Protective neutralizing influenza antibody response in the absence of T follicular helper cells.

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.

Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection.

Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore, there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1 domain of the viral L protein to the Na+/taurocholate cotransporting polypeptide (NTCP). Thus, NTCP should be a key target for the development of anti-HBV therapeutics. Indeed, myrcludex B, a synthetic form of the myristoylated preS1 peptide, effectively reduces HBV/hepatitis D virus (HDV) infection and has been approved as Hepcludex in Europe for the treatment of patients with chronic HDV infection. We established a monoclonal antibody (MAb), N6HB426-20, that recognizes the extracellular domain of human NTCP and blocks HBV entry in vitro into human liver cells but has much less of an inhibitory effect on bile acid uptake. In vivo, administration of the N6HB426-20 MAb prevented HBV viremia for an extended period of time after HBV inoculation in a mouse model system without strongly inhibiting bile acid absorption. Among the extracellular loops (ECLs) of NTCP, regions of amino acids (aa) 84 to 87 in ECL1 and aa 157 to 165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope mapping and the three-dimensional (3D) model of the NTCP structure suggested that the N6HB426-20 MAb may recognize aa 276/277 at the tip of ECL4 and interfere with binding of HBV to the region from aa 84 to 87. In summary, we identified an in vivo neutralizing NTCP-targeting antibody capable of preventing HBV infection. Further improvements in efficacy of this drug will pave the way for its clinical applications. IMPORTANCE A number of entry inhibitors are being developed to enhance the treatment of HBV patients with oral nucleoside/nucleotide analogues (NA). To amplify the effectiveness of NA therapy, several efforts have been made to develop therapeutic MAbs with neutralizing activity against HBs antigens. However, the neutralizing effect of these MAbs may be muted by a large excess of HBsAg-positive noninfectious particles in the blood of infected patients. The advantage of NTCP-targeted HBV entry inhibitors is that they remain effective regardless of viral genotype, viral mutations, and the presence of subviral particles. Although N6HB426-20 requires a higher dose than myrcludex to obtain equivalent suppression of HBV in a model mouse system, it maintained the inhibitory effect for a long time postadministration in proportion to the half-life of an IgG MAb. We believe that further improvements will make this antibody a promising treatment option for patients with chronic hepatitis B.

Bim establishes the B-cell repertoire from early to late in the immune response.

In T cell-dependent antibody responses, some of the activated B cells differentiate along extrafollicular pathways into low-affinity memory and plasma cells, whereas others are involved in subsequent germinal center (GC) formation in follicular pathways, in which somatic hypermutation and affinity maturation occur. The present study demonstrated that Bim, a proapoptotic BH3-only member of the Bcl-2 family, contributes to the establishment of the B-cell repertoire from early to late stages of immune responses to T cell-dependent antigens. Extrafollicular plasma cells grew in the spleen during the early immune response, but their numbers rapidly declined with the appearance of GC-derived progeny in wild-type mice. By contrast, conditional Bim deficiency in B cells resulted in expansion of extrafollicular IgG1+ antibody-forming cells (AFCs) and this expansion was sustained during the late response, which hampered the formation of GC-derived high-affinity plasma cells in the spleen. Approximately 10% of AFCs in mutant mice contained mutated VH genes; thus, Bim deficiency appears not to impede the selection of high-affinity AFC precursor cells. These results suggest that Bim contributes to the replacement of low-affinity antibody by high-affinity antibody as the immune response progresses.

Repression of the transcription factor Bach2 contributes to predisposition of IgG1 memory B cells toward plasma cell differentiation.

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.

Bcl6 middle domain repressor function is required for T follicular helper cell differentiation and utilizes the corepressor MTA3.

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.

CD4 memory T cells develop and acquire functional competence by sequential cognate interactions and stepwise gene regulation.

