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The efficacy of current coronavirus disease 2019 (COVID-19) vaccines has been demonstrated; however, emerging evidence suggests insufficient protection in certain immunocompromised cancer patients. We previously developed a cell-based anti-cancer vaccine platform involving artificial adjuvant vector cells (aAVCs) capable of inducing a strong adaptive response by enhancing the innate immunity. aAVCs are target antigen-transfected allogenic cells that simultaneously express the natural killer T-cell ligand-CD1d complex on their surface. In the present study, we applied this system for targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (CoV-2-S) using CoV-2-S-expressing aAVCs (aAVC-CoV-2) and evaluated the immune response in a murine model. A single dose of aAVC-CoV-2 induced a large amount of CoV-2-S-specific, multifunctional CTLs in addition to CD4

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

One of the primary goals in tumor immunotherapy is to reset the immune system from tolerogenic to immunogenic - a process in which invariant natural killer T (iNKT) cells are implicated. iNKT cells develop in the thymus and perform immunosurveillance against tumor cells peripherally. When optimally stimulated, iNKT cells differentiate and display more efficient immune functions. Some cells survive and act as effector memory cells. We discuss the putative roles of iNKT cells in antitumor immunity, and posit that it may be possible to develop novel therapeutic strategies to treat cancers using iNKT cells. In particular, we highlight the challenge of uniquely energizing iNKT cell-licensed dendritic cells to serve as effective immunoadjuvants for both arms of the immune system, thus coupling immunological networks.

CCL5 is a unique chemokine with distinct stage and cell-type specificities for regulating inflammation, but how these specificities are achieved and how CCL5 modulates immune responses is not well understood. Here we identify two stage-specific enhancers: the proximal enhancer mediates the constitutive CCL5 expression during the steady state, while the distal enhancer located 1.35 Mb from the promoter induces CCL5 expression in activated cells. Both enhancers are antagonized by RUNX/CBFβ complexes, and SATB1 further mediates the long-distance interaction of the distal enhancer with the promoter. Deletion of the proximal enhancer decreases CCL5 expression and augments the cytotoxic activity of tissue-resident T and NK cells, which coincides with reduced melanoma metastasis in mouse models. By contrast, increased CCL5 expression resulting from RUNX3 mutation is associated with more tumor metastasis in the lung. Collectively, our results suggest that RUNX3-mediated CCL5 repression is critical for modulating anti-tumor immunity., CCL5 is an important chemokine for modulation of inflammatory responses, but how CCL5 expression is regulated is still unclear. Here the authors show that the CCL5 locus contains two enhancers, with the proximal enhancer being responsible for homeostatic expression and the distal enhancer enforcing inducibility, while both enhancers are modulated by RUNX3.

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 Vβ-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.

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/β 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.

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.

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.

A new humanized mouse expressing human IL-7 and IL-15 facilitates development and maturation of human NK cells and can be useful as a preclinical in vivo model for testing new treatment modalities., The immune system encompasses acquired and innate immunity that matures through interaction with microenvironmental components. Cytokines serve as environmental factors that foster functional maturation of immune cells. Although NOD/SCID/IL2rgKO (NSG) humanized mice support investigation of human immunity in vivo, a species barrier between human immune cells and the mouse microenvironment limits human acquired as well as innate immune function. To study the roles of human cytokines in human acquired and innate immune cell development, we created NSG mice expressing hIL-7 and hIL-15. Although hIL-7 alone was not sufficient for supporting human NK cell development in vivo, increased frequencies of human NK cells were confirmed in multiple organs of hIL-7 and hIL-15 double knockin (hIL-7xhIL-15 KI) NSG mice engrafted with human hematopoietic stem cells. hIL-7xhIL-15 KI NSG humanized mice provide a valuable in vivo model to investigate development and function of human NK cells.

