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Vaccines against influenza and many other infectious diseases require multiple boosters in addition to the primary dose to improve efficacy, but this approach is not ideal for compliance. The multiple doses could potentially be replaced by sustained or pulsatile release of antigens encapsulated in degradable microparticles (MPs). The efficacy of a vaccine is improved by adding an adjuvant, which can be co-delivered from the particles to enhance immunogenicity. Here, we developed degradable poly-lactic-co-glycolic acid (PLGA) (7-17 kDa) MPs capable of sustained release of ultraviolet killed influenza virus (A/PR/8/34) (kPR8) vaccine and the natural killer T (NKT) cell agonist alpha-galactosylceramide (α-GalCer) and tested their effectiveness at providing long-term protection against influenza virus infection in mice. Multiple formulations were developed for encapsulating the virus and adjuvant separately, and in combination. The MPs exhibited sustained release of both the virus and the adjuvant lasting more than a month. Co-encapsulation significantly increased the encapsulation efficiency (EE) of the vaccine but reduced the release duration. On the other hand, co-encapsulation led to a reduction in EE for the α-GalCer and a change in release profile to a higher initial burst followed by a linear release compared to a low initial burst and slower linear release. The α-GalCer also had considerably longer release duration compared to the vaccine. Mice injected with particle formulations co-encapsulating kPR8 and α-GalCer were protected from a lethal influenza virus infection 30 weeks after vaccination. This study demonstrates that PLGA MP based vaccines are promising for providing effective vaccination and possibly for replacing multiple doses with a single injection., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although various types of mRNA-based vaccines have been explored, the optimal conditions for induction of both humoral and cellular immunity remain rather unknown. In this study, mRNA vaccines of nucleoside-modified mRNA in lipoplexes (LPXs) or lipid nanoparticles (LNPs) were evaluated after administration in mice through different routes, assessing mRNA delivery, tolerability and immunogenicity. In addition, we investigated whether mRNA vaccines could benefit from the inclusion of the adjuvant alpha-galactosylceramide (αGC), an invariant Natural Killer T (iNKT) cell ligand. Intramuscular (IM) vaccination with ovalbumin (OVA)-encoding mRNA encapsulated in LNPs adjuvanted with αGC showed the highest antibody- and CD8 + T cell responses. Furthermore, we observed that addition of signal peptides and endocytic sorting signals of either LAMP1 or HLA-B7 in the OVA-encoding mRNA sequence further enhanced CD8 + T cell activation although reducing the induction of IgG antibody responses. Moreover, mRNA LNPs with the ionizable lipidoid C12-200 exhibited higher pro-inflammatory- and reactogenic activity compared to mRNA LNPs with SM-102, correlating with increased T cell activation and antitumor potential. We also observed that αGC could further enhance the cellular immunity of clinically relevant mRNA LNP vaccines, thereby promoting therapeutic antitumor potential. Finally, a Listeria monocytogenes mRNA LNP vaccine supplemented with αGC showed synergistic protective effects against listeriosis, highlighting a key advantage of co-activating iNKT cells in antibacterial mRNA vaccines. Taken together, our study offers multiple insights for optimizing the design of mRNA vaccines for disease applications, such as cancer and intracellular bacterial infections., Competing Interests: Declaration of competing interest R.V., I.L. and S.D.S. are contributors to patent applications no. WO2020058239A1; Therapeutic nanoparticles and methods of use thereof. and no. WO2023209103; Prevention and treatment of infections with intracellular bacteria., together with I.A., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Influenza A viruses (IAV) are a major cause of respiratory diseases in pigs. Invariant natural killer T (iNKT) cells are an innate-like T cell subset that contribute significantly to IAV resistance in mice. In the current work, we explored whether expanding and activating iNKT cells with the iNKT cell superagonist α-galactosylceramide (α-GalCer) would change the course of an IAV infection in pigs. In one study, α-GalCer was administered to pigs intramuscularly (i.m.) 9 days before infection, which systemically expanded iNKT cells. In another study, α-GalCer was administered intranasally (i.n.) 2 days before virus infection to activate mucosal iNKT cells. Despite a synergistic increase in iNKT cells when α-GalCer i.m. treated pigs were infected with IAV, neither approach reduced disease signs, lung pathology, or virus replication. Our results indicate that prophylactic use of iNKT cell agonists to prevent IAV infection is ineffective in pigs. This is significant because this type of approach has been considered for humans whose iNKT cell levels and IAV infections are more similar to those of pigs than mice., (Copyright © 2020. Published by Elsevier Ltd.)

Murine and human invariant natural killer T (iNKT) lymphocytes are activated by α-galactosylceramide (α-GalCer) presented on CD1d. α-GalCer was first described as a lipid that had strong anti-metastatic effects in a mouse melanoma model, and it has subsequently been shown to induce efficient iNKT cell dependent tumor immunity in several tumor models. We have shown that α-GalCer treatment leads to a weak reduction of polyp burden in the autochthonous Apc Min/+ mouse model for human colon cancer, however this treatment resulted in upregulation of the inhibitory receptor PD-1 on iNKT cells. While anti-PD-1 treatment can prevent immune-suppression in other cancer types, human colon cancer is generally resistant to this treatment. Here we have used the Apc Min/+ model to investigate whether a combined treatment with α-GalCer and PD-1 blockade results in improved effects on polyp development. We find that PD-1 expression was high on T cells in polyps and lamina propria (LP) of Apc Min/+ mice compared to polyp free Apc+/+ littermates. Anti-PD-1 treatment alone promoted Tbet expression in iNKT cells and CD4 T cells, but did not significantly reduce polyp numbers. However, the combined treatment with anti-PD-1 and α-GalCer had synergistic effects, resulting in highly significant reduction of polyp numbers in the small and large intestine. Addition of PD-1 blockade to α-GalCer treatment prevented loss of iNKT cells that were skewed towards a TH1-like iNKT1 phenotype specifically in polyps. It also resulted in TH1 skewing and increased granzyme B expression of CD4 T cells. Taken together this demonstrates that a combination of immune stimulation targeting iNKT cells and checkpoint blockade may be a promising approach to develop for improved tumor immunotherapy., (Copyright © 2020 Wang, Bhave, Yagita and Cardell.)

Active co-delivery of tumor antigens (Ag) and α-galactosylceramide (α-GalCer), a potent agonist for invariant Natural Killer T ( i NKT) cells, to cross-priming CD8α + dendritic cells (DCs) was previously shown to promote strong anti-tumor responses in mice. Here, we designed a nanoparticle-based vaccine able to target human CD141 + (BDCA3 + ) DCs - the equivalent of murine CD8α + DCs - and deliver both tumor Ag (Melan A) and α-GalCer. This nanovaccine was inoculated into humanized mice that mimic the human immune system (HIS) and possess functional i NKT cells and CD8 + T cells, called HIS-CD8/NKT mice. We found that multiple immunizations of HIS-CD8/NKT mice with the nanovaccine resulted in the activation and/or expansion of human CD141 + DCs and i NKT cells and ultimately elicited a potent Melan-A-specific CD8 + T cell response, as determined by tetramer staining and ELISpot assay. Single-cell proteomics further detailed the highly polyfunctional CD8 + T cells induced by the nanovaccine and revealed their predictive potential for vaccine potency. This finding demonstrates for the first time the unique ability of human i NKT cells to license cross-priming DCs in vivo and adds a new dimension to the current strategy of cancer vaccine development., (Copyright © 2020 Huang, Zhou, Ghinnagow, Seki, Iketani, Soulard, Paczkowski, Tsuji, MacKay, Cruz, Trottein and Tsuji.)

