Self-Adjuvanting Lipoprotein Conjugate αGalCer-RBD Induces Potent Immunity against SARS-CoV-2 and its Variants of Concern.

Safe and effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants are the best approach to successfully combat the COVID-19 pandemic. The receptor-binding domain (RBD) of the viral spike protein is a major target to develop candidate vaccines. α-Galactosylceramide (αGalCer), a potent invariant natural killer T cell (iNKT) agonist, was site-specifically conjugated to the N-terminus of the RBD to form an adjuvant-protein conjugate, which was anchored on the liposome surface. This is the first time that an iNKT cell agonist was conjugated to the protein antigen. Compared to the unconjugated RBD/αGalCer mixture, the αGalCer-RBD conjugate induced significantly stronger humoral and cellular responses. The conjugate vaccine also showed effective cross-neutralization to all variants of concern (B.1.1.7/alpha, B.1.351/beta, P.1/gamma, B.1.617.2/delta, and B.1.1.529/omicron). These results suggest that the self-adjuvanting αGalCer-RBD has great potential to be an effective COVID-19 vaccine candidate, and this strategy might be useful for designing various subunit vaccines.

Reduced dose of PTCy followed by adjuvant α-galactosylceramide enhances GVL effect without sacrificing GVHD suppression.

Posttransplantation cyclophosphamide (PTCy) has become a popular option for haploidentical hematopoietic stem cell transplantation (HSCT). However, personalized methods to adjust immune intensity after PTCy for each patient's condition have not been well studied. Here, we investigated the effects of reducing the dose of PTCy followed by α-galactosylceramide (α-GC), a ligand of iNKT cells, on the reciprocal balance between graft-versus-host disease (GVHD) and the graft-versus-leukemia (GVL) effect. In a murine haploidentical HSCT model, insufficient GVHD prevention after reduced-dose PTCy was efficiently compensated for by multiple administrations of α-GC. The ligand treatment maintained the enhanced GVL effect after reduced-dose PTCy. Phenotypic analyses revealed that donor-derived B cells presented the ligand and induced preferential skewing to the NKT2 phenotype rather than the NKT1 phenotype, which was followed by the early recovery of all T cell subsets, especially CD4+Foxp3+ regulatory T cells. These studies indicate that α-GC administration soon after reduced-dose PTCy restores GVHD-preventing activity and maintains the GVL effect, which is enhanced by reducing the dose of PTCy. Our results provide important information for the development of a novel strategy to optimize PTCy-based transplantation, particularly in patients with a potential relapse risk.

KRN7000 Reduces Airway Inflammation via Natural Killer T Cells in Obese Asthmatic Mice.

Although natural killer T cells (NKT cells) are altered in obese asthmatic mice, their function remains completely unclear. To further explore the potential mechanism of NKT cells in airway inflammation of obesity-associated asthma, we examined the effects of α-galactosylceramide (KRN7000) on airway inflammation in obese asthmatic mice. Male C57BL/6J mice were divided into five groups: (1) control; (2) asthma; (3) A + KRN, asthma with KRN7000; (4) obese asthma; and (5) OA + KRN, obese asthma with KRN7000. Cytometric bead array (CBA) was used to detect interleukin-4 (IL-4), IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the serum. Flow cytometry was used to detect NKT cells and CD69+ NKT cells. Airway inflammation was observed in pathological sections, and calmodulin (CaM) expression was observed by immunohistochemistry in lung tissues. Airway inflammation in the obese asthma group was more severe than that of the asthma group. Airway inflammation of the OA + KRN group was reduced more than that of the A + KRN group. CD69+ NKT cells were only significantly reduced in the OA + KRN group. The levels of serum IFN-γ and TNF-α increased more in the OA + KRN group than in the A + KRN group. CaM is widely expressed in the cytoplasm of the lung tissues and was sharply decreased in the OA + KRN group. KRN7000 can significantly reduce airway inflammation in obesity-associated asthma by regulating NKT cell cytokine secretion and intracellular calcium. These results may contribute to the development of novel therapeutic approaches.

Using agonists for iNKT cells in cancer therapy.