Both innate and adaptive immunity are crucial for cancer immunosurveillance, but precise therapeutic equations to restore immunosurveillance in patients with cancer patients have yet to be developed. In murine models, α-galactosylceramide (α-GalCer)–loaded, tumor antigen–expressing syngeneic or allogeneic cells can act as cellular adjuvants, linking the innate and adaptive immune systems. In the current study, we established human artificial adjuvant vector cells (aAVC) consisting of human HEK293 embryonic kidney cells stably transfected with the natural killer T (NKT) immune cell receptor CD1d, loaded with the CD1d ligand α-GalCer and then transfected with antigen-encoding mRNA. When administered to mice or dogs, these aAVC-activated invariant NKT (iNKT) cells elicited antigen-specific T-cell responses with no adverse events. In parallel experiments, using NOD/SCID/IL-2rγcnull–immunodeficient (hDC-NOG) mouse model, we also showed that the human melanoma antigen, MART-1, expressed by mRNA transfected aAVCs can be cross-presented to antigen-specific T cells by human dendritic cells. Antigen-specific T-cell responses elicited and expanded by aAVCs were verified as functional in tumor immunity. Our results support the clinical development of aAVCs to harness innate and adaptive immunity for effective cancer immunotherapy. Cancer Res; 73(1); 62–73. ©2012 AACR.

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.

Reprogramming of antigen-specific T lymphocytes into induced pluripotent stem cells (iPSCs) and their subsequent re-differentiation has enabled expansion of functional T lymphocytes in vitro, thus opening up new approaches for immunotherapy of cancer and other diseases. In this study, we have established a robust protocol to reprogram human invariant NKT (Vα24

Immunotherapy using dendritic cells (DCs) has the potential to activate both T cells and NK cells. We previously demonstrated the long-lasting antitumor responses by NK cells following immunization with bone marrow-derived DCs. In the current study, we demonstrate that long-term antitumor NK responses require endogenous DCs and a subset of effector memory CD4+ T (CD4+ TEM) cells. One month after DC immunization, injection of a tumor into DC-immunized mice leads to an increase in the expression of CXCL10 by endogenous DCs, thus directing NK cells into the white pulp where the endogenous DCs bridged CD4+ TEM cells and NK cells. In this interaction, CD4+ TEM cells express CD40L, which matures the endogenous DCs, and produce cytokines, such as IL-2, which activates NK cells. These findings suggest that DC vaccination can sustain long-term innate NK cell immunity but requires the participation of the adaptive immune system.

Establishment of a system with efficient generation of natural killer T (NKT) cells from embryonic stem (ES) cells would enable us to identify the cells with NKT-cell potential and obtain NKT cells with desired function. Here, using cloned ES (NKT-ES) cells generated by the transfer of nuclei from mature NKT cells, we have established a culture system that preferentially developed functional NKT cells and also identified early NKT progenitors, which first appeared on day 11 as a c-kit+ population in the cocultures on OP9 cells with expression of Notch ligand, delta-like1 (OP9/Dll-1) and became c-kitlo/− on day 14. Interestingly, in the presence of Notch signals, NKT-ES cells differentiated only to thymic CD44lo CD24hi NKT cells producing mainly interleukin-4 (IL-4), whereas NKT cells resembling CD44hi CD24lo liver NKT cells producing mainly interferon γ (IFN-γ) and exhibiting strong adjuvant activity in vivo were developed in the switch culture starting at day 14 in the absence of Notch. The cloned ES culture system offers a new opportunity for the elucidation of the molecular events on NKT-cell development and for the establishment of NKT-cell therapy.

Because of their unique role in linking the innate and adaptive immune systems, dendritic cells (DCs) have been a logical focus for novel immunotherapies. However, strategies employing active immunization with ex vivo generated and antigen-pulsed DCs have shown limited efficacy in clinical trials. These past approaches did not take into account the complex interactions between cells of the innate immune system and DCs during DC maturation, antigen processing, and presentation to naïve T cells. By better understanding the natural sequence of events occurring in vivo during an effective immune response, we can tailor antitumor immunotherapeutic strategies to augment aspects of this response from the activation of innate immune cells to antigen uptake and DC maturation to priming of naïve T cells and, ultimately, to the establishment of antitumor immunity. Current DC vaccination strategies utilize a number of methods to recapitulate the cascade of events that culminate in a protective antitumor immune response.