We conducted a phase I study of the trans-bronchial injection of α-galactosylceramide (αGalCer)-pulsed antigen presenting cells (APCs) to evaluate their safety, immune responses, and anti-tumor activities. Patients with advanced or recurrent non-small cell lung cancer (NSCLC) refractory to standard treatments were eligible. αGalCer-pulsed APCs were administered intratumorally or intranodally by bronchoscopy. Twenty-one patients were enrolled in this study. No severe adverse events related to the cell therapy were observed during this study in any patient. After αGalCer-pulsed APCs were administrated, increased iNKT cell numbers were observed in PBMCs from eight cases, and IFN-γ producing cells were increased in the peripheral blood of 10 cases. Regarding clinical responses, one case exhibited a partial response and eight were classified as stable disease. In the tumor microenvironment, IFN-γ expression was upregulated after treatment in partial response or stable disease cases and TGF-β was upregulated in progressive disease cases., Competing Interests: Declaration of Competing Interest The authors declare no potential conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Some studies have shown that maturation of dendritic cells (DCs) is modulated directly by pathogen components via pattern recognition receptors such as Toll-like receptors, but also by signal like CD40 ligand (CD40 L or CD154) mediated by activated T cells. Several reports indicate that invariant natural killer T (iNKT) cells up-regulate CD40 L upon stimulation and thereby induce activation and maturation of DCs through crosslink with CD40. Our previous findings indicated that iNKT cells promote Th2 cell responses through the induction of immunogenic maturation of lung DCs (LDCs) in the asthmatic murine, but its mechanism remains unclear. Therefore, we investigated the immunomodulatory effects of blockade of CD40 L using anti-CD40 L treatment on Th2 cell responses and immunogenic maturation of LDCs, and further analyzed whether these influences of blockade of CD40 L were related to lung iNKT cells using iNKT cell-deficient mice and the combination treatment of specific iNKT cell activation with anti-CD40 L treatment in murine models of asthma. Our findings showed that blockade of CD40 L using anti-CD40 L treatment attenuated Th2 cell responses in wild-type (WT) mice, but not in CD1d-deficient mice sensitized and challenged with ovalbumin (OVA) or house dust mite (HDM). Meanwhile, blockade of CD40 L down-regulated immunogenic maturation of LDCs in WT mice, but not in CD1d-deficient mice sensitized and challenged with OVA. Additionally, agonistic anti-CD40 treatment reversed the inhibitory effects of anti-CD40 L treatment on Th2 cell responses and LDC activation in an OVA-induced mouse model of asthma. Furthermore, LDCs from asthmatic mice treated with anti-CD40 L could significantly reduce the influence on Th2 cell responses in vivo and in vitro. Finally, α-Galactosylceramide plus anti-CD40 L treatment stimulated lung iNKT cells, but suppressed Th2 cell responses in the asthmatic mice. Taken together, our data raise an evidence that blockade of CD40 L attenuates Th2 cell responses through the inhibition of immunogenic maturation of LDCs, which may be at least partially related to lung iNKT cells in murine models of asthma., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Constructing an effective therapeutic cancer vaccine is very attractive and promising for cancer immunotherapy. However, the poor immunogenicity of tumor antigens and suppression of the immune system in the tumor microenvironment are two major obstacles for developing effective cancer vaccines. Invariant NKT cells (iNKT cells), which are essential bridges between the innate and adaptive immune systems, can be rapidly activated by their agonists and, consequently, evoke whole immune systems. Herein, we conjugated a potent agonist of the iNKT cell, α-galactosylceramide (α-GalCer), with the tumor-associated MUC1 glycopeptide antigens as novel self-adjuvanting cancer vaccines through click chemistry. Immunological studies revealed that the mouse immune system was potently evoked and that high levels of tumor-specific IgG antibodies were elicited by vaccine conjugates without an external adjuvant. The produced antibodies could specifically recognize and bind to antigen-expressing cancer cells and, subsequently, induce cytotoxicity through complement-dependent cytotoxicity. Thus, the insertion of α-GalCer significantly improved the immunogenicity of the MUC1 glycopeptide and induced strong antigen-specific antitumor responses, indicating that α-GalCer is an effective built-in adjuvant for constructing potent chemical synthetic antitumor vaccines.

Whether different injection modes of α-galactosylceramide (α-GalCer) affect the activation of different subsets of invariant natural killer T (iNKT) cells in different tissues and organs of mice is unclear. This study included healthy control, subcutaneous injection, and intraperitoneal injection groups (n=10 in each group). The subcutaneous and intraperitoneal injection groups were injected with α-Galcer (0.1 mg/kg weight), and then the changes in thymus, spleen, and liver iNKT cell frequencies and subsets were observed. The intraperitoneal injection of α-GalCer could increase the frequency of splenic iNKT cells, but the subcutaneous injection did not affect the frequency. Neither injection had any effect on the frequency of iNKT cells in the thymus and liver. The subcutaneous injection of α-GalCer increased the rate of iNKT2 subsets in the thymus but did not affect the rate of iNKT1 subsets. However, the intraperitoneal injection of α-GalCer did not affect thymus iNKT1 and iNKT2 subsets. Interestingly, the subcutaneous injection of α-GalCer significantly increased the proportion of iNKT1 in the spleen and liver but did not significantly change the proportion of iNKT2. The intraperitoneal injection of α-GalCer significantly increased the rate of iNKT2 in spleen and liver but decreased the rate of iNKT1. Subsets of iNKT1 or iNKT2 cells in the spleen and liver were selectively activated by the subcutaneous or intraperitoneal injection of α-GalCer. It provides a valuable means for treating tumors and certain autoimmune diseases. Further exploration of the activation mechanism may provide new ideas about the development of related vaccines.

Type 1 diabetes (T1D) is an autoimmune disease caused by the destruction of pancreatic β cells by autoantigen-reactive diabetogenic cells. Antigen-specific therapies using islet autoantigens for restoring immune tolerance have emerged as promising approaches for the treatment of T1D but have been unsuccessful in humans. Herein, we report that RGI-3100-iB, a novel liposomal formulation carrying both α -galactosylceramide ( α -GalCer), which is a representative ligand for invariant natural killer T (iNKT) cells, and insulin B chain 9-23 peptide, which is an epitope for CD4 + T cells, could induce the accumulation of regulatory T cells (Tregs) in islets in a peptide-dependent manner, followed by the remarkable prevention of diabetes onset in nonobese diabetic (NOD) mice. While multiple administrations of a monotherapy using either α -GalCer or insulin B peptide in a liposomal formulation was confirmed to delay/prevent T1D in NOD mice, RGI-3100-iB synergistically enhanced the prevention effect of each monotherapy and alleviated insulitis in NOD mice. Immunopathological analysis showed that Foxp3 + Tregs accumulated in the islets in RGI-3100-iB-treated mice. Cotransfer of diabetogenic T cells and splenocytes of NOD mice treated with RGI-3100-iB, but not liposomal α -GalCer encapsulating an unrelated peptide, to NOD-SCID mice resulted in the prevention of diabetes and elevation of Foxp3 mRNA expression in the islets. These data indicate that the migration of insulin B-peptide-specific Tregs to islet of NOD mice that are involved in the suppression of pathogenic T cells related to diabetes onset and progression could be enhanced by the administration of liposomes containing α -GalCer and insulin B peptide., Competing Interests: Akimoto H., Fukuda-Kawaguchi E., Duramad O., Ishii Y., and Tanabe K. declare no conflict of interest., (Copyright © 2019 Hidetoshi Akimoto et al.)