Memory CD4(+) T cells promote protective humoral immunity; however, how memory T cells acquire this activity remains unclear. This study demonstrates that CD4(+) T cells develop into antigen-specific memory T cells that can promote the terminal differentiation of memory B cells far more effectively than their naive T-cell counterparts. Memory T cell development requires the transcription factor B-cell lymphoma 6 (Bcl6), which is known to direct T-follicular helper (Tfh) cell differentiation. However, unlike Tfh cells, memory T cell development did not require germinal center B cells. Curiously, memory T cells that develop in the absence of cognate B cells cannot promote memory B-cell recall responses and this defect was accompanied by down-regulation of genes associated with homeostasis and activation and up-regulation of genes inhibitory for T-cell responses. Although memory T cells display phenotypic and genetic signatures distinct from Tfh cells, both had in common the expression of a group of genes associated with metabolic pathways. This gene expression profile was not shared to any great extent with naive T cells and was not influenced by the absence of cognate B cells during memory T cell development. These results suggest that memory T cell development is programmed by stepwise expression of gatekeeper genes through serial interactions with different types of antigen-presenting cells, first licensing the memory lineage pathway and subsequently facilitating the functional development of memory T cells. Finally, we identified Gdpd3 as a candidate genetic marker for memory T cells.

Cutting edge: T follicular helper cell differentiation is defective in the absence of Bcl6 BTB repressor domain function.

T follicular helper (Tfh) cells are essential for germinal centers (GCs) and most long-term humoral immunity. Differentiation of Tfh cells depends on the transcriptional repressor B cell CLL/lymphoma 6 (Bcl6). Bcl6 mediates gene repression via the recruitment of corepressors. Currently, it is unknown how Bcl6 recruits corepressors to regulate gene expression of Tfh cells. In this article, we demonstrate, using a mutant form of Bcl6 with two BTB (bric-a-brac, tramtrack, broad-complex) mutations that abrogate corepressor binding, that the Bcl6 BTB domain is required for proper differentiation of Tfh and GC-Tfh cells in vivo. Importantly, we also observe a significant defect in GC B cell development. These results are consistent in multiple contexts, including a novel lymphocytic choriomeningitis virus nucleoprotein-specific TCR-transgenic mouse model. Taken together, these data suggest that the Bcl6 BTB domain is a key mediator of the differentiation of Tfh cells.

Generation of memory B cells inside and outside germinal centers.

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.

Both mutated and unmutated memory B cells accumulate mutations in the course of the secondary response and develop a new antibody repertoire optimally adapted to the secondary stimulus.

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.

Lyn signaling to upregulate GANP is critical for the survival of high-affinity B cells in germinal centers of lymphoid organs.

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.

HIV-1 Nef impairs multiple T-cell functions in antigen-specific immune response in mice.

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.

Augmented antibody response with premature germinal center regression in CD40L transgenic mice.

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.

Increase of regulatory T cells and the ratio of specific IgE to total IgE are candidates for response monitoring or prognostic biomarkers in 2-year sublingual immunotherapy (SLIT) for Japanese cedar pollinosis.

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.

The induced regulatory T cell level, defined as the proportion of IL-10(+)Foxp3(+) cells among CD25(+)CD4(+) leukocytes, is a potential therapeutic biomarker for sublingual immunotherapy: a preliminary report.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Development of a simple new flow cytometric antibody-dependent cellular cytotoxicity (ADCC) assay with excellent sensitivity.

Antibody-based therapeutic strategies have become recognized as useful clinical options in several types of cancer, often with the expectation that such therapies will trigger target cell elimination via antibody-dependent cellar cytotoxicity (ADCC) by natural killer cells. The successful development of therapeutic monoclonal antibodies (mAbs) requires an assay system that permits a critical evaluation of their physicochemical and biological characteristics. At present a number of ADCC assay systems have been reported, however, there is still room for improvement in terms of usability, operability and sensitivity. Here we report a novel flow cytometric ADCC assay that uses a human natural killer cell line stably transfected with mouse FcγRIII, and Fc receptor common-γ chain (FcRγ) and a reporter gene as effector cells. This assay relies on discriminating effector and target cells by their differential immunofluorescence, which allows for clear-cut gating and accurate calculation of the number of surviving cells in a target population. This assay is easy and quick to perform and provides reliable data even for low frequency target cells in assay samples and with low concentrations of mAbs. Furthermore, our approach allows us to identify synergistic ADCC activity of mAbs with different epitope specificities on the same target antigen.

