[Show abstract][Hide abstract] ABSTRACT: T follicular helper (Tfh) cells are essential providers of help to B cells. The transcription factor B-cell CLL/lymphoma 6 (Bcl6) is a lineage-defining regulator of Tfh cells and germinal center B cells. In B cells, Bcl6 has the potential to recruit distinct transcriptional corepressors through its BTB domain or its poorly characterized middle domain (also known as RDII), but in Tfh cells the roles of the Bcl6 middle domain have yet to be clarified. Mimicked acetylation of the Bcl6 middle domain (K379Q) in CD4 T cells results in significant reductions in Tfh differentiation in vivo. Blimp1 (Prdm1) is a potent inhibitor of Tfh cell differentiation. Although Bcl6 K379Q still bound to the Prdm1 cis-regulatory elements in Tfh cells, Prdm1 expression was derepressed. This was a result of the failure of Bcl6 K379Q to recruit metastasis-associated protein 3 (MTA3). The loss of Bcl6 function in Bcl6 K379Q-expressing CD4 T cells could be partially rescued by abrogating Prdm1 expression. In addition to Prdm1, we found that Bcl6 recruits MTA3 to multiple genes involved in Tfh cell biology, including genes important for cell migration, cell survival, and alternative differentiation pathways. Thus, Bcl6 middle domain mediated repression is a major mechanism of action by which Bcl6 controls CD4 T-cell fate and function.
Proceedings of the National Academy of Sciences 10/2015; 112(43). DOI:10.1073/pnas.1507312112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Germinal centers (GCs) are generally considered to be the sole site of memory B-cell generation. However, recent studies demonstrate that memory B cells can also develop in response to a T-cell-dependent (TD) antigen before the onset, and independently of, the GC reaction. These two classes of memory cells persist equally over long periods of time and attain functional maturation through distinct but related transcriptional programs. Although the development of both memory B-cell types requires classical T-cell help, the generation of GC-dependent memory B cells requires TFH -cell help, while the generation of GC-independent memory cells does not. These findings led to the conclusion that B-cell memory is generated along two fundamentally distinct cellular differentiation pathways. In this review, we focus on the GC-independent pathway of memory B-cell development, and discuss how the unique features of memory B cells are maintained in the GC-independent pathway. This article is protected by copyright. All rights reserved.
European Journal of Immunology 05/2014; 44(5). DOI:10.1002/eji.201343716 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: High-affinity memory B cells are preferentially selected during secondary responses and rapidly differentiate into antibody-producing cells. However, it remains unknown whether only high-affinity, mutated memory B cells simply expand to dominate the secondary response or if in fact memory B cells with a diverse VH repertoire, including those with no mutations, accumulate somatic mutations to create a new repertoire through the process of affinity maturation. In this report, we took a new approach to address this question by analyzing the VH gene repertoire of IgG1(+) memory B cells before and after antigen re-exposure in a host unable to generate IgG(+) B cells. We show here that both mutated and unmutated IgG1(+) memory B cells respond to secondary challenge and expand while accumulating somatic mutations in their VH genes in a stepwise manner. Both types of memory cells subsequently established a VH gene repertoire dominated by two major clonotypes, which are distinct from the original repertoire before antigen re-exposure. In addition, heavily mutated memory B cells were excluded from the secondary repertoire. Thus, both mutated and unmutated IgG1(+) memory cells equally contribute to establish a new antibody repertoire through a dynamic process of mutation and selection, becoming optimally adapted to the recall challenge.
International Immunology 09/2013; 25(12). DOI:10.1093/intimm/dxt030 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Memory B cells are essential for generating rapid and robust secondary antibody responses. It has been thought that the unique cytoplasmic domain of IgG causes the prompt activation of antigen-experienced IgG memory B cells. To assess this model, we have generated a mouse containing IgG1 B cells that have never encountered antigen. We found that, upon challenge, antigen-experienced IgG1 memory B cells rapidly differentiated into plasma cells, whereas nonexperienced IgG1 B cells did not, suggesting the importance of the stimulation history. In addition, our results suggest that repression of the Bach2 transcription factor, which results from antigen experience, contributes to predisposition of IgG1 memory B cells to differentiate into plasma cells.
