Tumor epitope spreading by a novel multivalent therapeutic cellular vaccine targeting cancer antigens to invariant NKT-triggered dendritic cells in situ.

Introduction: Cancer is categorized into two types based on the microenvironment: cold and hot tumors. The former is challenging to stimulate through immunity. The immunogenicity of cancer relies on the quality and quantity of cancer antigens, whether recognized by T cells or not. Successful cancer immunotherapy hinges on the cancer cell type, antigenicity and subsequent immune reactions. The T cell response is particularly crucial for secondary epitope spreading, although the factors affecting these mechanisms remain unknown. Prostate cancer often becomes resistant to standard therapy despite identifying several antigens, placing it among immunologically cold tumors. We aim to leverage prostate cancer antigens to investigate the potential induction of epitope spreading in cold tumors. This study specifically focuses on identifying factors involved in secondary epitope spreading based on artificial adjuvant vector cell (aAVC) therapy, a method established as invariant natural killer T (iNKT) -licensed DC therapy. Methods: We concentrated on three prostate cancer antigens (prostate-specific membrane antigen (PSMA), prostate-specific antigen (PSA), and prostatic acid phosphatase (PAP)). By introducing allogeneic cells with the antigen and murine CD1d mRNA, followed by α-galactosylceramide (α-GalCer) loading, we generated five types of aAVCs, i.e, monovalent, divalent and trivalent antigen-expressing aAVCs and four types of prostate antigen-expressing cold tumors. We evaluated iNKT activation and antigen-specific CD8+ T cell responses against tumor cells prompted by the aAVCs. Results: Our study revealed that monovalent aAVCs, expressing a single prostate antigen, primed T cells for primary tumor antigens and also induced T cells targeting additional tumor antigens by triggering a tumor antigen-spreading response. When we investigated the immune response by trivalent aAVC (aAVC-PROS), aAVC-PROS therapy elicited multiple antigen-specific CD8+ T cells simultaneously. These CD8+ T cells exhibited both preventive and therapeutic effects against tumor progression. Conclusions: The findings from this study highlight the promising role of tumor antigen-expressing aAVCs, in inducing efficient epitope spreading and generating robust immune responses against cancer. Our results also propose that multivalent antigen-expressing aAVCs present a promising therapeutic option and could be a more comprehensive therapy for treating cold tumors like prostate cancer.

Zeb2 regulates differentiation of long-lived effector of invariant natural killer T cells.

After activation, some invariant natural killer T (iNKT) cells are differentiated into Klrg1+ long-lived effector NKT1 cells. However, the regulation from the effector phase to the memory phase has not been elucidated. Zeb2 is a zinc finger E homeobox-binding transcription factor and is expressed in a variety of immune cells, but its function in iNKT cell differentiation remains also unknown. Here, we show that Zeb2 is dispensable for development of iNKT cells in the thymus and their maintenance in steady state peripheral tissues. After ligand stimulation, Zeb2 plays essential roles in the differentiation to and maintenance of Klrg1+ Cx3cr1+GzmA+ iNKT cell population derived from the NKT1 subset. Our results including single-cell-RNA-seq analysis indicate that Zeb2 regulates Klrg1+ long-lived iNKT cell differentiation by preventing apoptosis. Collectively, this study reveals the crucial transcriptional regulation by Zeb2 in establishment of the memory iNKT phase through driving differentiation of Klrg1+ Cx3cr1+GzmA+ iNKT population.

Detection of mutant antigen-specific T cell receptors against multiple myeloma for T cell engineering.