CD160 and BTLA both bind to herpes virus entry mediator. Although a negative regulatory function of BTLA in natural killer T (NKT) cell activation has been reported, whether CD160 is also involved is unclear. By analyzing CD160 -/- mice and mixed bone marrow chimeras, we show that CD160 is not essential for NKT cell development. However, CD160 -/- mice exhibit severe liver injury after in vivo challenge with α-galactosylceramide (α-GalCer). Moreover, CD160 -/- mice are more susceptible to Concanavalin A challenge, and display elevated serum AST and ALT levels, hyperactivation of NKT cells, and enhanced IFN-γ, TNF, and IL-4 production. Lastly, inhibition of BTLA by anti-BTLA mAb aggravates α-GalCer-induced hepatic injury in CD160 -/- mice, suggesting that both CD160 and BTLA serve as non-overlapping negative regulators of NKT cells. Our data thus implicate CD160 as a co-inhibitory receptor that delivers antigen-dependent signals in NKT cells to dampen cytokine production during early innate immune activation.

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3-4 million cases globally with 100,000-150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral ® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill ® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

CD8 + T cell immune response plays a critical role in the clearance of human papillomavirus (HPV)-infected cells. During the natural history of HPV infection, the E1 protein, an early-expressed helicase highly conserved among papillomaviruses, is involved in the replication of HPV genomes. We have previously shown, in a murine model, that immunization with HPV18 E1 protein combined with α -galactosylceramide elicits a specific CD8 + T cell response. We further proved those findings by analyzing whether CD8 + T cells from mice immunized with α -galactosylceramide plus HPV18 E1 protein could have a cytotoxic effect on cells expressing the carboxyl-terminal domain from the E1 proteins of other HPV types. Interestingly, CD8 + T cells raised against HPV18 E1 antigen presented cross-reactivity against the E1 protein from HPV53, 33, 16, and 31. Poor cross-reactivity was observed for HPV11, and none for HPV6. This outcome may be relevant for the design of broad-spectrum immune-protective agents against HPV infections.

The recognition of α-galactosylceramide (αGC), a high-affinity CD1d antigen, by the invariant Natural Killer T (iNKT) lymphocytes results in potent immunostimulatory responses that have been exploited in advanced cancer patients. Therefore, to improve αGC biological activity, several studies vectorized this agonist in PLGA and/or PEG-based nanoparticles. Despite promising findings, these approaches require several steps, from organic solvent decontamination through extrusion in membrane systems. Using a nano spray dryer, we vectorized αGC into a cationic copolymer (dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate - DBM) in a single step process, free of organic solvent. This methodology allowed the production of stable αGC-vectorized nanoparticles (DBM + αGC) with a more potent biological activity than the free agonist. DBM nanoparticles improved in vivo αGC loading into the CD1d molecule and induced a higher frequency of IFN-γ-expressing iNKT cells. Consequently, mice treated with DBM + αGC presented higher levels of serum IFN-γ than those treated with free agonist. Also, vectorized nanoparticles improved αGC ability to control the growth of murine lung metastatic carcinoma. Thus, this is the first study showing that nano spray dryer technology is a simple and alternative approach to enhance iNKT responses., (Copyright © 2019 Elsevier B.V. All rights reserved.)

NKT cells are CD1d-restricted innate-like T cells expressing both T cell receptor and NK cell markers. The major group of NKT cells in both human and mice is the invariant NKT (iNKT) cells and the best-known function of iNKT cells is their potent anti-tumor function in mice. Since its discovery 25 years ago, the prototype ligand of iNKT cells, α-galactosylceramide (α-GalCer) has been used in over 30 anti-tumor clinical trials with mostly suboptimal outcomes. To realize its therapeutic potential, numerous preclinical models have been developed to optimize the scheme and strategies for α-GalCer-based cancer immunotherapies. Nevertheless, since there is no standard protocol for α-GalCer delivery, we reviewed the preclinical studies with a focus on B16 melanoma model in the goal of identifying the best treatment schemes for α-GalCer treatment. We then reviewed the current progress in developing more clinically relevant mouse models for these preclinical studies, most notably the generation of new mouse models with a humanized CD1d/iNKT cell system. With ever-emerging novel iNKT cell ligands, invention of novel α-GalCer delivery strategies and significantly improved preclinical models for optimizing these new strategies, one can be hopeful that the full potential of anti-tumor potential for α-GalCer will be realized in the not too distant future.

CD8+ T cell-mediated immune response plays a major role in the clearance of virus-infected cells, including human papillomavirus (HPV). The effective treatment of women with normal cytology but persistent high risk-HPV infection or with low-grade intraepithelial lesions could take advantage of novel strategies based on vaccination with viral immunological targets with a wide spectrum of cross-protection. The helicase E1, expressed early during viral replication in HPV infection, is among the most conserved papillomavirus proteins, which makes it a good vaccine candidate. In the present study, we examined E1-specific CD8+ T cell and NK immune responses in a mouse model with α-galactosylceramide (α-GalCer) as an adjuvant. We found that mice immunized with E1 combined with α-GalCer elicited an E1-specific CD8+ T and NK cell cytotoxic responses, which correlated with growth inhibition of grafted melanoma B16-F0 cells expressing E1, both in prophylactic and therapeutic protocols., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Chemoimmunotherapy with chemotherapeutics and immunoadjuvant inhibits tumor growth by activating cytotoxic T cells. However, this process also upregulates the expression of PD-1/PD-L1 and consequently leads to immune suppression. To maximize the anti-tumor immune responses and alleviate immunosuppression, PD-L1 antibody was combined with paclitaxel (PTX) and the immunoadjuvant α-galactosylceramide (αGC), which were coencapsulated into pH-sensitive TH peptide-modified liposomes (PTX/αGC/TH-Lip) to treat melanoma and lung metastasis. Compared to treatment with PD-L1 antibody or PTX/αGC/TH-Lip alone, the combination of PD-L1 antibody and PTX/αGC/TH-Lip further elevated the tumor-specific cytotoxic T cell responses and promoted apoptosis in tumor cells, leading to enhanced anti-tumor and anti-metastatic effects. In adoptive therapy, PD-L1 antibody further alleviated immunosuppression and enhanced the anti-tumor effect of CD8 + T cells. The combination of PD-L1 antibody and chemoimmunotherapy PTX/αGC/TH-Lip provides a promising strategy for enhancing treatment for melanoma and lung metastasis.