[Show abstract][Hide abstract] ABSTRACT: Infection with single strand RNA (ssRNA) viruses, such as influenza A virus, is known to induce protective acquired immune responses, including the production of neutralizing antibodies. Vaccination also causes a reduction in the number of peripheral blood leukocytes (PBL) shortly after inoculation, a result which may have undesirable adverse effects. The cellular mechanisms for this response have not been elucidated so far. Here we report that formalin-inactivated influenza A whole virus vaccine (whole virion) induces a significant decrease in PBL in mice 5-16h after administration, whereas an ether-split vaccine (HA split) made from the same influenza virus strain does not induce a similar loss of PBL. Concordant with this reduction in the number of PBL, a rapidly induced and massive production of interferon (IFN)-α is observed when mice are injected with whole virion, but not with HA split vaccines. The role of Toll-like receptors (TLR), which are involved in signal transduction of influenza virus, and the subsequent induction of IFNα were confirmed using mice lacking TLR7, MyD-88, or IFNα/β receptor. We further demonstrated that the observed PBL loss is caused by apoptosis in an IFNα-dependent manner, and not by leukocyte redistribution s due to chemokine signaling failure. These findings indicate that RNA-encapsulated whole virion vaccines can rapidly induce a loss of leukocytes from peripheral blood by apoptosis, which may modulate the subsequent immune response.
[Show abstract][Hide abstract] ABSTRACT: One component of memory in the antibody system is long-lived memory B cells selected for the expression of somatically mutated, high-affinity antibodies in the T cell-dependent germinal center (GC) reaction. A puzzling observation has been that the memory B cell compartment also contains cells expressing unmutated, low-affinity antibodies. Using conditional Bcl6 ablation, we demonstrate that these cells are generated through proliferative expansion early after immunization in a T cell-dependent but GC-independent manner. They soon become resting and long-lived and display a novel distinct gene expression signature which distinguishes memory B cells from other classes of B cells. GC-independent memory B cells are later joined by somatically mutated GC descendants at roughly equal proportions and these two types of memory cells efficiently generate adoptive secondary antibody responses. Deletion of T follicular helper (Tfh) cells significantly reduces the generation of mutated, but not unmutated, memory cells early on in the response. Thus, B cell memory is generated along two fundamentally distinct cellular differentiation pathways. One pathway is dedicated to the generation of high-affinity somatic antibody mutants, whereas the other preserves germ line antibody specificities and may prepare the organism for rapid responses to antigenic variants of the invading pathogen.
Journal of Experimental Medicine 10/2012; 209(11):2079-97. DOI:10.1084/jem.20120127 · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Signals through BCR and costimulatory molecules play essential roles in selecting high-affinity B cells with Ig V-region mutations in the germinal centers (GCs) of peripheral lymphoid organs. Lyn-deficient (lyn(-/-)) mice show impaired BCR signal triggering for cell proliferation and GC formation, causing hyper-IgM, and display autoimmunity after aging. In this study, we demonstrate that Lyn-mediated signaling to upregulate GANP is essential for the survival of mature GC-like (mGC) B cells with high-affinity type BCR mutations upon Ag immunization. Transgenic ganp expression into lyn(-/-) mice did not recover the Lyn-deficient phenotype with regard to B cell differentiation, serum Igs, and impaired GC formation in spleens after immunization with nitrophenyl-chicken γ-globulin, but it markedly rescued cell survival of mGC B cells by suppressing DNA damage, thereby increasing the frequency of the Trp(33)-to-Leu mutation in the IgV(H)-186.2 region and affinity maturation of nitrophenyl-binding B cells. GANP may play a critical role in Lyn-mediated signaling for the selection of high-affinity B cells in peripheral lymphoid organs.