Multiple myeloma (MM) remains an incurable hematological neoplasm. Neoantigen-specific T cell receptor (TCR)-engineered T (TCR-T) cell therapy is a potential alternative treatment. Particularly, TCRs derived from a third-party donor may cover broad ranges of neoantigens, whereas TCRs in patients suffering from immune disorders are limited. However, the efficacy and feasibility of treating MM have not been evaluated thoroughly. In this study, we established a system for identifying immunogenic mutant antigens on MM cells and their corresponding TCRs using healthy donor-derived peripheral blood mononuclear cells (PBMCs). Initially, the immune responses to 35 candidate peptides predicted by the immunogenomic analysis were investigated. Peptide-reactive T lymphocytes were enriched, and subsequently, TCR repertoires were determined by single-cell TCR sequencing. Eleven reconstituted TCRs showed mutation-specific responses against 4 peptides. Particularly, we verified the HLA-A∗24:02-binding QYSPVQATF peptide derived from COASY S55Y as the naturally processed epitope across MM cells, making it a promising immune target. Corresponding TCRs specifically recognized COASY S55Y+HLA-A∗24:02+ MM cells and augmented tumoricidal activity. Finally, adoptive cell transfer of TCR-T cells showed objective responses in the xenograft model. We initiatively proposed the utility of tumor mutated antigen-specific TCR genes to suppress MM. Our unique strategy will facilitate further identification of neoantigen-specific TCRs.

Natural Killer T and Natural Killer Cell-Based Immunotherapy Strategies Targeting Cancer.

Both natural killer T (NKT) and natural killer (NK) cells are innate cytotoxic lymphoid cells that produce inflammatory cytokines and chemokines, and their role in the innate immune response to tumors and microorganisms has been investigated. Especially, emerging evidence has revealed their status and function in the tumor microenvironment (TME) of tumor cells. Some bacteria producing NKT cell ligands have been identified to exert antitumor effects, even in the TME. By contrast, tumor-derived lipids or metabolites may reportedly suppress NKT and NK cells in situ. Since NKT and NK cells recognize stress-inducible molecules or inhibitory molecules on cancer cells, their status or function depends on the balance between inhibitory and activating receptor signals. As a recent strategy in cancer immunotherapy, the mobilization or restoration of endogenous NKT or NK cells by novel vaccines or therapies has become a focus of research. As a new biological evidence, after activation, effector memory-type NKT cells lasted in tumor-bearing models, and NK cell-based immune checkpoint inhibition potentiated the enhancement of NK cell cytotoxicity against cancer cells in preclinical and clinical trials. Furthermore, several new modalities based on the characteristics of NKT and NK cells, including artificial adjuvant vector cells, chimeric antigen receptor-expressing NK or NKT cell therapy, or their combination with immune checkpoint blockade have been developed. This review examines challenges and future directions for improving these therapies.

Association of cellular immunity with severity of COVID-19 from the perspective of antigen-specific memory T cell responses and cross-reactivity.

Coronaviruses regularly cause outbreaks of zoonotic diseases characterized by severe pneumonia. The new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused the global pandemic disease COVID-19 that began at the end of 2019 and spread rapidly owing to its infectious nature and rapidly progressing pneumonia. Although the infectivity of SARS-CoV-2 is high, indicated by the worldwide spread of the disease in a very short period, many individuals displayed only subclinical infection, and some of them transmitted the disease to individuals who then developed a severe symptomatic infection. Furthermore, there are differences in the severity of infection across countries, which can be attributed to factors such as the emergence of viral mutations in a short period of time as well as to the immune responses to viral factors. Anti-viral immunity generally consists of neutralizing antibodies that block viral infection and cytotoxic CD8+ T cells that eliminate the virus-infected cells. There is compelling evidence for the role of neutralizing antibodies in protective immunity in SARS-CoV-2 infection. However, the role of CD4+ and CD8+ T cells after the viral entry is complex and warrants a comprehensive discussion. Here, we discuss the protection afforded by cellular immunity against initial infection and development of severe disease. The initial failure of cellular immunity to control the infection worsens the clinical outcomes and functional profiles that inflict tissue damage without effectively eliminating viral reservoirs, while robust T cell responses are associated with mild outcomes. We also discuss persistent long-lasting memory T cell-mediated protection after infection or vaccination, which is rather complicated as it may involve SARS-CoV-2-specific cytotoxic T lymphocytes or cross-reactivity with previously infected seasonal coronaviruses, which are largely related to HLA genotypes. In addition, cross-reactivity with mutant strains is also discussed. Lastly, we discuss appropriate measures to be taken against the disease for immunocompromised patients. In conclusion, we provide evidence and discuss the causal relationship between natural infection- or vaccine-mediated memory T cell immunity and severity of COVID-19. This review is expected to provide a basis to develop strategies for the next generation of T cell-focused vaccines and aid in ending the current pandemic.