Background & Aims: Chemokine-mediated immune cell recruitment plays pivotal roles in liver inflammation. C-C motif chemokine ligand 5 (CCL5) has been shown to be responsible for the recruitment of monocytes/macrophages and has been implicated in various liver diseases, including nonalcoholic fatty liver disease, fibrosis, and hepatocellular carcinoma. Previous studies have also shown that inhibition of CCL5 appears to be a promising therapeutic approach for several chronic liver diseases. However, whether blocking CCL5 could benefit immune cell-mediated hepatitis remains largely elusive., Methods: By adopting a specific agonist, alpha-galactosylceramide (α-Galcer), of invariant natural killer T cells (iNKTs), we investigated the function and mechanism of CCL5 in the iNKT induced murine hepatitis model., Results: We found significantly increased CCL5 expression in α-Galcer-induced hepatitis murine model. Such an increase in CCL5 is mainly enriched in non-parenchymal cells such as macrophages and iNKTs but not in hepatocytes. Surprisingly, CCL5 blockage by genetic deletion of Ccl5 does not affect the α-Galcer-induced iNKT activation but greatly worsens α-Galcer-induced liver injury accompanied by an increased hepatic neutrophil infiltration. Mechanistically, we demonstrated that greater neutrophil accumulation in the liver is responsible for the enhanced liver injury in Ccl5 -/- mice. Such an increased hepatic neutrophil infiltration is mainly caused by an enhanced CXCL1-CXCR2 signal in Ccl5 -/- mice. Therapeutically, either antibody-mediated neutrophil depletion or a CXCR2 antagonist, SB225002, mediated CXCR2 signaling blockage significantly ameliorated α-Galcer-induced liver injury in Ccl5 -/- mice., Conclusions: Our present study demonstrates that (1) α-Galcer-induced murine hepatitis could greatly induce CCL5 production in macrophages and iNKT cells; (2) loss of CCL5 could enhance CXCL1 expression in hepatocytes and activate CXCL1-CXCR2 axis in neutrophils to augment their hepatic infiltration; and (3) neutrophil depletion or blockage of CXCL1-CXCR2 axis greatly improves α-Galcer-induced liver injury in Ccl5 -/- mice. This study suggests that clinical utilization of CCL5 blockage may compensatorily induce the activation of other chemokine pathways to enhance neutrophil recruitment and liver injury in hepatitis., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Graphene oxide (GO) modulates the functions of antigen-presenting cells including dendritic cells (DCs). Although carbon nanotubes affect expression of the MHC class I-like CD1d molecule, whether GO can influence immune responses of CD1d-dependent invariant natural killer T (iNKT) cells remains unclear. Here, we investigated the impact of GO on inflammatory responses mediated by α-galactosylceramide (α-GalCer), an iNKT cell agonist. We found that in vivo GO treatment substantially inhibited the capacity of α-GalCer to induce the iNKT cell-mediated trans-activation of and cytokine production by innate and innate-like cells, including DCs, macrophages, NK cells, and γδ T cells. Such effects of GO on α-GalCer-induced inflammatory responses closely correlated with iNKT cell polarization towards TGFβ production, which also explains the capacity of GO to expand regulatory T cells. Interestingly, the absence of TLR4, a receptor for GO, failed to downregulate, and instead partially enhanced the anti-inflammatory activity of GO against α-GalCer-elicited responses, implying negative effects of TLR4 signaling on the anti-inflammatory properties of GO. By employing an α-GalCer-induced sepsis model, we further demonstrated that GO treatment significantly protected mice from α-GalCer-induced lethality. Taken together, we provide strong evidence that GO holds promise as an adjuvant to modulate iNKT cell responses for immunotherapy.

Increased expression of T cell immunoglobulin and mucin domain-3 (Tim-3) on invariant natural killer T (iNKT) cells is reported in chronic hepatitis B virus (HBV) infection. However, whether Tim-3 regulates iNKT cells in chronic HBV condition remains unclear. In this study, our results showed that the expression of Tim-3 was up-regulated on hepatic iNKT cells from HBV-transgenic (Tg) mice or iNKT cells stimulated with α-galactosylceramide (α-Galcer). Compared with Tim-3 - iNKT cells, Tim-3 + iNKT cells expressed more IFN-γ, IL-4 and CD107a, indicating a strong relationship between Tim-3 and iNKT cell activation. Constantly, treatment of Tim-3 blocking antibodies significantly enhanced the production of IFN-γ, TNF-α, IL-4 and CD107a in iNKT cells both in vivo and in vitro. This Tim-3 - mediated suppression of iNKT cells was further confirmed in Tim-3 knockout (KO) mice. Moreover, Tim-3 blockade promoted α-Galcer-triggered inhibition of HBV replication, displaying as the decreased HBV DNA and HBsAg level in serum, and down-regulated pgRNA expression in liver tissues. Collectively, our data, for the first time, demonstrated the potential role of Tim-3 blockade in promoting iNKT cell-mediated HBV inhibition. Therefore, combination of α-Galcer with Tim-3 blockade might be a promising approach in chronic hepatitis B therapy., (© 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Lupus nephritis is a crucial complication of systemic lupus erythematosus. In this study, we investigated the roles of mouse natural killer T (NKT) cells in lupus nephritis. From 24 weeks of age, NZB/NZW F1 mice were injected with alpha-galactosylceramide (α-GalCer) or vehicle once a week for four weeks. In the α-GalCer group, the levels of proteinuria and blood urea nitrogen were significantly lower than those in the vehicle group. The histological evaluation showed a decrease in glomerular immune complex deposits and an alleviation of podocyte injury. The proportion of NKT cells in the mononuclear cell (MNC) fraction in the α-GalCer group was significantly decreased in the liver, kidney, and spleen. The proliferation and cytokine production in α-GalCer-stimulated liver MNCs were markedly diminished in the α-GalCer group (anergy). The IFN-γ production in liver MNCs stimulated by concanavalin A or an anti-CD3 antibody did not differ between the two groups, whereas the IL-4 production was significantly lower in the α-GalCer group. In addition, the IgM production in CpG-oligodeoxynucleotide-stimulated spleen MNCs was significantly lower in the α-GalCer group. These results suggest that α-GalCer suppressed Th2 immune responses in NKT cells and B cell function, thereby slowing the progression of lupus nephritis.

Sepsis is the third leading cause of death in the neonatal population, due to susceptibility to infection conferred by immaturity of both the innate and adaptive components of the immune system. Invariant natural killer T (iNKT) cells are specialized adaptive immune cells that possess important innate-like characteristics and have not yet been well-studied in septic neonates. We hypothesized that iNKT cells would play an important role in mediating the neonatal immune response to sepsis. To study this, we subjected 5- to 7-day-old neonatal C57BL/6 mice to sepsis by intraperitoneal (i.p.) cecal slurry (CS) injection. Thirty hours prior to or immediately following sepsis induction, pups received i.p. injection of the iNKT stimulator KRN7000 (KRN, 0.2 µg/g) or vehicle. Ten hours after CS injection, blood and tissues were collected for various analyses. Thirty-hour pretreatment with KRN resulted in better outcomes in inflammation, lung injury, and survival, while immediate treatment with KRN resulted in worse outcomes compared to vehicle treatment. We further analyzed the activation status of neonatal iNKT cells for 30 h after KRN administration, and showed a peak in frequency of CD69 expression on iNKT cells and serum IFN-γ levels at 5 and 10 h, respectively. We then used CD1d knockout neonatal mice to demonstrate that KRN acts through the major histocompatibility complex-like molecule CD1d to improve outcomes in neonatal sepsis. Finally, we identified that KRN pretreatment exerts its protective effect by increasing systemic levels of TGF-β1. These findings support the importance of iNKT cells for prophylactic immunomodulation in neonates susceptible to sepsis.

pH-Sensitive fusogenic polymer-modified (pH-sensitive) liposomes co-loaded with tumor model antigen, ovalbumin (OVA), and adjuvant, α-galactosylceramide (α-GalCer) were fabricated and administered subcutaneously into mice. The ability of pH-sensitive liposomes containing OVA and α-GalCer to stimulate cellular and humoral immune responses in vivo was compared with OVA-encapsulating pH-sensitive liposomes as well as with OVA alone. After immunization, significant OVA-specific antibodies were detected in the serum. When sera were analyzed for isotype distribution, antigen-specific IgG1 antibody responses were noted in mice immunized with OVA alone, whereas immunization with OVA-containing pH-sensitive liposomes and with pH-sensitive liposomes containing OVA and α-GalCer resulted in the induction of OVA-specific IgG1 and IgG2b antibody responses. Moreover, more substantial production of IFN-γ and IL-4 was demonstrated in spleen cells from mice immunized with pH-sensitive liposomes having OVA and α-GalCer than OVA-containing pH-sensitive liposomes in vitro. Spleen cells from the immunized mice showed strong cytotoxic activity against E.G7-OVA tumor cells. In addition, prophylactic vaccination efficacy against tumor formation was evaluated. In all mice immunized with pH-sensitive liposomes having OVA and α-GalCer, immunization provided substantial protection from tumor formation. The therapeutic efficacy of pH-sensitive liposomes containing OVA and α-GalCer against already established E.G7-OVA tumors was also investigated. Tumor growth was reduced significantly in all mice treated with pH-sensitive liposomes having OVA and α-GalCer. The provided evidence on the advantage of antigen and α-GalCer co-encapsulation into pH-sensitive liposomes should be considered in the design of future cancer vaccines for prophylactic and therapeutic purposes.