The Journal of Immunology 08/2012; 189(7):3472-9. DOI:10.4049/jimmunol.1200649 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The viral protein Nef is a key element for the progression of HIV disease. Previous in vitro studies suggested that Nef expression in T-cell lines enhanced TCR signaling pathways upon stimulation with TCR cross-linking, leading to the proposal that Nef lowers the threshold of T-cell activation, thus increasing susceptibility to viral replication in immune response. Likewise, the in vivo effects of Nef transgenic mouse models supported T-cell hyperresponse by Nef. However, the interpretation is complicated by Nef expression early in the development of T cells in these animal models. Here, we analyzed the consequence of Nef expression in ovalbumin-specific/CD4(+) peripheral T cells by using a novel mouse model and demonstrate that Nef inhibits antigen-specific T-cell proliferation and multiple functions required for immune response in vivo, which includes T-cell helper activity for the primary and memory B-cell response. However, Nef does not completely abrogate T-cell activity, as defined by low levels of cytokine production, which may afford the virus a replicative advantage. These results support a model, in which Nef expression does not cause T-cell hyperresponse in immune reaction, but instead reduces the T-cell activity, that may contribute to a low level of virus spread without viral cytopathic effects.
International Immunology 07/2011; 23(7):433-41. DOI:10.1093/intimm/dxr031 · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aims of this study were to examine the therapeutic effects of sublingual immunotherapy (SLIT) and to identify potential biomarkers that would predict the therapeutic response in a randomized, double-blind, placebo-controlled clinical trial. The trial was carried out over two pollinosis seasons in 2007 and 2008. Carry-over therapeutic effects were analyzed in 2009. SLIT significantly ameliorated the symptoms of pollinosis during the 2008 and 2009 pollen seasons. Cry j 1-specific cytokine production in a subgroup of patients with mild disease in the SLIT group was significantly attenuated. The ratio of specific IgE to total IgE before treatment correlated with the symptom-medication score in the SLIT group in 2008. Patients with increased Cry j 1-iTreg in the SLIT group had significantly improved QOL and QOL-symptom scores. In summary, the specific IgE to total IgE ratio and upregulation of Cry j 1-iTreg are candidates for biomarker of the clinical response to SLIT.
[Show abstract][Hide abstract] ABSTRACT: Japanese cedar (Cryptomeria japonica) pollinosis is one of the most prevalent allergies in Japan. Recently, two reports described the positive effects of sublingual immunotherapy (SLIT) against Japanese cedar pollinosis. However, the therapeutic biomarkers for SLIT are still unclear. We performed this unblinded, nonrandomized, open-label study to identify therapeutic biomarkers for SLIT against Japanese cedar pollinosis.
We performed an open-label study during one pollinosis season in 2007, enrolling 19 patients from in-house volunteers suffering from Japanese cedar pollinosis. Peripheral blood was obtained from all participants before SLIT treatment as well as before and after the pollen season. The plasma levels of an immunoglobulin specific to a major allergen (Cry j 1) were determined. We analyzed the induction of regulatory T cells (iTregs), namely IL-10(+)Foxp3(+) cells in CD25(+)CD4(+) leukocytes, by flow cytometry. The Th2-type responses were analyzed by cytokine production from peripheral blood mononuclear cells after stimulation with Cry j 1. Clinical symptoms were estimated using a quality of life questionnaire in the middle of the pollen season.
The difference in numbers of iTregs between the medium-only control cell culture and cells stimulat- ed with Cry j 1 was significantly decreased in the non-SLIT group but was unchanged in the SLIT group after the pollen season. The subgroup of the SLIT group with increased iTregs showed more attenuated Th2-type cytokine profiles, and symptom scores in the subgroup with increased iTregs were significantly lower than those in the subgroup with decreased iTregs.
The antigen-specific iTreg level is a potential therapeutic biomarker that correlates with clinical pollinosis symptoms and may be involved in the therapeutic mechanisms of SLIT.
International Archives of Allergy and Immunology 11/2010; 153(4):378-87. DOI:10.1159/000316349 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although CD40 signaling is required for activation and differentiation of B cells, including germinal center (GC) formation and generation of memory B cells, in vivo generation of CD40 signaling augments plasma cell differentiation but disrupts GCs. Thus, CD40 signaling is thought to direct B cells to extrafollicular plasma cell fate rather than GC formation. In this study, we analyzed CD40L transgenic (CD40LTg) mice that constitutively express CD40L on B cells. After immunization, activation of B cells, but not dendritic cells, was augmented, although dendritic cells can be activated by CD40 ligation. Bone marrow chimera carrying CD40LTg and nontransgenic B cells showed increased Ab production from transgenic, but not from coexisting nontransgenic, B cells, suggesting that CD40L on a B cell preferentially stimulates the same B cell through an autocrine pathway, thereby augmenting Ab production. Although GCs rapidly regressed after day 5 of immunization and failed to generate late-appearing high-affinity Ab, CD40LTg mice showed normal GC formation up to day 5, as well as normal generation of long-lived plasma cells and memory B cell responses. This observation suggests that CD40 signaling does not block GC formation or differentiation of GC B cells, but it inhibits sustained expansion of GC B cells and augments B cell differentiation.