The capacity of α-galactosylceramide (α-GalCer) to act as an anti-cancer agent in mice through the specific stimulation of type I NKT (iNKT) cells has prompted extensive investigation to translate this finding to the clinic. However, low frequencies of iNKT cells in cancer patients and their hypo-responsiveness to repeated stimulation have been seen as barriers to its efficacy. Currently the most promising clinical application of α-GalCer, or its derivatives, is as stimuli for ex vivo expansion of iNKT cells for adoptive therapy, although some encouraging clinical results have recently been reported using α-GalCer pulsed onto large numbers of antigen presenting cells (APCs). In on-going preclinical studies, attempts to improve efficacy of injected iNKT cell agonists have focussed on optimising presentation in vivo, through encapsulation in particulate vectors, making structural changes that help binding to the presenting molecule CD1d, or injecting agonists covalently attached to recombinant CD1d. Variations on these same approaches are being used to enhance the APC-licencing function of iNKT cells in vivo to induce adaptive immune responses to associated tumour antigens. Looking ahead, a unique capacity of in vivo-activated iNKT cells to facilitate formation of resident memory CD8+ T cells is a new observation that could find a role in cancer therapy.

A combination of check-point blockade and α-galactosylceramide elicits long-lasting suppressive effects on murine hepatoma cell growth in vivo.

Immunotherapy for cancer cells induced by interfering with PD-1/PD-L1 engagement via check-point blockades was initiated by tumour-specific PD-1+ CD8+ cytotoxic T lymphocytes (CTLs) within a tumour mass and eliminate the tumour. Here, we used C57BL/6 (B6) mice implanted with the syngeneic hepatoma cell line Hepa1-6-1, and confirmed that the dendritic cells (DCs) within Hepa1-6-1 tumour mass were tolerogenic with downmodulated co-stimulatory molecules by tumour-derived factors. Although Hepa1-6-1 cells did not prime tumour-specific CTLs within the tumour, specific CTLs primed in the regional lymph nodes seemed to be invaded into the tumour mass. The specific CTLs gained PD-1+ expression when associated with PD-L1+ Hepa1-6-1 cells within the tumour mass. Their cytotoxic activity in vivo was revitalised after intraperitoneal (i.p.) administration of the anti-PD-1 monoclonal antibody (mAb), indicating that PD-1/PD-L1 engagement within the tumour was abrogated by check-point blockade. Nonetheless, the tolerogenic DCs within the Hepa1-6-1 tumour mass remained tolerogenic even after three shots of PD-1-blockade administration, and the suppressed Hepa1-6-1 growth was revisited. In this study, we show here an excellent therapeutic effect consisting of three injections of anti-PD1 mAb and the sequential administration of the CD1d molecule-restricted ligand α-galactosylceramide (α-GalCer), an immuno-potent lipid/glycolipid, which converts tolerogenic DCs into immunogenic DCs with upregulated expression of co-stimulatory molecules. The α-GalCer-activated DCs secreted a large amount of IL-12, which can activate tumour-specific CTLs in vivo. The check-point blockade was not sufficiently effective, but the dose needed for tumour eradication was reduced by 90% when tumour-bearing mice were also administered i.p. α-GalCer.

Sublingual administration of liposomes enclosing alpha-galactosylceramide as an effective adjuvant of allergen immunotherapy in a murine model of allergic rhinitis.

BACKGROUND: Sublingual immunotherapy (SLIT) is an established efficacious approach for the treatment of allergic rhinitis (AR). However, SLIT requires a long administration period to establish stable and adequate responses. This study investigated the efficacy of the sublingual administration of an allergen with liposomes enclosing α-GalCer (α-GC-liposome) as a potential adjuvant in mice with AR. METHODS: Mice with AR induced by OVA received the sublingual administration of OVA, α-GC-liposomes, or OVA plus α-GC-liposomes for 7 days. After nasal re-challenge with OVA, nasal symptoms were evaluated. The serum levels of OVA-specific Ig, the cytokine production of CD4+ T cells in the cultures of cervical lymph node (CLN) cells, and the gene expression of CLNs were analyzed. RESULTS: Although IL-4, IL-5 and IL-13 production from CD4+ T cells in CLN cells was significantly inhibited by the sublingual administration of OVA alone in mice with AR induced by OVA, their nasal symptoms were not significantly diminished. However, the combined sublingual administration of α-GC-liposomes and OVA completely suppressed nasal symptoms, downregulated Th2 and Th17 type cytokine production in CD4+ T cells as well as Th2 and Th17 gene expressions, and upregulated Th1 type cytokine production as well as Th1 gene expressions in CLN cells. Additionally, the serum levels of specific IgG2a were promoted, and specific IgE and IgG1 were inhibited. CONCLUSIONS: Our findings suggest that the sublingual administration of an allergen with α-GC-liposomes as an adjuvant might increase the therapeutic efficacy and effectiveness of this treatment method.