Alpha-galactosylceramide (αGalCer) is a glycolipid derived from a marine sponge that is a potent activator of both mouse and human invariant natural killer T (iNKT) cells. For that reason, αGalCer is a promising vaccine adjuvant that has been shown to improve both humoral and cellular immunity when co-administered with various vaccines, including candidate vaccines for biodefense. In the current study, we tested the effectiveness of αGalCer as an adjuvant for the clinically-relevant ricin toxin subunit vaccine, RiVax. αGalCer had a potent adjuvant effect, as shown by a rapid onset of anti-ricin IgG titers, accelerated development of serum toxin-neutralizing activity, and enhanced protection from lethal ricin challenge in a mouse model. These results underscore the potential of αGalCer to augment the protective immune response to a vaccine designed to counteract ricin toxin, a fast-acting biothreat agent.

Dendritic cell (DC) vaccine-based immunotherapy for glioblastoma multiforme (GBM) has shown apparent benefit in animal experiments and early-phase clinical trials, but the survival benefit is variable. In this work, we analyzed the mechanism of the potent antitumor immune response induced in vivo by tumor-associated antigen (TAA)-specific DCs with an invariant natural killer T (iNKT) cell adjuvant in orthotopic glioblastoma-bearing rats vaccinated with tumor-derived exosomes and α-galactosylceramide (α-GalCer) -pulsed DCs. Compared with traditional tumor lysate, exosomes were utilized as a more potent antigen to load DCs. iNKT cells, as an effective cellular adjuvant activated by α-GalCer, strengthened TAA presentation through their interaction with DCs. Co-delivery of tumor-derived exosomes with α-GalCer on a DC-based vaccine showed powerful effects in glioblastoma immunotherapy. This vaccine induced strong activation and proliferation of tumor-specific cytotoxic T lymphocytes, synergistically breaking the immune tolerance and improving the immunosuppressive environment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objectives: α-Galactosylceramide (α-GalCer), a synthetic glycosphingolipid that exhibits potent immunostimulatory effects through activation of natural killer T (NKT) cells, can be used to treat conditions such as atopy, cancer, infection and autoimmunity. Administration of therapeutics through the oral route has advantages such as patient convenience, safety and reduced cost; however, there has been little research to investigate whether oral delivery of α-GalCer is possible. The aim of this study was therefore to determine whether α-GalCer formulated in either DMSO/Tween 80 or in liposomes, could access lymphoid tissue and stimulate immune activation following oral administration., Methods: Fluorescently labelled cationic liposomes incorporating α-GalCer were prepared, characterized and administered by oral gavage to fasted mice., Key Findings: Liposomes were detected inside the Peyer's patches (PPs), in the subepithelial dome just under the follicle-associated epithelium. CD11b + cells and CD11c + were shown to have taken up the formulation in a higher proportion compared to the total cell proportion in the PPs, suggesting that cells with these markers may be the prominent antigen-presenting cells involved in selective uptake. Finally, the liposomal formulation demonstrated a higher degree of immune stimulation compared to the DMSO/Tween 80 solubilized α-GalCer in the PPs, mesenteric lymph nodes and spleen as shown by the increased expression of IL-4 mRNA expression and increased proportion of NKT cells at 6 h and 3 days after administration., Conclusions: These results show that oral delivery of a liposomal α-GalCer can stimulate local and systemic immune responses to a different degree compared to the non-liposomal form., (© 2017 Royal Pharmaceutical Society.)

Intradermal vaccine delivery is a promising alternative to the conventional intramuscular route. The skin layer is immunologically supported by a densely network of antigen presenting cells, while the skeletal muscle is loaded with a relatively sparse population of APCs. Nevertheless, the vaccine to be suitable for intradermal delivery needs a new formulation to facilitate either smaller injection volumes or the introduction into new delivery devises as micro-needles. This study presents a proof of concept for intradermal delivery of the MenC-CRM 197 glycoconjugate vaccine using a mouse model. Tangential flow filtration allowed obtaining a 20-fold concentrated vaccine formulation suitable for intradermal injection. Importantly the intradermal delivery of non-adjuvanted MenC glycoconjugate vaccine showed a quicker on-set and superiority in terms of immunogenicity compared to intramuscular administration of the respective vaccine and comparable immunogenicity to the aluminum adjuvanted vaccine formulation given intramuscular. Subsequently, the use of adjuvants allowed to further increase the immunogenicity and to modulate the quality of the immune response towards a more beneficial Th1 response. As adjuvants two Toll like receptor agonists (TLR4a and TLR7a), a mutant of the heat-labile enterotoxin from Escherichia coli (LT), a α-GalactosylCeramide analogue and an oil in water emulsion were investigated in order to target skin-resident antigen-presenting cells. This approach has the potential to be extended to other meningococcal serogroups, representing a promising strategy for the development of dermally administered multivalent glycoconjugate vaccines., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cancer immunity is mediated through the effective priming and activation of tumour-specific class I MHC molecule-restricted CD8 + cytotoxic T lymphocytes (CTLs). DEC-205 + dendritic cells (DCs) can cross-present the epitope(s) of captured tumour antigens associated with class I MHC molecules alongside co-stimulatory molecules to prime and activate tumour-specific CD8 + CTLs. Immunosuppressive tolerogenic DCs with reduced co-stimulatory molecules may be a cause of impaired CTL induction. Hepa1-6-1 cells were established from the mouse hepatoma cell line Hepa1-6; these cells grow continuously after subcutaneous implantation into syngeneic C57BL/6 (B6) mice and do not prime CD8 + CTLs. In this study, we show that the growth of ongoing tumours was suppressed by activated CD8 + CTLs with tumour-specific cytotoxicity through the administration of the glycolipid α-galactosylceramide (α-GalCer), which is a compound known to stimulate invariant natural killer T (iNKT) cells and selectively activate DEC-205 + DCs. Moreover, we demonstrated that sequential repetitive intraperitoneal inoculation with α-GalCer every 48 hr appeared to convert tolerogenic DEC-205 + DCs into immunogenic DCs with a higher expression of co-stimulatory molecules and a stronger cross-presentation capacity, which primed CTL precursors and induced tumour-specific CD8 + CTLs within the tumour environment without activating iNKT cells. These findings provide a new basis for cancer immunotherapy to convert tolerogenic DEC-205 + DCs within tumours into immunogenic DCs through the sequential administration of an immuno-potent lipid/glycolipid, and then activated immunogenic DCs with sufficient expression of co-stimulatory molecules prime and activate tumour-specific CD8 + CTLs within the tumour to control tumour growth., (© 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.)