The Journal of Immunology 07/2010; 185(1):211-9. DOI:10.4049/jimmunol.0901694 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Resting antigen-experienced memory B cells are thought to be responsible for the more rapid and robust antibody responses after antigen reencounter, which are the hallmark of memory humoral responses. The molecular basis for the development and survival of memory B cells remains largely unknown. We report that phospholipase C (PLC) gamma2 is required for efficient formation of germinal center (GC) and memory B cells. Moreover, memory B cell homeostasis is severely hampered by inducible loss of PLC-gamma2. Accordingly, mice with a conditional deletion of PLC-gamma2 in post-GC B cells had an almost complete abrogation of the secondary antibody response. Collectively, our data suggest that PLC-gamma2 conveys a survival signal to GC and memory B cells and that this signal is required for a productive secondary immune response.
Journal of Experimental Medicine 04/2009; 206(3):681-9. DOI:10.1084/jem.20082100 · 12.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Group A streptococcus (GAS) causes variety of diseases ranging from common pharyngitis to life-threatening severe invasive diseases, including necrotizing fasciitis and streptococcal toxic shock-like syndrome. The characteristic of invasive GAS infections has been thought to attribute to genetic changes in bacteria, however, no clear evidence has shown due to lack of an intriguingly study using serotype-matched isolates from clinical severe invasive GAS infections. In addition, rare outbreaks of invasive infections and their distinctive pathology in which infectious foci without neutrophil infiltration hypothesized us invasive GAS could evade host defense, especially neutrophil functions. Herein we report that a panel of serotype-matched GAS, which were clinically isolated from severe invasive but not from non-invaive infections, could abrogate functions of human polymorphnuclear neutrophils (PMN) in at least two independent ways; due to inducing necrosis to PMN by enhanced production of a pore-forming toxin streptolysin O (SLO) and due to impairment of PMN migration via digesting interleukin-8, a PMN attracting chemokine, by increased production of a serine protease ScpC. Expression of genes was upregulated by a loss of repressive function with the mutation of csrS gene in the all emm49 severe invasive GAS isolates. The csrS mutants from clinical severe invasive GAS isolates exhibited high mortality and disseminated infection with paucity of neutrophils, a characteristic pathology seen in human invasive GAS infection, in a mouse model. However, GAS which lack either SLO or ScpC exhibit much less mortality than the csrS-mutated parent invasive GAS isolate to the infected mice. These results suggest that the abilities of GAS to abrogate PMN functions can determine the onset and severity of invasive GAS infection.
PLoS ONE 02/2008; 3(10):e3455. DOI:10.1371/journal.pone.0003455 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The demand for rapid and simple development of a vaccine against a newly emerging infectious disease is increasing worldwide. We previously revealed that UV-inactivated severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) virions (UV-V) elicited high levels of humoral immunity and a weak Th0 response in mice immunized subcutaneously. To ensure the safety of such a whole inactivated SARS-CoV vaccine, we additionally treated the UV-V vaccine with formalin, resulting in the UV-F-V vaccine. Analysis of the immunogenicity of the UV-F-V+alum vaccine in mice revealed that it generated comparable neutralizing serum anti-SARS-CoV IgG antibody levels as the UV-V+alum vaccine. Moreover, both vaccines induced similar frequencies of anti-SARS-CoV IgG antibody-producing cells in bone marrow. Interestingly, the UV-F-V vaccine induced fewer IgG(2a) subtype antibodies and higher interleukin-4 production in vaccinated mice than did UV-V. Thus, UV-F-V imposes a Th2-type bias on the immune response, unlike UV-V. We propose here that doubly-inactivated SARS-CoV virions by UV and formalin constitute a safe vaccine that may effectively induce neutralizing antibodies in humans.