Formalin-treated UV-inactivated SARS coronavirus vaccine retains its immunogenicity and promotes Th2-type immune responses.

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.

Selective expansion of perforin-positive CD8+ T cells by immature dendritic cells infected with live Bacillus Calmette-Guérin mycobacteria.

Live, but not dead Bacillus Calmette-Guérin (BCG) is partially protective against infection by Mycobacterium tuberculosis, which causes a disease with high mortality in immune compromised individuals. We have shown that uptake of BCG induces maturation of immature dendritic cells (DCs) regardless of the viability of the bacteria. Importantly, when T cells are cocultured with live BCG-infected DCs, the proportion of CD45RA(-) perforin(+) CD8+ T cells is markedly expanded markedly; however, little expansion is seen when T cells are cocultured with DCs harboring heat-killed BCG. The direct contact of T cells with live BCG-infected DCs was required for the expansion of perforin(+) CD8+ T cells. These CD8+ T cells demonstrated a high level of killing activity against BCG-infected macrophages. There was little contribution of cytokines, including IFN-gamma, TNF-alpha, and IL-12, to the expansion of CD8+ T cells by live BCG-infected DCs. We found that the interaction between BCG-infected DCs and CD8+ T cells through CD40/CD40L was crucial for the expansion and maturation of CD8+ T cells, the process of which was CD4-independent. In contrast, blocking the CD58/CD2 but not the CD40/CD40L interaction reduced production of IFN-gamma without affecting the maturation of CD8+ T cells. This indicates that the production of IFN-gamma and perforin by CD8+ T cells is mediated by distinct signals delivered from BCG-infected DCs. Thus, BCG-specific CD8+ CTL memory cells may be maintained for a long period of time in BCG-vaccinated hosts, and these cells could mature rapidly into effectors through the potent antigen-presenting function of DCs upon mycobacterial infection.

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.

Studies on the generation and maintenance of mucosal cytotoxic T lymphocytes against human immunodeficiency virus type 1 Gag in mice.

Aspects of the generation and maintenance of mucosal immunity against human immunodeficiency virus type 1 (HIV-1) were examined. Mice were immunized either intranasally or intrarectally with recombinant HIV-1 Gag p24 protein plus cholera toxin. Nasal immunization generated strong nasal IgA responses but low vaginal IgA, whereas rectal immunization yielded good vaginal IgA responses but poor nasal responses. Nasal immunization resulted in strong Gag-specific cytotoxic T lymphocyte (CTL) activity in nasal-associated lymphoid tissue (NALT), posterior cervical lymph nodes (pCLNs), and the spleen, but not in mesenteric lymph nodes (MLNs). Rectal immunization induced weak Gag-specific CTLs in the MLNs only, indicating distinct compartmentalization of the upper and lower mucosa. Combining nasal and rectal immunizations overcame their respective deficiencies. CTL memory after the third nasal immunization was found to persist for up to 6 months in the draining pCLNs, but was gradually lost from the NALT induction site. Analysis of the T cell receptor Vbeta usage of Gag-specific CD8(+) T cells in lymphoid tissues of intranasally immunized mice indicated that the memory CTLs in the pCLNs are generated from a few clones in NALT. The memory CTL clones also appear to be poor killers whereas the NALT clones from which the pCLN clones appear to originate are potent killers. Our results support the view that CTL activity is determined by the level and duration of antigen stimulation and that in NALT, CTLs develop as effector memory T cells with high avidity, whereas the pCLNs sequester the memory T cells with low avidity but longer survival.