Regulatory T (Treg) cells play a central role in immune tolerance and prevention of aberrant immune responses. Several studies have suggested that the risk of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) can be ameliorated by increasing Tregs. We have developed an approach of in vivo expansion of Tregs with RGI-2001, a novel liposomal formulation of a synthetic derivative of alpha-galactosylceramide, a naturally occurring ligand that binds to CD1 and activates and expands invariant natural killer cells. In preclinical studies, a single intravenous infusion of RGI-2001 expanded Treg and could ameliorate GVHD in a mouse model of allogeneic HCT. To explore the role of RGI-2001 in clinical HCT, we initiated a phase 2A clinical trial (n = 29), testing 2 different doses of RGI-2001 administered as a single infusion on day 0 of allogeneic HCT. RGI-2001 was well tolerated and without infusion reactions or cytokine release syndrome. A subset of patients (8 of 29, 28%) responded to RGI-2001 by inducing a markedly increased number of cells with a Treg phenotype. The Treg had a high Ki-67 index and were almost exclusively Helios + and Foxp3 + , indicating that their accumulation was due to expansion of natural Treg. Notably, the incidence of grade 2 to 4 GVHD in the 8 patients who responded to RGI-2001 was 12.5%, compared with 52.4% in the 21 patients who did not respond. No grade 3 or 4 GVHD was observed in the responder group, compared with a 9.5% incidence among nonresponders. Immunosuppression with sirolimus was also associated with a low incidence of GVHD, suggesting that RGI-2001 may have synergized with sirolimus to promote Treg expansion., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Combinations of mAbs that target various components of T-cell activation/inhibition may work synergistically to improve antitumor immunity against cancer. In this study, we investigated the therapeutic potential of combining an anticancer vaccination strategy with antibodies targeting an immune stimulatory (4-1BB) and immune inhibitory (PD-1) receptor, in a preclinical model of spontaneously arising c-Myc-driven B-cell lymphoma. In Eμ-myc transgenic mice, we reveal that 4-1BB agonistic mAb treatment alone was sufficient to drive antitumor immunity and prevent disease progression in 70% of mice. When combined with an α-GalCer-loaded, irradiated tumor cell vaccine, 4-1BB mAb treatment led to increased expansion of effector CD8 T-cell populations and protection of long-term surviving mice against tumor rechallenge. Unexpectedly, PD-1 blockade did not provide therapeutic benefit. The T-cell-promoting effects and antitumor activity of 4-1BB mAb were diminished when used simultaneously with a PD-1-blocking mAb. This was associated with a rapid and dramatic reduction in effector CD8 + T-cell subsets in the presence of PD-1 blockade. These findings reveal that supporting T-cell activation therapeutically is effective for controlling B-cell lymphomas; however, caution is required when combining antibody-mediated modulation of both costimulatory and coinhibitory T-cell receptors. Cancer Immunol Res; 5(3); 191-7. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Objectives: A CD1d-binding invariant natural killer T (iNKT)-cell stimulatory glycolipid, namely 7DW8-5, is shown to enhance the efficacy of radiation-attenuated sporozoites (RAS)-based malaria vaccine in mice. In the current study, we aim to determine whether 7DW8-5 can display a potent adjuvant effect in human immune system (HIS) mice., Methods: HIS-A2/hCD1d mice, which possess both functional human iNKT cells and CD8+ T cells, were generated by the transduction of NSG mice with adeno-associated virus serotype 9 expressing genes that encode human CD1d molecules and HLA-A*0201, followed by the engraftment of human hematopoietic stem cells. The magnitudes of human iNKT-cell response against 7DW8-5 and HLA-A*0201-restricted human CD8+ T-cell response against a human malaria antigen in HIS-A2/hCD1d mice were determined by using human CD1d tetramer and human HLA-A*0201 tetramer, respectively., Results: We found that 7DW8-5 stimulates human iNKT cells in HIS-A2/hCD1d mice, as well as those derived from HIS-A2/hCD1d mice in vitro. We also found that 7DW8-5 significantly increases the level of a human malarial antigen-specific HLA-A*0201-restricted human CD8+ T-cell response in HIS-A2/hCD1d mice., Conclusions: Our study indicates that 7DW8-5 can display a potent adjuvant effect on RAS vaccine-induced anti-malarial immunity by augmenting malaria-specific human CD8+ T-cell response.

Innate immunity, which is unable to discriminate self from allo-antigens, is thought to be important players in the induction of miscarriages. Here, we show that the administration of IL-12 to syngeneic-mated C57BL/6 mice on gestation day 7.5 (Gd 7.5), drives significant miscarriages in pregnant females. Furthermore, the administration on Gd 7.5 of α-galactosylceramide (α-GalCer), which is known to activate invariant natural killer T (iNKT) cells, induced miscarriages in both syngeneic-mated C57BL/6 mice and allogeneic-mated mice (C57BL/6 (♀) × BALB/c (♂)). Surprisingly, the percentages of both DEC-205(+) DCs and CD1d-restricted NK1.1(+) iNKT cells were higher in the myometrium of pregnant mice treated i.p. with α-GalCer than in the decidua. IL-12 secreted from α-GalCer-activated DEC-205(+) DCs stimulated the secretion of cytokines, including IL-2, IL-4, IFN-γ, TNF-α, perforin, and granzyme B, from the NK1.1(+) iNKT cells in the myometrium, leading to fetal loss in pregnant mice. Finally, the i.p. administration of IL-12 and/or α-GalCer in iNKT-deficient Jα18(-/-) (Jα18 KO) mice did not induce miscarriages. This study provides a new perspective on the importance of the myometrium, rather than the decidua, in regulating pregnancy and a mechanism of miscarriage mediated by activated DEC-205(+) DCs and NK1.1(+) iNKT cells in the myometrium of pregnant mice., (© 2016 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Diarrhoeal infections are a major cause of morbidity and mortality with enterotoxigenic Escherichia coli (ETEC) and cholera imposing a significant global burden. There is currently no licensed vaccine for ETEC. Development of new nonliving oral vaccines has proven difficult due to the physicochemical and immunological challenges associated with the oral route. This demands innovative delivery solutions to protect antigens, control their release and build in immune-stimulatory activity. We describe the Single Multiple Pill® (SmPill®) vaccine formulation which combines the benefits of enteric polymer coating to protect against low gastric pH, a dispersed phase to control release and aid the solubility of non-polar components and an optimized combination of adjuvant and antigen to promote mucosal immunity. We demonstrate the effectiveness of this system with whole cell killed E. coli overexpressing colonization factor antigen I (CFA/I), JT-49. Alpha-galactosylceramide was identified as a potent adjuvant within SmPill® that enhanced the immunogenicity of JT-49. The bacteria associated with the dispersed phase were retained within the capsules at gastric pH but released at intestinal pH. Vaccination with an optimized SmPill® formulation promoted CFA/I-specific immunoglobulin A (IgA) responses in the intestinal mucosa in addition to serum IgG and a solubilized adjuvant was indispensable for efficacy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In the present study, the combined adjuvant effect of 7DW8-5, a potent α-GalCer-analog, and monophosphoryl lipid A (MPLA), a TLR4 agonist, on the induction of vaccine-induced CD8(+) T-cell responses and protective immunity was evaluated. Mice were immunized with peptides corresponding to the CD8(+) T-cell epitopes of a malaria antigen, a circumsporozoite protein of Plasmodium yoelii, and a tumor antigen, a Wilms Tumor antigen-1 (WT-1), together with 7DW8-5 and MPLA, as an adjuvant. These immunization regimens were able to induce higher levels of CD8(+) T-cell responses and, ultimately, enhanced levels of protection against malaria and tumor challenges compared to the levels induced by immunization with peptides mixed with 7DW8-5 or MPLA alone. Co-administration of 7DW8-5 and MPLA induces activation of memory-like effector natural killer T (NKT) cells, i.e. CD44(+)CD62L(-)NKT cells. Our study indicates that 7DW8-5 greatly enhances important synergistic pathways associated to memory immune responses when co-administered with MPLA, thus rendering this combination of adjuvants a novel vaccine adjuvant formulation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Natural killer T (NKT) -cells activated with the glycolipid ligand α-galactosylceramide (α-GalCer) stimulate a wide array of immune responses with many promising immunotherapeutic applications, including the enhancement of vaccines against infectious diseases and cancer. In the current study, we evaluated whether α-GalCer generates protective immunity against a swine influenza (SI) virus infection when applied as an intramuscular vaccine adjuvant. Immunization of newly weaned piglets with UV-killed pandemic H1N1 A/California/04/2009 (kCA04) SI virus and α-GalCer induced high titers of anti-hemagglutinin antibodies and generated virus-specific T cells that localized in intrapulmonary airways and in alveolar walls. Vaccination with α-GalCer resulted in a systemic increase in NKT-cell concentrations, including in the respiratory tract, which was associated with complete inhibition of viral replication in the upper and lower respiratory tract and much reduced viral shedding. These results indicate that NKT-cell agonists could be used to improve swine vaccine formulations in order to reduce the clinical signs of SI infection and limit the spread of influenza viruses amongst commercial pigs.