Dendritic cells cross-talk with tumour antigen-specific CD8+ T cells, Vγ9γδT cells and Vα24NKT cells in patients with glioblastoma multiforme and in healthy donors.

The finding that dendritic cells (DCs) orchestrate innate and adaptive immune responses has stimulated research on harnessing DCs for developing more effective vaccines for DC therapy. The expression of cytomegalovirus (CMV) antigens in glioblastoma multiforme (GBM) presents a unique opportunity to target these viral proteins for tumour immunotherapy. Here, we demonstrate that Vγ9γδT cells, innate immune cells activated by zoledronate (Z) and Vα24 natural killer (Vα24NK) cells, innate/adaptive immune cells activated by α-galactosylceramide (G) can link innate and adaptive immunities through cross-talk with interferon (IFN) DCs from patients with glioblastoma multiforme (GBM) and healthy donors in a manner that can amplify the activation and proliferation of CMVpp65-specific CD8+ T cells. The IFN DCs derived from patients with GBM used in this study express lower levels of programmed cell death ligand (PD)-L1 and PD-L2 and higher levels of C-C receptor 7 (CCR7) than the most commonly used mature interleukin (IL)-4 DCs. The expression level of programmed cell death 1 (PD-1) on CD8+ T cells, including CMVpp65-specific CD8+ T cells, expanded by IFN DCs pulsed with the CMVpp65-peptide and Z plus G (IFN DCs/P+Z+G), was lower than that expanded by IFN DCs pulsed with the peptide alone (IFN DCs/P). Multi-functional T cells, including human leucocyte antigen (HLA)-A*0201-restricted CMVpp65-specific CD8+ T cells, Vγ9γδT cells and Vα24NKT cells, efficiently kill the HLA-A*0201-positive GBM cell line expressing CMVpp65 protein (T98G). These findings indicate that DC therapy using IFN DCs/P+Z+G and/or CTL therapy using CMVpp65-specific CD8+ T cells expanded by IFN DCs/P+Z+G may lead to a good clinical outcome for patients with GBM.

Activation of Invariant Natural Killer T Cells Redirects the Inflammatory Response in Neonatal Sepsis.

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-ß1. These findings support the importance of iNKT cells for prophylactic immunomodulation in neonates susceptible to sepsis.

Repeated administration of alpha-galactosylceramide ameliorates experimental lupus nephritis in mice.

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.

Evaluation of pH-sensitive fusogenic polymer-modified liposomes co-loaded with antigen and α-galactosylceramide as an anti-tumor vaccine.

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.

Augmenting Influenza-Specific T Cell Memory Generation with a Natural Killer T Cell-Dependent Glycolipid-Peptide Vaccine.

The development of a universal vaccine for influenza A virus (IAV) that does not require seasonal modification is a long-standing health goal, particularly in the context of the increasing threat of new global pandemics. Vaccines that specifically induce T cell responses are of considerable interest because they can target viral proteins that are more likely to be shared between different virus strains and subtypes and hence provide effective cross-reactive IAV immunity. From a practical perspective, such vaccines should induce T cell responses with long-lasting memory, while also being simple to manufacture and cost-effective. Here we describe the synthesis and evaluation of a vaccine platform based on solid phase peptide synthesis and bio-orthogonal conjugation methodologies. The chemical approach involves covalently attaching synthetic long peptides from a virus-associated protein to a powerful adjuvant molecule, α-galactosylceramide (α-GalCer). Strain-promoted azide-alkyne cycloaddition is used as a simple and efficient method for conjugation, and pseudoproline methodology is used to increase the efficiency of the peptide synthesis. α-GalCer is a glycolipid that stimulates NKT cells, a population of lymphoid-resident immune cells that can provide potent stimulatory signals to antigen-presenting cells engaged in driving proliferation and differentiation of peptide-specific T cells. When used in mice, the vaccine induced T cell responses that provided effective prophylactic protection against IAV infection, with the speed of viral clearance greater than that seen from previous viral exposure. These findings are significant because the vaccines are highly defined, quick to synthesize, and easily characterized and are therefore appropriate for large scale affordable manufacture.