Cancer immunotherapy using artificial adjuvant vector cells to deliver NY-ESO-1 antigen to dendritic cells in situ.

NY-ESO-1 is a cancer/testis antigen expressed in various cancer types. However, the induction of NY-ESO-1-specific CTLs through vaccines is somewhat difficult. Thus, we developed a new type of artificial adjuvant vector cell (aAVC-NY-ESO-1) expressing a CD1d-NKT cell ligand complex and a tumor-associated antigen, NY-ESO-1. First, we determined the activation of invariant natural killer T (iNKT) and natural killer (NK) cell responses by aAVC-NY-ESO-1. We then showed that the NY-ESO-1-specific CTL response was successfully elicited through aAVC-NY-ESO-1 therapy. After injection of aAVC-NY-ESO-1, we found that dendritic cells (DCs) in situ expressed high levels of costimulatory molecules and produced interleukn-12 (IL-12), indicating that DCs undergo maturation in vivo. Furthermore, the NY-ESO-1 antigen from aAVC-NY-ESO-1 was delivered to the DCs in vivo, and it was presented on MHC class I molecules. The cross-presentation of the NY-ESO-1 antigen was absent in conventional DC-deficient mice, suggesting a host DC-mediated CTL response. Thus, this strategy helps generate sufficient CD8+ NY-ESO-1-specific CTLs along with iNKT and NK cell activation, resulting in a strong antitumor effect. Furthermore, we established a human DC-transferred NOD/Shi-scid/IL-2γcnull immunodeficient mouse model and showed that the NY-ESO-1 antigen from aAVC-NY-ESO-1 was cross-presented to antigen-specific CTLs through human DCs. Taken together, these data suggest that aAVC-NY-ESO-1 has potential for harnessing innate and adaptive immunity against NY-ESO-1-expressing malignancies.

NK and NKT Cell-Mediated Immune Surveillance against Hematological Malignancies.

Recent cancer treatment modalities have been intensively focused on immunotherapy. The success of chimeric antigen receptor T cell therapy for treatment of refractory B cell acute lymphoblastic leukemia has pushed forward research on hematological malignancies. Among the effector types of innate lymphocytes, natural killer (NK) cells show great importance in immune surveillance against infectious and tumor diseases. Particularly, the role of NK cells has been argued in either elimination of target tumor cells or escape of tumor cells from immune surveillance. Therefore, an NK cell activation approach has been explored. Recent findings demonstrate that invariant natural killer T (iNKT) cells capable of producing IFN-γ when optimally activated can promptly trigger NK cells. Here, we review the role of NKT and/or NK cells and their interaction in anti-tumor responses by highlighting how innate immune cells recognize tumors, exert effector functions, and amplify adaptive immune responses. In addition, we discuss these innate lymphocytes in hematological disorders, particularly multiple myeloma and acute myeloid leukemia. The immune balance at different stages of both diseases is explored in light of disease progression. Various types of innate immunity-mediated therapeutic approaches, recent advances in clinical immunotherapies, and iNKT-mediated cancer immunotherapy as next-generation immunotherapy are then discussed.

Granzyme A Stimulates pDCs to Promote Adaptive Immunity via Induction of Type I IFN.

Granzyme A (GzmA), together with perforin, are well-known for their cytotoxic activity against tumor or virus-infected cells. In addition to this cytotoxic function, GzmA stimulates several immune cell types and induces inflammation in the absence of perforin, however, its effect on the dendritic cell (DC) is unknown. In the current study, we showed that recombinant GzmA induced the phenotypic maturation of plasmacytoid DCs (pDCs) and conventional DCs (cDCs), but not their apoptosis. Particularly, GzmA made pDCs more functional, thus leading to production of type I interferon (IFN) via the TLR9-MyD88 pathway. We also demonstrated that GzmA binds TLR9 and co-localizes with it in endosomes. When co-administered with antigen, GzmA acted as a powerful adjuvant for eliciting antigen-specific cytotoxic CD8+ T lymphocytes (CTLs) that protected mice from tumor challenge. The induction of CTL was completely abolished in XCR1+ DC-depleted mice, whereas it was reduced to less than half in pDC-depleted or IFN-α/ß receptor knockout mice. Thus, CTL cross-priming was dependent on XCR1+cDC and also type I IFN, which was produced by GzmA-activated pDCs. These results indicate that GzmA -stimulated pDCs enhance the cross-priming activity of cDCs in situ. We also showed that the adjuvant effect of GzmA is superior to CpG-ODN and LPS. Our findings highlight the ability of GzmA to bridge innate and adaptive immune responses via pDC help and suggest that GzmA may be useful as a vaccine adjuvant.