The maturation of dendritic cells (DCs) in situ by danger signals plays a central role in linking innate and adaptive immunity. We previously demonstrated that the activation of invariant natural killer T (iNKT) cells by administration of α-galactosylceramide (α-GalCer)–loaded tumor cells can act as a cellular adjuvant through the DC maturation. In the current study, we used allogeneic fibroblasts loaded with α-GalCer and transfected with antigen-encoding mRNA, thus combining the adjuvant effects of iNKT-cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected into major histocompatibility complex (MHC)–mismatched mice, they elicited antigen-specific T-cell responses and provided tumor protection, suggesting that these immune responses depend on host DCs. In addition, antigen-expressing fibroblasts loaded with α-GalCer lead to a more potent T-cell response than those expressing NK cell ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to provide iNKT-cell activation, leading to DC maturation and T-cell immunity. By harnessing the innate immune system and generating an adaptive immune response to a variety of antigens, this unique tool could prove clinically beneficial in the development of immunotherapies against malignant and infectious diseases.

We vaccinated mice with DC loaded with or without invariant NKT-cell ligand alpha-galactosylceramide and evaluated long-term resistance against tumor challenge. When mice had been given either DC or DC/galactosylceramide and were challenged with tumor cells even 6-12 months later, both NK and NKT cells were quickly activated to express CD69 and produce IFN-gamma. The NK cells could resist a challenge with several different tumors in vivo. The activated NK and NKT cells could be depleted with anti-NK1.1 treatment. In spite of this, the activated cells recovered, indicating that tumor-responsive NK and NKT cells were being generated continuously as a result of vaccination with DC and were not true memory cells. The NK and NKT antitumor response in DC-vaccinated mice depended on CD4(+) T cells, but neither CD8(+)T cells nor CD4(+)CD25(+) regulatory T cells. However, both vaccine DC and host DC were required for the development of long-term, tumor reactive innate immunity. These results indicate that DC therapy in mice induces long-lasting innate NK- and NKT-cell activation through a pathway that requires host DC and CD4(+) T cells and that the continued generation of active NK cells resists the establishment of metastases in vivo.

The observation that the glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent stimulator of natural killer T (NKT) cells has provided an important means for investigating NKT cell biology. alpha-GalCer is presented on CD1d to the invariant NKT receptor, leading to interleukin-12 (IL-12) production by dendritic cells (DCs) and to NK cell activation. We review our research on the tumor-protective properties of alpha-GalCer, particularly the major role played by DCs. We compared administration of alpha-GalCer on mature DCs with soluble glycolipid and found that DCs induced more prolonged interferon-gamma (IFN-gamma) production by NKT cells and better protection against B16 melanoma. Human alpha-GalCer-loaded DCs also expanded NKT cell numbers in cancer patients. alpha-GalCer-activated NKT cells were then found to induce DC maturation in vivo. The maturing DCs produced IL-12, upregulated co-stimulatory molecules, and induced adaptive immunity to captured cellular antigens, including prolonged, combined CD4(+)/CD8(+) T-cell immunity to dying tumor cells. Surprisingly, co-stimulator-poor tumor cells, if directly loaded with alpha-GalCer ('tumor/Gal') and injected intravenously, also induced strong NKT- and NK-cell responses. The latter killed the tumor/Gal, which were subsequently cross presented by CD1d on DCs to elicit DC maturation and prolonged adaptive T-cell immunity, which lasted 6-12 months. These findings help explain tumor protection via alpha-GalCer and urge development of the DC-NKT axis to provide innate and adaptive immunity to human cancers.

We report a mechanism to induce combined and long-lived CD4(+) and CD8(+) T cell immunity to several mouse tumors. Surprisingly, the initial source of antigen is a single low dose of tumor cells loaded with alpha-galactosylceramide (alpha-GalCer) glycolipid (tumor/Gal) but lacking co-stimulatory molecules. After tumor/Gal injection intravenously (i.v.), innate NKT and NK cells reject the tumor cells, some of which are taken up by dendritic cells (DCs). The DCs in turn cross-present glycolipid on CD1d molecules to NKT cells and undergo maturation. For B16 melanoma cells loaded with alpha-GalCer (B16/Gal), interferon gamma-producing CD8(+) T cells develop toward several melanoma peptides, again after a single low i.v. dose of B16/Gal. In all four poorly immunogenic tumors tested, a single dose of tumor/Gal i.v. allows mice to become resistant to tumors given subcutaneously. Resistance requires CD4(+) and CD8(+) cells, as well as DCs, and persists for 6-12 mo. Therefore, several immunogenic features of DCs are engaged by the CD1d-mediated cross-presentation of glycolipid-loaded tumor cells, leading to particularly strong and long-lived adaptive immunity.