A randomized phase II study to assess the effect of adjuvant immunotherapy using α-GalCer-pulsed dendritic cells in the patients with completely resected stage II-IIIA non-small cell lung cancer: study protocol for a randomized controlled trial.

BACKGROUND: As the toxicity associated with the α-GalCer-pulsed dendritic cell (DC) therapy could be considered to be negligible, its addition to postoperative adjuvant chemotherapy would be expected to greatly improve the therapeutic effect, and could result in prolonged survival. The aim of the present study is to compare the therapeutic efficacy of alpha-galactosylceramide-pulsed DC therapy in patients who have undergone a complete resection of stage II-IIIA non-small-cell lung cancer (NSCLC) followed by postoperative adjuvant therapy with cisplatin plus vinorelbine, to that in patients who did not receive additional treatment (surgical resection plus postoperative adjuvant chemotherapy only). METHODS: Subsequent to the complete resection of NSCLC, followed by the administration of cisplatin plus vinorelbine dual-agent combination adjuvant chemotherapy, patients who satisfy the inclusion criteria will be randomly allocated to either the α-GalCer-pulsed DC immune therapy group, or the standard treatment group. In total, 56 patients will be included in the study. The primary endpoint is recurrence-free survival, and the secondary endpoints are natural killer T-cell-specific immune response, the frequency of toxic effects and safety, and overall survival. DISCUSSION: In order to determine the efficacy of α-GalCer-pulsed DC therapy, the present study compares patients with stage II-III NSCLC who underwent complete surgical resection followed by postoperative adjuvant therapy with cisplatin plus vinorelbine, to those who did not receive additional treatment (surgical resection plus postoperative adjuvant chemotherapy only). TRIAL REGISTRATION: UMIN000010386 ( R000012145 ). Registered on 1 April 2013. UMIN-CTR is officially recognized as a registration site which satisfies ICMJE criteria.

Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis.

Immune-modulating drugs that target myeloid-derived suppressor cells or stimulate natural killer T cells have been shown to reduce mycobacterial loads in tuberculosis (TB). We aimed to determine if a combination of these drugs as adjunct immunotherapy to conventional antibiotic treatment could also increase therapeutic efficacy against TB. In our model of pulmonary TB in mice, we applied treatment with isoniazid, rifampicin, and pyrazinamide for 13 weeks alone or combined with immunotherapy consisting of all-trans retinoic acid, 1,25(OH)2-vitamin D3, and α-galactosylceramide. Outcome parameters were mycobacterial load during treatment (therapeutic activity) and 13 weeks after termination of treatment (therapeutic efficacy). Moreover, cellular changes were analyzed using flow cytometry and cytokine expression was assessed at the mRNA and protein levels. Addition of immunotherapy was associated with lower mycobacterial loads after 5 weeks of treatment and significantly reduced relapse of disease after a shortened 13-week treatment course compared with antibiotic treatment alone. This was accompanied by reduced accumulation of immature myeloid cells in the lungs at the end of treatment and increased TNF-α protein levels throughout the treatment period. We demonstrate, in a mouse model of pulmonary TB, that immunotherapy consisting of three clinically approved drugs can improve the therapeutic efficacy of standard antibiotic treatment.

Estrogen Therapy Delays Autoimmune Diabetes and Promotes the Protective Efficiency of Natural Killer T-Cell Activation in Female Nonobese Diabetic Mice.

Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18(-/-)) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18(-/-) mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention.