Mast cells play role in wound healing through the ZnT2/GPR39/IL-6 axis.

Zinc (Zn) is an essential nutrient and its deficiency causes immunodeficiency and skin disorders. Various cells including mast cells release Zn-containing granules when activated; however, the biological role of the released Zn is currently unclear. Here we report our findings that Zn transporter ZnT2 is required for the release of Zn from mast cells. In addition, we found that Zn and mast cells induce IL-6 production from inflammatory cells such as skin fibroblasts and promote wound healing, a process that involves inflammation. Zn induces the production of a variety of pro-inflammatory cytokines including IL-6 through signaling pathways mediated by the Zn receptor GPR39. Consistent with these findings, wound healing was impaired in mice lacking IL-6 or GPR39. Thus, our results show that Zn and mast cells play a critical role in wound healing through activation of the GPR39/IL-6 signaling axis.

Optimal therapeutic strategy using antigen-containing liposomes selectively delivered to antigen-presenting cells.

Recent immunotherapies have shown clinical success. In particular, vaccines based on particulate antigen (Ag) are expected to be implemented based on their efficacy. In the current study, we describe a strategy entailing Ag-encapsulating PEG-modified liposomes (PGL-Ag) as antigen protein delivery devices and show that the success of the liposome depends on the antigen-presenting cell (APC) capacity; after administration of PGL-Ag, dendritic cells (DCs) in particular take up the Ag and subsequently prime T cells. For the generation of antitumor T cell responses in the lymphoid tissues, the function of encapsulated Ag-capturing DCs in vivo could be a biomarker. We next designed a prime-boost strategy to enhance the antitumor effects of the PGL-Ag. In the tumor sites, we show that Ag retention in nanoparticle-capturing DCs promotes a robust antitumor response. Thus, this efficient particulate Ag-based host antigen-presenting cell delivery strategy provides a bridge between innate and adaptive immune response and offers a novel therapeutic option against tumor cells.

Immune suppression and reversal of the suppressive tumor microenvironment.

Most tumors employ multiple strategies to attenuate T-cell-mediated immune responses. In particular, immune suppression surrounding the tumor is achieved by interfering with antigen-presenting cells and effector T cells. Controlling both the tumor and the tumor microenvironment (TME) is critical for cancer treatment. Checkpoint blockade therapy can overcome tumor-induced immune suppression, but more than half of the patients fail to respond to this treatment; therefore, more effective cancer immunotherapies are needed. Generation of an anti-tumor immune response is a multi-step process of immune activation against the tumor that requires effector T cells to recognize and exert toxic effects against tumor cells, for which two strategies are employed-inhibition of various types of immune suppressor cells, such as myeloid cells and regulatory T cells, and establishment of anti-tumor immune surveillance including, activation of natural killer cells and cytotoxic T cells. It was recently shown that anti-cancer drugs not only directly kill tumor cells, but also influence the immune response to cancer by promoting immunogenic cell death, enhancing antigen presentation or depleting immunosuppressive cells. Herein, we review the mechanisms by which tumors exert immune suppression as well as their regulation. We then discuss how the complex reciprocal interactions between immunosuppressive and immunostimulatory cells influence immune cell dynamics in the TME. Finally, we highlight the new therapies that can reverse immune suppression in the TME and promote anti-tumor immunity.

Vaccine Designs Utilizing Invariant NKT-Licensed Antigen-Presenting Cells Provide NKT or T Cell Help for B Cell Responses.