Japanese journal of infectious diseases 06/2007; 60(2-3):106-12. · 1.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Jawless vertebrates have acquired immunity but do not have immunoglobulin-type antigen receptors. Variable lymphocyte receptors (VLRs) have been identified in lamprey that consist of multiple leucine-rich repeat (LRR) modules. An active VLR gene is generated by the assembly of a series of variable gene segments, including many that encode LRRs. Stepwise assembly of the gene segments seems to occur by replacement of the intervening DNA between the 5' and 3' constant-region genes. Here we report that lamprey (Lethenteron japonicum) assemble their VLR genes by a process involving 'copy choice'. Regions of short homology seemed to prime copying of donor LRR-encoding sequences into the recipient gene. Those LRR-encoding germline sequences were abundant and shared extensive sequence homologies. Such genomic organization permits initiation of copying anywhere in an LRR-encoding module for the generation of various hybrid LRRs. Thus, a vast repertoire of recombinant VLR genes could be generated not only by copying of various LRR segments in diverse combinations but also by the use of multiple sites in an LRR gene segment for priming.
[Show abstract][Hide abstract] ABSTRACT: The programmed death-1 (PD-1) costimulatory pathway has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in establishing an immune privilege status of corneal allografts in mice. B7-H1, but not B7-DC or PD-1, was expressed constitutively in the eye, i.e., cornea, iris-ciliary body, and retina. After corneal allografting, PD-1(+)CD4(+) T cells infiltrated and adhered with B7-H1(+) corneal endothelium. Blockade of PD-1 or B7-H1, but not B7-DC, led to accelerated corneal allograft rejection. In B7-H1-expressing corneal allografts, apoptosis of the infiltrating PD-1(+)CD4(+) or CD8(+) T cells was observed, after which there was allograft acceptance. In contrast, B7-H1 blockade suppressed apoptosis of infiltrating PD-1(+) T cells, which led to allograft rejection. In vitro, destruction of corneal endothelial cells by alloreactive T cells was enhanced when the cornea was pretreated with anti-B7-H1 Ab. This is the first demonstration that the constitutive expression of B7-H1 plays a critical role in corneal allograft survival. B7-H1 expressed on corneal endothelial cells maintains long-term acceptance of the corneal allografts by inducing apoptosis of effector T cells within the cornea.
The Journal of Immunology 12/2006; 177(9):5928-35. DOI:10.4049/jimmunol.177.9.5928 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SARS-coronavirus (SARS-CoV) has recently been identified as the causative agent of SARS. We constructed a series of recombinant DIs (rDIs), a highly attenuated vaccinia strain, expressing a gene encoding four structural proteins (E, M, N and S) of SARS-CoV individually or simultaneously. These rDIs elicited SARS-CoV-specific serum IgG antibody and T-cell responses in vaccinated mice following intranasal or subcutaneous administration. Mice that were subcutaneously vaccinated with rDIs expressing S protein with or without other structural proteins induced a high level of serum neutralizing IgG antibodies and demonstrated marked protective immunity against SARS-CoV challenge in the absence of a mucosal IgA response. These results indicate that the potent immune response elicited by subcutaneous injection of rDIs containing S is able to control mucosal infection by SARS-CoV. Thus, replication-deficient DIs constructs hold promise for the development of a safe and potent SARS vaccine.
[Show abstract][Hide abstract] ABSTRACT: SARS-CoV is a new type of human coronavirus identified as a causative agent of severe acute respiratory syndrome (SARS). On the occasion of the SARS outbreak, various monoclonal antibodies (mAbs) against SARS-CoV have been developed and applied for diagnosis, clinical management and basic research. In this review, we overview the biochemical and functional properties and applications of these SARS-CoV mAbs. We also focus on a variety of vaccines currently under development and discuss the immune response elicited by these vaccines in animal models, hopefully to better understand what we need to do next to fight against newly emerging pathogens in the future.
Reviews in Medical Virology 03/2006; 16(2):117-31. DOI:10.1002/rmv.492 · 5.57 Impact Factor