas.

Expression of activation-induced cytidine deaminase (AID) is the hallmark of B cells engaged in an immune response in germinal centers. We designed an inducible fate-mapping reporter mouse in which AID-expressing B cells could be timely and irreversibly marked, by knockin at the Aicda locus of a tamoxifen-inducible Cre recombinase. This mouse model allows notably for the long-term follow-up of memory B cells and plasma cells engaged in an immune response. We describe here a protocol to generate hybridomas from small memory subsets that can be easily traced and identified in this mouse line through Cre-activated fluorescent reporters.

Double knockout mice show BASH and PKCdelta have different epistatic relationships in B cell maturation and CD40-mediated activation.

The development and survival of mature B cells requires an antigen-independent signal from the B cell receptor (BCR) through an adaptor protein containing an SH2 domain, BASH (BLNK/SLP-65). It also requires signaling through BAFF and the BAFF receptor (BAFF-R), and is negatively regulated by protein kinase Cdelta (PKCdelta). In PKCdelta-deficient mice, B cell maturation occurs independently of the BAFF receptor (BAFF-R), indicating that BAFF-R signaling promotes maturation by inhibiting the negative function of PKCdelta. To clarify which of the two signaling pathways plays the primary role in B cell maturation, we crossed BASH-deficient mice with PKCdelta-deficient mice to generate BASH/PKCdelta-double knockout (DKO) mice. In the DKO mice, B cell maturation was blocked at the transitional type 1 (T1) stage and B cells were prone to apoptosis, in common with BASH-deficient mice. This indicates that BASH-mediated BCR signaling primarily controls B cell survival and maturation, with BAFF-R signaling and its inhibition of PKCdelta acting as a secondary regulator. By contrast, CD40-mediated proliferation and antibody production, which are low in BASH-deficient mice, were rescued in the DKO mice, indicating that the suppression of CD40-mediated B cell activation by PKCdelta is epistatic to BASH-mediated promotion. The physiological relevance of these opposing hierarchical effects of BASH and PKCdelta in the regulation of B cell maturation and activation is discussed.

Ex vivo engineered immune organoids for controlled germinal center reactions.

Ex vivo engineered three-dimensional organotypic cultures have enabled the real-time study and control of biological functioning of mammalian tissues. Organs of broad interest where its architectural, cellular, and molecular complexity has prevented progress in ex vivo engineering are the secondary immune organs. Ex vivo immune organs can enable mechanistic understanding of the immune system and more importantly, accelerate the translation of immunotherapies as well as a deeper understanding of the mechanisms that lead to their malignant transformation into a variety of B and T cell malignancies. However, till date, no modular ex vivo immune organ has been developed with an ability to control the rate of immune reaction through tunable design parameter. Here we describe a B cell follicle organoid made of nanocomposite biomaterials, which recapitulates the anatomical microenvironment of a lymphoid tissue that provides the basis to induce an accelerated germinal center (GC) reaction by continuously providing extracellular matrix (ECM) and cell-cell signals to naïve B cells. Compared to existing co-cultures, immune organoids provide a control over primary B cell proliferation with ∼100-fold higher and rapid differentiation to the GC phenotype with robust antibody class switching.

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.

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.

BASH-novel PKC-Raf-1 pathway of pre-BCR signaling induces kappa gene rearrangement.

The pre-B-cell receptor (pre-BCR) is thought to signal transcriptional activation of the immunoglobulin light (L) chain gene locus, proceeding to its V-J rearrangement. The pre-BCR signaling pathway for this process is largely unknown but may involve the adaptor protein BASH (BLNK/SLP-65). Here we report that the pre-B leukemia cell lines established from affected BASH-deficient mice rearrange kappaL-chain gene locus and down-regulate pre-BCR upon PMA treatment or BASH reconstitution. Analyses with specific inhibitors revealed that activation of novel PKC (nPKC) and MEK, but not Ras, is necessary for the rearrangement. Accordingly, retroviral transduction of active PKCeta, PKCepsilon, or Raf-1, but not Ras, induced the kappa gene rearrangement and expression in the pre-B-cell line. Tamoxifen-mediated BASH reconstitution resulted in the translocation of PKCeta to the plasma membrane and kappa chain expression. These data make evident that the Ras-independent BASH-nPKC-Raf-1 pathway of pre-BCR signaling induces the L-chain gene rearrangement and expression.

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 Iglambda chain expression, recombination sequence rearrangements at Igkappa loci, and presence of in-frame VkappaJkappa joins in the Igkappa loci inactivated by the recombination sequence rearrangements, were all diminished in BASH-deficient mice with unmanipulated Ig loci. BCR ligation-induced Iglambda 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.

Distinct signaling requirements for Dmu 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.