Vaccines against a variety of infectious diseases have been developed and tested. Although there have been some notable successes, most are less than optimal or have failed outright. There has been discussion about whether either B cells or dendritic cells (DCs) could be useful for the development of antimicrobial vaccines with the production of high titers of antibodies. Invariant (i)NKT cells have direct antimicrobial effects as well as adjuvant activity, and iNKT-stimulated antigen-presenting cells (APCs) can determine the form of the ensuing humoral and cellular immune responses. In fact, upon activation by ligand, iNKT cells can stimulate both B cells and DCs as via either cognate or non-cognate help. iNKT-licensed DCs generate antigen-specific follicular helper CD4+ T cells, which in turn stimulate B cells, thus leading to long-term antigen-specific antibody production. Follicular helper iNKT cell-licensed B cells generally produce rapid, but short-term antibody. However, under some conditions in the presence of Th cells, the antibody production can be prolonged. With regards to humoral immunity, the quality and quantity of Ab produced depends on the APC type and the form of the vaccine. In terms of cellular immunity and, in particular, the induction of cytotoxic CD8+ T cells, iNKT-licensed DCs show prominent activity. In this review, we discuss differences in iNKT-stimulated APC types and the quality of the ensuing immune response, and also discuss their application in vaccine models to develop successful preventive immunotherapy against infectious diseases.

Amelioration of NK cell function driven by Vα24+ invariant NKT cell activation in multiple myeloma.

NK cells represent a first line of immune defense, but are progressively dysregulated in multiple myeloma (MM) patients. To restore and facilitate their antitumor effect, NK cells are required in sufficient quantities and must be stimulated. We initially assessed the proportions of NKT and NK cells in 34 MM patients. The frequencies of both in PBMC populations correlated with those in BMMNCs irrespective of low BMMNC numbers. We then assessed the adjunctive effect of stimulating NKT cells with CD1d and α-GalCer complexes on the NK cells. The expression of NKG2D on CD56dimCD16+ NK cells and DNAM-1 on CD56brightCD16- NK cells increased after NKT cell activation. Apparently, NK cell-mediated anti-tumor effects were dependent on NKG2D and DNAM-1 ligands on myeloma cells. Thus, NK cell function in patients could be ameliorated, beyond the effect of immunosuppression, by NKT cell activation. This NKT-driven NK cell therapy could represent a potential new treatment modality.

In vivo dendritic cell targeting cellular vaccine induces CD4+ Tfh cell-dependent antibody against influenza virus.

An induction of long-term cellular and humoral immunity is for the goal of vaccines, but the combination of antigens and adjuvant remain unclear. Here, we show, using a cellular vaccine carrying foreign protein antigen plus iNKT cell glycolipid antigen, designated as artificial adjuvant vector cells (aAVCs), that mature XCR1- DCs in situ elicit not only ordinal antigen-specific CD4+T cells, but also CD4+ Tfh and germinal center, resulted in inducing long-term antibody production. As a mechanism for leading the long-term antibody production by aAVC, memory CD4+ Tfh cells but not iNKTfh cells played an important role in a Bcl6 dependent manner. To develop it for influenza infection, we established influenza hemagglutinin-carrying aAVC (aAVC-HA) and found that all the mice vaccinated with aAVC-HA were protected from life-threatening influenza infection. Thus, the in vivo DC targeting therapy by aAVC would be useful for protection against viral infection.

Systemic DC Activation Modulates the Tumor Microenvironment and Shapes the Long-Lived Tumor-Specific Memory Mediated by CD8+ T Cells.

Strategies to reprogram the tumor microenvironment are being explored to improve cancer immunotherapy. In one approach, we have targeted dendritic cells (DC) to improve their function with adjuvant vector cells (aAVC) that are engineered from NKT ligand-loaded CD1d(+) allogeneic cells transfected with tumor antigen mRNAs. Here, we report the finding that this approach also programs local immune responses by establishing tertiary lymphoid structures (TLS), which include expanded antigen-specific CD8(+) T-cell clones, mobilized DCs, and normalized tumor vasculature. aAVC therapy also expanded specific Vß-expressing antitumor T-cell clones, leading to the formation of long-term memory T cells. When combined with PD-1 blockade, aAVC infusion triggered regression of poorly immunogenic tumor cells that did not respond to PD-1 blockade alone, as well as expansion of antigen-specific CD8(+) T-cell clones in the tumor. The findings of this study help to inform a next-generation platform for the generation of efficacious cancer vaccines. Cancer Res; 76(13); 3756-66. ©2016 AACR.