NKT cells play a role in immunological regulation of certain diseases, and their frequency and/or function may be related to disease prognosis. However, it is often difficult to evaluate NKT cell function in patients with malignancies due to reduced numbers of NKT cells as well as the dysfunction of the APCs used as stimulators. We found that NKT cell function could not be evaluated by conventional ELISPOT assays, confirming the impaired function of APCs in chronic myelogenous leukemia (CML)-chronic phase patients. To overcome this problem, we have established a sensitive assay using murine dendritic cells to evaluate the function of small numbers of human NKT cells independent of autologous APCs. We found that imatinib-treated CML-chronic phase patients showing a complete cytogenetic response had NKT cells capable of producing IFN-γ, whereas NKT cells from patients who were only partially responsive to imatinib treatment did not produce IFN-γ. Functional NKT cells found in imatinib-treated, CML-complete cytogenetic response patients may offer the promise of effective immunotherapy with ex vivo-generated α-galactosylceramide-pulsed dendritic cells. This new approach should be available for evaluating the functions of NKT cells and APCs in cancer patients.

The maturation of dendritic cells (DCs) allows these antigen-presenting cells to initiate immunity. We pursued this concept in situ by studying the adjuvant action of alpha-galactosylceramide (alphaGalCer) in mice. A single i.v. injection of glycolipid induced the full maturation of splenic DCs, beginning within 4 h. Maturation was manifest by marked increases in costimulator and major histocompatibility complex class II expression, interferon (IFN)-gamma production, and stimulation of the mixed leukocyte reaction. These changes were not induced directly by alphaGalCer but required natural killer T (NKT) cells acting independently of the MyD88 adaptor protein. To establish that DC maturation was responsible for the adjuvant role of alphaGalCer, mice were given alphaGalCer together with soluble or cell-associated ovalbumin antigen. Th1 type CD4+ and CD8+ T cell responses developed, and the mice became resistant to challenge with ovalbumin-expressing tumor. DCs from mice given ovalbumin plus adjuvant, but not the non-DCs, stimulated ovalbumin-specific proliferative responses and importantly, induced antigen-specific, IFN-gamma producing, CD4+ and CD8+ T cells upon transfer into naive animals. In the latter instance, immune priming did not require further exposure to ovalbumin, alphaGalCer, NKT, or NK cells. Therefore a single dose of alphaGalCer i.v. rapidly stimulates the full maturation of DCs in situ, and this accounts for the induction of combined Th1 CD4+ and CD8+ T cell immunity to a coadministered protein.

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 CDR3β 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.

Complex interactions among multiple cell types contribute to the immunosuppressive milieu of the tumor microenvironment. Using a murine model of adoptive T-cell immunotherapy (ACT) for B16 melanoma, we investigated the impact of tumor infiltrating cells on this complex regulatory network in the tumor. Transgenic pmel-1-specific cytotoxic T lymphocytes (CTLs) were injected intravenously into tumor-bearing mice and could be detected in the tumor as early as on day 1, peaking on day 3. They produced IFN-γ, exerted anti-tumor activity and inhibited tumor growth. However, CTL infiltration into the tumor was accompanied by the accumulation of large numbers of cells, the majority of which were CD11b(+) Gr1(+) myeloid-derived suppressor cells (MDSCs). Notably, CD11b(+) Gr1(int) Ly6G(-) Ly6C(+) monocytic MDSCs outnumbered the CTLs by day 5. They produced nitric oxide, arginase I and reactive oxygen species, and inhibited the proliferation of antigen-specific CD8(+) T cells. The anti-tumor activity of the adoptively-transferred CTLs and the accumulation of MDSCs both depended on IFN-γ production on recognition of tumor antigens by the former. In CCR2(-/-) mice, monocytic MDSCs did not accumulate in the tumor, and inhibition of tumor growth by ACT was improved. Thus, ACT triggered counter-regulatory immunosuppressive mechanism via recruitment of MDSCs. Our results suggest that strategies to regulate the treatment-induced recruitment of these MDSCs would improve the efficacy of immunotherapy.