The interaction between tissue-resident mast cells (MCs) and recruited immune cells contributes to tissue immunosurveillance. However, the cells, mechanisms, and receptors involved in this crosstalk remain ill defined. Invariant natural killer T (iNKT) cells are CD1d-restricted innate lymphocytes that recognize glycolipid antigens and have emerged as critical players in immunity. Here, we show that primary mouse peritoneal MCs express surface CD1d, which is upregulated in vivo following administration of alpha-galactosylceramide. In contrast, in BM-derived MCs CD1d was found to be stored intracellularly and to relocate at the cell surface upon IgE-mediated degranulation. Activated BM-derived MCs expressing surface CD1d and loaded with alpha-galactosylceramide were found to induce iNKT-cell proliferation and the release of IFN-γ, IL-13, and IL-4 in a CD1d-restricted manner. Moreover, the costimulatory molecules CD48, CD137L, CD252, CD274, and CD275 affected MC-induced IFN-γ release and iNKT-cell proliferation. Interestingly, among the costimulatory molecules, CD48 and CD252 exhibited a distinctly regulatory activity on iNKT-cell release of both IFN-γ and IL-13. In conclusion, we demonstrate that the crosstalk between MCs and iNKT cells may regulate inflammatory immune responses., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Alpha-galactosylceramide (GC) represents a potentially new class of adjuvant because GC strongly induces interferon (IFN) gamma production from natural killer T (NKT) cells, leading to the induction of strong antitumor immunity. Interleukin (IL)-12 is another stimulating signal that induces IFN-γ production by NKT cells. We report herein on an investigation of the effect of recombinant IL-12 on NKT cell activation, when used in combination with GC-loaded octaarginine modified liposomes (GC-Lip). IFN-γ production from splenocytes simulated with GC-Lip was dose dependently enhanced in the presence of IL-12 in vitro. In contrast, IFN-γ production in vivo was enhanced at a low dose of IL-12. Enhanced IFN-γ production was observed in the case of low doses (0.5 μg and 2.5 μg) of GC-Lip but not a high dose (5 μg), that is, the IL-12 combination enhanced NKT cell activation at a 10-fold lower GC dose. The use of the above combination also enhanced the expansion of the NKT cell population. These findings indicate that in vivo IFN-γ production is inversely correlated with the dose of IL-12 during dual signal stimulation of NKT cells via both GC-Lip and IL-12, indicating that the dose of GC-Lip can be reduced without weakening NKT cell activation., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Dendritic cells (DCs) and invariant natural killer T (iNKT) cells play important roles in linking innate immunity and adaptive immunity. Mature DCs activated by Toll-like receptor (TLR) agonists directly activate iNKT cells and the iNKT ligand α-galactosylceramide (α-Galcer) can induce DC maturation, resulting in enhanced protective immune responses. In the present study, we aimed to boost anti-tumour immunity in a murine colon cancer model by synergizing DCs and iNKT cells using α-Galcer-loaded tumour cells (tumour-Gal) and the TLR9 agonist cytosine-phosphorothioate-guanine (CpG1826). The vaccine strategy was sufficient to inhibit growth of established tumours and prolonged survival of tumour-bearing mice. Importantly, the immunization induced an adaptive memory immune response as the survivors from primary tumour inoculations were resistant to a tumour re-challenge. Furthermore, injection of tumour-Gal with CpG1826 resulted in iNKT cell activation and DC maturation as defined by interferon (IFN)-γ secretion by iNKT, natural killer (NK) cells and interleukin (IL)-12 by DCs. Immunohistochemistry analysis revealed that cluster of differentiation (CD)4(+) T-cells and CD8(+) T-cells played important roles in anti-tumour immunity. Additionally, the vaccine redirected Th2 (T-helper cell type 2) responses toward Th1 (T-helper cell type 1) responses with increases in IL-2, IFN-γ expression and decreases in IL-4 and IL-5 expression after immunization with tumour-Gal with CpG1826. Taken together, our results demonstrated a novel vaccination by synergizing tumour-Gal and CpG1826 against murine colon cancer, which can be further developed as tumour-specific immunotherapy against human cancer., (© 2016 Authors; published by Portland Press Limited.)

The lipid antigen α-galactosylceramide (α-GalCer) is a potent activator of invariant natural killer T-cells (iNKT cells) and can stimulate cytotoxic and anti-tumour immune responses. However optimal responses appear to be induced by α-GalCer when cell-based vaccines are delivered intravenously. Here we investigated if co-delivery of protein and peptide antigens along with α-GalCer in a liposomal formulation could stimulate therapeutic anti-tumour immune responses. Cationic liposomes were inherently immune-stimulatory and induced cytotoxic immune responses when delivered both by intravenous and subcutaneous injection. However, only vaccine delivered intravenously stimulated therapeutic anti-tumour immune responses to a peptide antigen. Surface modification with polyethylene glycol (PEG) did not improve immune responses to either intravenously or subcutaneously delivered vaccines. Immune responses to short and long peptide sequences (CD8 and CD4 epitopes) of the self-antigen tyrosinase-related protein 2 (TRP2) as a vaccine antigen, co-delivered with α-GalCer in either cationic liposomes or PBS were further examined. Enhanced production of IFN-γ, increased cytotoxic T-cell responses and tumour survival were observed when a long TRP2-peptide was delivered with α-GalCer in cationic liposomes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

NKT cells are a subgroup of T cells, which express a restricted TCR repertoire and are critical for the innate immune responses to viral infections. Activation of NKT cells depends on the major histocompatibility complex-related molecule CD1d, which presents bioactive lipids to NKT cells. The marine sponge derived lipid αGalCer has recently been demonstrated as a specific agonist for activation of human and murine NKT cells. In the present study we investigated the applicability of αGalCer pre-treatment for immune protection against intra-vaginal HSV-2 infection. We found that C57BL/6 WT mice that received local pre-treatment with αGalCer prior to intra-vaginal HSV-2 infection had a lower mean disease score, mortality and viral load in the vagina following infection, compared to mice that did not receive αGalCer pre-treatment. Further, we found increased numbers of CD45 and NK1.1 positive cells in vaginal tissue and elevated levels of IFN-γ in the vaginal tissue and in vaginal fluids 24h after αGalCer pre-treatment. Collectively our data demonstrate a protective effect of αGalCer induced activation of NKT cells in the innate immune protection against viral infection., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Objective: To investigate the cytoprotective effects of high dose of α-galactosylceramide (α-GC) on the activation-induced CD4+ T and CD8+ T cell death., Methods: Experimental autoimmune encephalomyelitis (EAE) was induced using adoptive transfer of MOGCD4+ cells treated using α-GC into recipient C57BL/6 mice while the MOGCD4+ cells treated using 0.5% polysorbate were set as vehicle group, based on which to investigate the effects of α-GC on activation induced CD4+ T cell death. Additionally, an EG7 tumor-bearing mice model is established using adoptive transfer of CD8+ T cells, based on which to investigate the effect of α-GC on the apoptosis of CD8+ T cells., Results: A higher induction rate was noticed after adoptive transfer of MOGCD4+ cells treated using α-GC together with the severity of EAE compared with the conventional methods. Longer survival duration was noted in the green fluorescent protein (GFP) labeled MOGT in the α-GC group compared with the vehicle group (P < 0.05). Severe inflammatory cell infiltration and myelinoclasis was noted in the white matter of nervous system in the α-GC group. In the EG7 tumor model, more adoptive CD8+ T cells were survived in α-GC group compared with that of vehicle group. The growth of tumor mass was significantly inhibited in α-GC group., Conclusions: high dose of α-GC could be used as an adjuvant for inhibiting activation-induced CD4+ T and CD8+ T cell death. Our study could provide helpful information for the development of adoptive cell therapy with reduced programmed cell death.