Natural killer T cells in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic inflammatory disease that causes demyelination of neurons in the central nervous system. Traditional therapies for MS have involved anti-inflammatory and immunosuppressive drugs with significant side effects that often only provide short-term relief. A more desirable outcome of immunotherapy would be to protect against disease before its clinical manifestation or to halt disease after its initiation. One attractive approach to accomplish this goal would be to restore tolerance by targeting immunoregulatory cell networks. Although much of the work in this area has focused on CD4(+) Foxp3(+) regulatory T cells, other studies have investigated natural killer T (NKT) cells, a subset of T cells that recognizes glycolipid antigens in the context of the CD1d glycoprotein. Studies with human MS patients have revealed alterations in the numbers and functions of NKT cells, which have been partially supported by studies with the experimental autoimmune encephalomyelitis model of MS. Additional studies have shown that activation of NKT cells with synthetic lipid antigens can, at least under certain experimental conditions, protect mice against the development of MS-like disease. Although mechanisms of this protection remain to be fully investigated, current evidence suggests that it involves interactions with other immunoregulatory cell types such as regulatory T cells and immunosuppressive myeloid cells. These studies have provided a strong foundation for the rational design of NKT-cell-based immunotherapies for MS that induce tolerance while sparing overall immune function. Nevertheless, additional pre-clinical and clinical studies will be required to bring this goal to fruition.

A self-adjuvanting vaccine induces cytotoxic T lymphocytes that suppress allergy.

Epitope-based peptide vaccines encompass minimal immunogenic regions of protein antigens to allow stimulation of precisely targeted adaptive immune responses. However, because efficacy is largely determined by the functional status of antigen-presenting cells (APCs) that acquire and present peptides to cells of the adaptive immune system, adjuvant compounds are needed to enhance immunogenicity. We present here a vaccine consisting of an allergen-derived peptide conjugated to a prodrug of the natural killer-like T (NKT) cell agonist α-galactosylceramide, which is highly effective in reducing inflammation in a mouse model of allergic airway inflammation. Unlike other peptide-adjuvant conjugates that directly activate APCs through pattern recognition pathways, this vaccine encourages third-party interactions with NKT cells to enhance APC function. Therapeutic efficacy was correlated with marked increases in the number and functional activity of allergen-specific cytotoxic T lymphocytes (CTLs), leading to suppression of immune infiltration into the lungs after allergen challenge in sensitized hosts.

α-Galactosylceramide but not phenyl-glycolipids induced NKT cell anergy and IL-33-mediated myeloid-derived suppressor cell accumulation via upregulation of egr2/3.

Strategies for cancer immunotherapy include activating immune system for therapeutic benefit or blockade of immune checkpoints. To harness innate immunity to fight cancer, α-galactosylceramide (α-GalCer) has been used to activate NKT cells. Unfortunately, administration of α-GalCer causes long-term NKT cell anergy, but the molecular mechanism is unclear. In this study, we showed that α-GalCer-triggered egr2/3, which induced programmed death 1 and cbl-b in NKT cells, leading to NKT cell anergy. We also uncovered the induction of the immunosuppressive myeloid-derived suppressor cells (MDSCs) in the spleen by α-GalCer that might attenuate its antitumor efficacy. The accumulation of MDSC was accompanied by 20-fold rise in their arg-1 mRNAs and enhanced expression of programmed death 1/programmed death ligand 1. Furthermore, α-GalCer-induced egr-2/3 in hepatic NKT cells upregulated their TRAIL in addition to Fas ligand (FasL) and induced alarm signaling molecule IL-33 in Kupffer cells, presumably because of liver damage triggered by TRAIL/FasL. We further demonstrated that IL-33-stimulated macrophages produce G-CSF, which in turn, boosted MDSCs. Thus, α-GalCer-induced FasL/TRAIL and IL-33 provided a novel mechanism underlying α-GalCer-induced hepatotoxicity and MDSC accumulation. In contrast, analogs of α-GalCer containing phenyl group in the lipid tail could neither induce NKT anergy nor enhance MDSCs accumulation. Furthermore, tumor-infiltrating MDSCs in mice injected repeatedly with α-GalCer were 2-fold higher than those treated with phenyl-glycolipids. These results not only revealed the induction of MDSC via IL-33 as a new mechanism for α-GalCer-elicited immunosuppression but also provided one of the mechanisms underlying the superior antitumor potency of phenyl-glycolipids. Our findings have important implications for the development of NKT-stimulatory glycolipids as vaccine adjuvants and anticancer therapeutics.

[Regulatory role of NKT cells in the prevention of type 1 diabetes]. / Rôle régulateur des lymphocytes NKT dans la prévention du diabète de type 1.