A key goal of vaccine immunotherapy is the generation of long-term memory CD8+ T cells capable of mediating immune surveillance. We discovered a novel intercellular pathway governing the development of potent memory CD8+ T cell responses against cell-associated Ags that is mediated through cross-presentation by XCR1+ dendritic cells (DCs). Generation of CD8+ memory T cells against tumor cells pulsed with an invariant NKT cell ligand depended on cross-talk between XCR1+ and plasmacytoid DCs that was regulated by IFN-α/IFN-αR signals. IFN-α production by plasmacytoid DCs was stimulated by an OX40 signal from the invariant NKT cells, as well as an HMGB1 signal from the dying tumor cells. These findings reveal a previously unknown pathway of intercellular collaboration for the generation of tumor-specific CD8+ memory T cells that can be exploited for strategic vaccination in the setting of tumor immunotherapy.

Intracerebroventricular (ICV) administration of gonadotropin-releasing hormone II (GnRH II), which plays a crucial role in the regulation of reproduction in vertebrates, markedly reduces food intake in goldfish. However, the neurochemical pathways involved in the anorexigenic action of GnRH II and its interaction with other neuropeptides have not yet been identified. Alpha-melanocyte-stimulating hormone (α-MSH), corticotropin-releasing hormone (CRH) and CRH-related peptides play a major role in feeding control as potent anorexigenic neuropeptides in goldfish. However, our previous study has indicated that the GnRH II-induced anorexigenic action is not blocked by treatment with melanocortin 4 receptor (MC4R) and CRH receptor antagonists. Therefore, in the present study, we further examined whether the anorexigenic effects of α-MSH and CRH in goldfish could be mediated through the GnRH receptor neuronal pathway. ICV injection of the MC4R agonist, melanotan II (80 pmol/g body weight; BW), significantly reduced food intake, and its anorexigenic effect was suppressed by ICV pre-administration of the GnRH type I receptor antagonist, antide (100 pmol/gBW). The CRH-induced (50 pmol/gBW) anorexigenic action was also blocked by treatment with antide. ICV injection of CRH (50 pmol/gBW) induced a significant increase of the GnRH II mRNA level in the hypothalamus, while ICV injection of melanotan II (80 pmol/gBW) had no effect on the level of GnRH II mRNA. These results indicate that, in goldfish, the anorexigenic actions of α-MSH and CRH are mediated through the GnRH type I receptor-signaling pathway, and that the GnRH II system regulates feeding behavior.

NKT cells are characterized by their production of both T(h)1 and T(h)2 cytokines immediately after stimulation with alpha-galactosylceramide (alpha-GalCer), which is composed of alpha-galactopyranose linked to ceramide (itself composed of sphingosine and fatty-acyl chains); the chain length of the ceramide varies and this affects the ability of alpha-GalCer to stimulate cytokine production. However, the contribution of its galactopyranose sugar moiety remains unclear. We synthesized alpha-carba-GalCer, which has an alpha-linked carba-galactosyl moiety; here, the 5a'-oxygen atom of the D-galactopyranose ring of alpha-GalCer is replaced by a methylene group. The alpha-carba-GalCer was more stable and showed higher affinity to the NKT receptor. It thus enhanced and prolonged production of IL-12 and IFN-gamma compared with alpha-GalCer, resulting in augmented NKT cell-mediated adjuvant effects in vivo. The alpha-carba-GalCer, which has an ether linkage, was more resistant to degradation by liver microsomes than was alpha-GalCer, which has an acetal bond. Modulation of the sugar moiety in glycolipids might therefore provide optimal therapeutic reagents for protective immune responses against tumor or pathogens.

Invariant NKT cells (NKT) cells become activated after stimulation with antigen-presenting cells (APCs) loaded with the NKT cell ligand, alpha-galactosylceramide (alpha-GalCer). In this study, we investigated whether human APCs loaded with alpha-GalCer have the ability to activate NKT cells in mice. We found that human dendritic cells (DCs) loaded with alpha-GalCer (hDC/Gal) and injected into C57BL/6 mice stimulated the secretion of IFN-gamma by activated murine NKT cells. Furthermore, the number of transferred hDC/Gal correlated with the number of recovered IFN-gamma-producing spleen cells, indicating that the capacity of APCs to load alpha-GalCer can be measured by IFN-gamma release in an ELISPOT assay. Finally, alpha-GalCer-loaded human leukemic cell lines and primary leukemic cells injected into C57BL/6 mice also had the capacity to stimulate murine NKT cells in vivo. These results indicate that in vivo murine NKT cell responses can be used to quantitate the alpha-GalCer-loading capacity of human APCs. This method could be utilized to develop future immunotherapies in which NKT cells are targeted for activation.