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and T cells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids., (© The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Natural Killer T (NKT) cells are a unique type of innate immune cells which exert paradoxical roles in animal models through producing either Th1 or Th2 cytokines and activating dendritic cells. Alpha-galactosylceramide (αGalCer), a synthetic antigen for NKT cells, was found to be safe and immune stimulatory in cancer and hepatitis patients. We recently developed microparticle-formulated αGalCer, which is selectively presented by dendritic cells and macrophages, but not B cells, and thus can avoid the anergy of NKT cells. In this study, we have examined the immunogenicity of microparticles containing αGalCer and protein vaccine components through sublingual injection in mice. The results showed that sublingual injection of microparticles containing αGalCer and ovalbumin triggered IgG responses in serum (titer >1:100,000), which persisted for more than 3months. Microparticles containing ovalbumin alone also induced comparable level of IgG responses. However, immunoglobulin subclass analysis showed that sublingually injected microparticles containing αGalCer and ovalbumin induced 20 fold higher Th1 biased antibody (IgG2c) than microparticles containing OVA alone (1:20,000 as compared to 1:1000 titer). Sublingual injection of microparticles containing αGalCer and ovalbumin induced secretion of both IgG (titer >1:1000) and IgA (titer=1:80) in saliva secretion, while microparticles containing ovalbumin alone only induced secretion of IgG in saliva. Our results suggest that sublingual injection of microparticles and their subsequent trafficking to draining lymph nodes may induce adaptive immune responses in mucosal compartments. Ongoing studies are focused on the mechanism of antigen presentation and lymphocyte biology in the oral cavity, as well as the toxicity and efficacy of these candidate microparticles for future applications., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

α-galactosylceramide (GalCer), which is a natural killer T (NKT) cell ligand, has been reported to exert therapeutic effects against cancer in humans and mice. Toll-like receptor (TLR) agonists systemically or locally boost antitumor efficacy in mouse cancer models. In our previous study, the co-administration of GalCer and a TLR agonist synergistically enhanced interferon-γ (IFN-γ) production in mouse splenocytes in vitro and in vivo. The increased IFN-γ production promoted a tumor antigen-specific Th1 response. Therefore, co-treatment with GalCer and a TLR agonist is expected to exert an enhanced antitumor effect. In the present study, we examined the effect of GalCer and lipopolysaccharide (LPS) combination therapy in a mouse lung-metastasis model. GalCer and LPS combination therapy markedly decreased the number of lung metastatic tumor nodes. Co-treatment with GalCer and LPS enhanced the mRNA expression of CXCL9 and CXCL10 in mediastinal lymph nodes (MLNs) and increased the number of CD8+ cells in the MLNs. Furthermore, the depletion of CD8+ T cells canceled the antitumor effect of GalCer and LPS combination therapy. Thus, GalCer and LPS combination therapy significantly enhanced tumor antigen-specific immune responses and suppressed tumor growth in a mouse lung-metastasis model.

Background: Two major distinct subsets of dendritic cells (DCs) are arranged to regulate immune responses: DEC-205+ DCs drive Th1 polarization and 33D1+ DCs establish Th2 dominancy. Th1 polarization can be achieved either by depletion of 33D1+ DCs with a 33D1-specific monoclonal antibody (mAb) or by activation of DEC-205+ DCs via intraperitoneal injection of α-galactosylceramide (α-GalCer). We studied the effect of 33D1+ DC depletion or DEC-205+ DC activation in vivo using an established mouse model of allergic rhinitis (AR)., Methods: Mice were injected intraperitoneally with OVA plus alum and challenged 4 times with daily intranasal administration of OVA. Immediately after the last challenge, allergic symptoms such as sneezing and nasal rubbing as well as the number of cells in the bronchoalveolar lavage fluid (BALF) and nasal lavage fluid (NALF) were counted. The levels of serum OVA-specific IgG1, IgG2a, and IgE were also determined by ELISA., Results: The allergic symptom scores were significantly decreased in 33D1+ DC-depleted or DEC-205+ DC-activated AR mice. The levels of OVA-specific IgG1, IgG2a, and IgE, and the number of NALF cells, but not BALF cells, were reduced in 33D1+ DC-depleted but not in DEC-205+ DC-activated AR mice. Moreover, the activated DEC-205+ DCs suppressed histamine release from IgE-sensitized mast cells, probably through IL-12 secretion., Conclusions: The manipulation of innate DC subsets may provide a new therapeutic strategy for controlling various allergic diseases by reducing histamine release from IgE-sensitized mast cells by driving the immune response towards Th1 dominancy via activation of DEC-205+ DCs in vivo., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

The vast majority of HIV-1 infections occur at mucosa during sexual contact. It may therefore be advantageous to provide mucosal barrier protection against this entry by mucosal vaccination. While a number of mucosal routes of vaccination are possible, many like enteric oral vaccines or intranasal vaccines have significant impediments that limit vaccine efficacy or pose safety risks. In contrast, immunogens applied to the sublingual region of the mouth could provide a simple route for mucosal vaccination. While sublingual immunization is appealing, this site does not always drive strong immune responses, particularly when using protein antigens. To address this issue, we have tested the ability of two mucosal adjuvants: alpha-galactosylceramide (αGalCer) that is a potent stimulator of natural killer T cells and CpG-oligodeoxynucleotide (CpG-ODN) a TLR9 agonist for their ability to amplify immune responses against clade C gp140 HIV-1 envelope protein antigen. Immunization with envelope protein alone resulted in a weak T cell and antibody responses. In contrast, CD4(+) and CD8(+) T cells responses in systemic and mucosal tissues were significantly higher in mice immunized with gp140 in the presence of either αGalCer or CpG-ODN and these responses were further augmented when the two adjuvants were used together. While both the adjuvants effectively increased gp140-specific serum IgG and vaginal IgA antibody levels, combining both significantly improved these responses. Memory T cell responses 60 days after immunization revealed αGalCer to be more potent than CpG-ODN and the combination of the αGalCer and CpG-ODN adjuvants was more effective than either alone. Serum and vaginal washes collected 60 days after immunization with gp140 with both αGalCer and CpG-ODN adjuvants had significant neutralization activity against Tier 1 and Tier 2 SHIVs. These data support the utility of the sublingual route for mucosal vaccination particularly in combination with αGalCer and CpG-ODN adjuvants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We have assessed the immune-regulatory and adjuvant activities of a synthetic glycolipid, ABX196, a novel analog of the parental compound α-GalCer. As expected, ABX196 demonstrated a measurable and significant adjuvant effect in mice and monkeys with no appreciable toxicity at the doses used to promote immune responses. We performed a phase I/II dose escalation study of ABX196 in healthy volunteers, with the objectives to evaluate its safety profile, as well as its ability to be utilized as an adjuvant in the context of a prophylactic vaccine against hepatitis B. ABX196 was administered at three doses: 0.2, 0.4, and 2.0μg, in 44 subjects. In all the individuals injected with ABX196, peripheral blood NKT cells displayed hallmarks of activation, and 45% of them had measurable circulating IFN-γ 24h after the first administration. More importantly, the addition of ABX196 to the very poorly immunogenic HBs antigen resulted in protective anti-HBs antibody responses in majority of patients, demonstrating the adjuvant properties of ABX196 in human. Further analysis of the cohort of subjects receiving ABX196 with HBs antigen also indicates that a single injection appears sufficient to provide protection. A limited set of adverse events linked to the systemic delivery of ABX196 and access to the liver, is discussed in the context of formulation and the need to limit transport of ABX196 to secondary lymphoid tissues for maximal efficacy (Eudra-CT 2012-001566-15)., (Copyright © 2014 Elsevier Ltd. All rights reserved.)