Type 1 diabetes is an autoimmune disease resulting from the destruction of pancreatic ß cells by the immune system. NKT cells are innate-like T cells that can exert potent immuno-regulatory functions. The regulatory role of NKT cells was initially proposed after the observed decreased frequency of this subset in mouse models of type 1 diabetes, as well as in patients developing various autoimmune pathologies. Increasing NKT cell frequency and function prevent the development of type 1 diabetes in mouse models. Several mechanisms including IL-4 and IL-10 production by NKT cells and the accumulation of tolerogenic dendritic cells are critical for the dampening of pathogenic anti-islet T cell responses by NKT cells. Importantly, these cells can at the same time prevent diabetes and promote efficient immune responses against infectious agents. These results strengthen the potential role of NKT cells as a key target for the development of therapeutic strategies against type 1 diabetes.

Intra-peritoneal hyperthermia combining α-galactosylceramide in the treatment of ovarian cancer.

The purpose of this study was to investigate the anti-tumor effect and potential mechanisms of i.p. hyperthermia in combination with α-galactosylceramide (α-GalCer) for the treatment of ovarian cancer. In this study, immuno-competent tumor models were established using murine ovarian cancer cell lines and treated with i.p. hyperthermia combining α-GalCer. Th1/Th2 cytokine expression profiles in the serum, NK cell cytotoxicity and phagocytic activities of dendritic cells (DCs) were assayed. We also analyzed the number of CD8(+)/IFN-γ(+) tumor specific cytotoxic T cells, as well as the tumor growth based on depletion of lymphocyte sub-population. Therapeutic effect on those ovarian tumors was monitored by a non-invasive luminescent imaging system. Intra-peritoneal hyperthermia induced significant pro-inflammatory cytokines expression, and sustained the response of NK and DCs induced by α-GalCer treatment. The combination treatment enhanced the cytotoxic T lymphocyte (CTL) immune response in two mouse ovarian cancer models. This novel treatment modality by combination of hyperthermia and glycolipid provides a pronounced anti-tumor immune response and better survival. In conclusion, intra-peritoneal hyperthermia enhanced the pro-inflammatory cytokine secretion and phagocytic activity of DCs stimulated by α-GalCer. The subsequent CTL immune response induced by α-GalCer was further strengthened by combining with i.p. hyperthermia. Both innate and adaptive immunities were involved and resulted in a superior therapeutic effect in treating the ovarian cancer.

Activation of invariant natural killer T cells by α-galactosylceramide ameliorates myocardial ischemia/reperfusion injury in mice.

Invariant natural killer T (iNKT) cells orchestrate tissue inflammation via regulating various cytokine productions. However the role of iNKT cells has not been determined in myocardial ischemia/reperfusion (I/R) injury. The purpose of this study was to examine whether the activation of iNKT cells by α-galactosylceramide (α-GC), which specifically activates iNKT cells, could affect myocardial I/R injury. I/R or sham operation was performed in male C57BL/6J mice. I/R mice received the injection of either αGC (I/R+αGC, n=48) or vehicle (I/R+vehicle, n=49) 30 min before reperfusion. After 24h, infarct size/area at risk was smaller in I/R+αGC than in I/R+vehicle (37.8 ± 2.7% vs. 47.1 ± 2.5%, P<0.05), with no significant changes in area at risk. The numbers of infiltrating myeloperoxidase- and CD3-positive cells were lower in I/R+αGC. Apoptosis evaluated by TUNEL staining and caspase-3 protein was also attenuated in I/R+αGC. Myocardial gene expression of tumor necrosis factor-α and interleukin (IL)-1ß in I/R+αGC was lower to 46% and 80% of that in I/R+vehicle, respectively, whereas IL-10, IL-4, and interferon (IFN)-γ were higher in I/R+αGC than I/R+vehicle by 2.0, 4.1, and 9.6 folds, respectively. The administration of anti-IL-10 receptor antibody into I/R+αGC abolished the protective effects of αGC on I/R injury (infarct size/area at risk: 53.1 ± 5.2% vs. 37.4 ± 3.5%, P<0.05). In contrast, anti-IL-4 and anti-IFN-γ antibodies did not exert such effects. In conclusion, activated iNKT cells by αGC play a protective role against myocardial I/R injury through the enhanced expression of IL-10. Therapies designed to activate iNKT cells might be beneficial to protect the heart from I/R injury.