Dendritic cells (DCs) work as a natural adjuvant to elicit T cell immunity. Though DCs have been widely used in immunotherapy, little is known about their number and function in patients with cancer or autoimmune disease. In recent studies, antigen has been targeted to DCs through DC-specific receptors, such as DEC205, the mannose receptor and dying cell receptors. However, antigen captured by DCs in the absence of danger signals induces tolerance. Therefore, the duration and/or magnitude of danger signals plays a crucial role in generating an immunogeneic response. Various danger signals, i.e., pathogen-associated molecular pattern (PAMP), damage-associated molecular pattern (DAMP) and the activation of innate lymphocytes, serve as maturation signals for DCs. An immunotherapeutic approach which delivers both maturation signals and antigen to DCs would link the innate and adaptive arms of the immune system for a more effective and global immune response. It is therefore crucial to determine optimal conditions for antigen delivery to DCs in an environment suited to maximally stimulate the immune system.

Dendritic cells (DCs) loaded with α-galactosylceramide (α-GalCer) are known to be active APCs for the stimulation of innate NKT and NK cell responses in vivo. In this study, we evaluated the capacity of non-DCs to present α-GalCer in vitro and in vivo, particularly tumor cells loaded with α-GalCer (tumor/Gal). Even though the tumor cells lacked expression of CD40, CD80, and CD86 costimulatory molecules, the i.v. injection of tumor/Gal resulted in IFN-γ secretion by NKT and NK cells. These innate responses to tumor/Gal, including the induction of IL-12p70, were comparable to or better than α-GalCer-loaded DCs. B16 melanoma cells that were stably transduced to express higher levels of CD1d showed an increased capacity relative to wild-type B16 cells to present α-GalCer in vivo. Three different tumor cell lines, when loaded with α-GalCer, failed to establish tumors upon i.v. injection, and the mice survived for at least 6 mo. The resistance against tumor cells was independent of CD4 and CD8 T cells but dependent upon NKT and NK cells. Mice were protected from the development of metastases if the administration of live B16 tumor cells was followed 3 h or 3 days later by the injection of CD1dhigh-α-GalCer-loaded B16 tumor cells with or without irradiation. Taken together, these results indicate that tumor/Gal are effective APCs for innate NKT and NK cell responses, and that these innate immune responses are able to resist the establishment of metastases in vivo.

Interleukin-10 (IL-10) is a multifunctional cytokine that can exert suppressive and stimulatory effects on T cells. It was investigated whether IL-10 could serve as an immunostimulant for specific CD8(+) cytotoxic T cell (CTL) in vivo after vaccination and, if so, under what conditions. In tumor prevention models, administration of IL-10 before, or soon after, peptide-pulsed primary dendritic cell immunization resulted in immune suppression and enhanced tumor progression. Injection of IL-10, however, just after a booster vaccine significantly enhanced antitumor immunity and vaccine efficacy. Analysis of spleen cells derived from these latter animals 3 weeks after IL-10 treatment revealed that the number of CD8(+) CD44(hi) CD122(+) T cells had increased and that antigen-specific proliferation in vitro was enhanced. Although cytotoxicity assays did not support differences between the various treatment groups, 2 more sensitive assays measuring antigen-specific interferon-gamma production at the single-cell level demonstrated increases in the number of antigen-specific responder T cells in animals in the vaccine/IL-10 treatment group. Thus, IL-10 may maintain the number of antitumor CD8(+) T cells. In adoptive transfer studies, the ability of IL-10 to maintain CTL function could be enhanced by the depletion of CD4(+) T cells. This suggests that IL-10 mediates contrasting effects on both CD4(+) and CD8(+) T cells that result in either immune dampening or immune potentiation in situ, respectively. Appreciation of this dichotomy in IL-10 immunobiology may allow for the design of more effective cancer vaccines designed to activate and maintain specific CD8(+) T-cell effector function in situ.