Transfer of mRNA Encoding Invariant NKT Cell Receptors Imparts Glycolipid Specific Responses to T Cells and γδT Cells.

Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and ß chain mRNA (the Vα24 and Vß11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide (α-GalCer), they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and ß chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer.

Characterization of the myeloid-derived suppressor cell subset regulated by NK cells in malignant lymphoma.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population with the ability to suppress immune responses and are currently classified into three distinct MDSC subsets: monocytic, granulocytic and non-monocytic, and non-granulocytic MDSCs. Although NK cells provide an important first-line defense against newly transformed cancer cells, it is unknown whether NK cells can regulate MDSC populations in the context of cancer. In this study, we initially found that the frequency of MDSCs in non-Hodgkin lymphoma (NHL) patients was increased and inversely correlated with that of NK cells, but not that of T cells. To investigate the regulation of MDSC subsets by NK cells, we used an EL4 murine lymphoma model and found the non-monocytic and non-granulocytic MDSC subset, i.e., Gr1+CD11b+Ly6GmedLy6Cmed MDSC, is increased after NK cell depletion. The MDSC population that expresses MHC class II, CD80, CD124, and CCR2 is regulated mainly by CD27+CD11b+NK cells. In addition, this MDSC subset produces some immunosuppressive cytokines, including IL-10 but not nitric oxide (NO) or arginase. We also examined two subsets of MDSCs (CD14+HLA-DR- and CD14- HLA-DR- MDSC) in NHL patients and found that higher IL-10-producing CD14+HLA-DR-MDSC subset can be seen in lymphoma patients with reduced NK cell frequency in peripheral blood. Our analyses of MDSCs in this study may enable a better understanding of how MDSCs manipulate the tumor microenvironment and are regulated by NK cells in patients with lymphoma.

Zinc-binding metallothioneins are key modulators of IL-4 production by basophils.

Zinc (Zn) is an essential nutrient, and Zn deficiency causes immunodeficiency and skin disorders. Basophils express FcɛRI on their surface and release multiple mediators after receptor cross-linking, including large amounts of IL-4. However, the mechanisms involved in the FcɛRI-mediated regulation of basophil IL-4 production are currently unclear. Here, we show that the Zn-binding metallothionein (MT) proteins are essential for the FcɛRI-induced basophil production of IL-4. Basophils from MT-I/II(-/-) mice produced significantly less FcɛRI-induced IL-4 than did wild-type basophils. The MTs were involved in maintaining intracellular Zn levels, thereby regulated the calcineurin activity and nuclear factor of activated T-cell (NFAT)-mediated IL-4 production. These results suggest that the MT-dependent control of Zn homeostasis is a novel mechanism for regulating basophil IL-4 production.

KLRG+ invariant natural killer T cells are long-lived effectors.

Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and γδT cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)-mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d(+) cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-γ-producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1(+) (Killer cell lectin-like receptor subfamily G, member 1-positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3ß by RNA deep sequencing demonstrated that some particular KLRG1(+) iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1(+) iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells.

Signal-transducing adaptor protein-2 controls the IgE-mediated, mast cell-mediated anaphylactic responses.

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that regulates immune and inflammatory responses through interactions with a variety of signaling and transcriptional molecules. In the current study, we clarified the physiological role of STAP-2 in mast cell function, a key mediator of IgE-associated allergic responses. STAP-2 is constitutively expressed in mast cells. STAP-2 deficiency in mast cells greatly enhances FcεRI-mediated signals, resulting in the increased tyrosine phosphorylation of the phospholipase C-γ isoform, calcium mobilization, and degranulation. Of importance, STAP-2-deficient mice challenged with DNP-BSA after passive sensitization with anti-DNP IgE show more severe rectal temperature decrease than do wild-type mice. STAP-2-deficient mice also show increased vascular permeability and more severe cutaneous anaphylaxis after DNP-BSA injection. These regulatory functions performed by STAP-2 indicate that there is an interaction between STAP-2 and FcεRI. In addition, our previous data indicate that STAP-2 binds to the phospholipase C-γ isoform and IκB kinase-ß. Therefore, our data described in this article strongly suggest that manipulation of STAP-2 expression in mast cells may control the pathogenesis of allergic diseases and have the potential for treating patients with allergy.