Transnuclear mice reveal Peyer's patch iNKT cells that regulate B-cell class switching to IgG1.

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.

A phase I study of loco-regional immunotherapy by transbronchial injection of α-galactosylceramide-pulsed antigen presenting cells in patients with lung cancer.

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-ß was upregulated in progressive disease cases.

Fully Synthetic Invariant NKT Cell-Dependent Self-Adjuvanting Antitumor Vaccines Eliciting Potent Immune Response in Mice.

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.

Tim-3 blockade promotes iNKT cell function to inhibit HBV replication.

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.

Suppression of murine tumour growth through CD8+ cytotoxic T lymphocytes via activated DEC-205+ dendritic cells by sequential administration of α-galactosylceramide in vivo.

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.

Increased Foxp3+Helios+ Regulatory T Cells and Decreased Acute Graft-versus-Host Disease after Allogeneic Bone Marrow Transplantation in Patients Receiving Sirolimus and RGI-2001, an Activator of Invariant Natural Killer T Cells.

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.

α-Galactosylceramide-activated murine NK1.1(+) invariant-NKT cells in the myometrium induce miscarriages in mice.

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.

CD252 regulates mast cell mediated, CD1d-restricted NKT-cell activation in mice.

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.

Co-operation of α-galactosylceramide-loaded tumour cells and TLR9 agonists induce potent anti-tumour responses in a murine colon cancer model.

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.

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

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

Co-administration of α-GalCer analog and TLR4 agonist induces robust CD8(+) T-cell responses to PyCS protein and WT-1 antigen and activates memory-like effector NKT cells.

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.

RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells.

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.

CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation.

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.

Targeted delivery of α-galactosylceramide to CD8α+ dendritic cells optimizes type I NKT cell-based antitumor responses.

Immunotherapy aiming at enhancing innate and acquired host immunity is a promising approach for cancer treatment. The invariant NKT (iNKT) cell ligand α-galactosylceramide (α-GalCer) holds great promise in cancer therapy, although several concerns limit its use in clinics, including the uncontrolled response it promotes when delivered in a nonvectorized form. Therefore, development of delivery systems to in vivo target immune cells might be a valuable option to optimize iNKT cell-based antitumor responses. Using dendritic cell (DC)-depleted mice, DC transfer experiments, and in vivo active cell targeting, we show that presentation of α-GalCer by DCs not only triggers optimal primary iNKT cell stimulation, but also maintains secondary iNKT cell activation after challenge. Furthermore, targeted delivery of α-GalCer to CD8α(+) DCs, by means of anti-DEC205 decorated nanoparticles, enhances iNKT cell-based transactivation of NK cells, DCs, and γδ T cells. We report that codelivery of α-GalCer and protein Ag to CD8α(+) DCs triggers optimal Ag-specific Ab and cytotoxic CD8(+) T cell responses. Finally, we show that targeting nanoparticles containing α-GalCer and Ag to CD8α(+) DCs promotes potent antitumor responses, both in prophylactic and in therapeutic settings. Our data may have important implications in tumor immunotherapy and vaccine development.

Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury.

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.

Effects of Dendritic Cell Subset Manipulation on Airway Allergy in a Mouse 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.

BAFF- and APRIL-dependent maintenance of antibody titers after immunization with T-dependent antigen and CD1d-binding ligand.

CD1d-restricted invariant NKT (iNKT) cells boost humoral immunity to T-dependent Ags that are coadministered with the CD1d-binding glycolipid Ag α-galactosylceramide (α-GC). Observations that mice lacking iNKT cells have decaying Ab responses following vaccination have led to the hypothesis that iNKT cells express plasma cell (PC) survival factors that sustain specific Ab titers. Bone marrow chimeric mice in which the entire hematopoietic compartment or iNKT cells selectively lacked BAFF, a proliferation-inducing ligand (APRIL), or both BAFF and APRIL were created and immunized with nitrophenol hapten-conjugated keyhole limpet hemocyanin adsorbed to Imject aluminum hydroxide-containing adjuvant or mixed with α-GC. In comparison with BAFF- or APRIL-sufficient bone marrow chimeras, absence of hematopoietic compartment- and iNKT-derived BAFF and APRIL was associated with rapidly decaying Ab titers and reduced PC numbers. The iNKT cell-derived BAFF or APRIL assumed a greater role in PC survival when α-GC was used as the adjuvant for immunization. These results show that iNKT cell-derived BAFF and APRIL each contribute to survival of PCs induced by immunization. This study sheds new light on the mechanisms through which iNKT cells impact humoral immunity and may inform design of vaccines that incorporate glycolipid adjuvants.

Activation of invariant natural killer T cells by lipid excess promotes tissue inflammation, insulin resistance, and hepatic steatosis in obese mice.

Obesity triggers a low-grade systemic inflammation, which plays an important role in the development of obesity-associated metabolic diseases. In searching for links between lipid accumulation and chronic inflammation, we examined invariant natural killer T (iNKT) cells, a subset of T lymphocytes that react with lipids and regulate inflammatory responses. We show that iNKT cells respond to dietary lipid excess and become activated before or at the time of tissue recruitment of inflammatory leukocytes, and that these cells progressively increase proinflammatory cytokine production in obese mice. Such iNKT cells skew other leukocytes toward proinflammatory cytokine production and induce an imbalanced proinflammatory cytokine environment in multiple tissues. Further, iNKT cell deficiency ameliorates tissue inflammation and provides protection against obesity-induced insulin resistance and hepatic steatosis. Conversely, chronic iNKT cell stimulation using a canonical iNKT cell agonist exacerbates tissue inflammation and obesity-associated metabolic disease. These findings place iNKT cells into the complex network linking lipid excess to inflammation in obesity and suggest new therapeutic avenues for obesity-associated metabolic disorders.

A novel approach inducing transplant tolerance by activated invariant natural killer T cells with costimulatory blockade.

Invariant natural killer T (iNKT) cells are one of the innate lymphocytes that regulate immunity, although it is still elusive how iNKT cells should be manipulated for transplant tolerance. Here, we describe the potential of a novel approach using a ligand for iNKT cells and suboptimal dosage of antibody for CD40-CD40 ligand (L) blockade as a powerful method for mixed chimerism establishment after allogenic bone marrow transplantation in sublethally irradiated fully allo recipients. Mixed-chimera mice accepted subsequent cardiac allografts in a donor-specific manner. High amounts of type 2 helper T cytokines were detected right after iNKT cell activation, while subsequent interferon-gamma production by NK cells was effectively inhibited by CD40/CD40L blockade. Tolerogenic components, such as CD11c(low) mPDCA1(+) plasmacytoid dendritic cells and activated regulatory T cells (Tregs) expressing CD103, KLRG-1 and PD-1, were subsequently augmented. Those activating Tregs seem to be required for the establishment of chimerism because depletion of the Tregs 1 day before allogenic cell transfer resulted in a chimerism brake. These results collectively suggest that our new protocol makes it possible to induce donor-specific tolerance by enhancement of the innate ability for immune tolerance in place of the conventional immunosuppression.

Innate immunity drives xenobiotic-induced murine autoimmune cholangitis.

Although primary biliary cirrhosis (PBC) is considered a model autoimmune disease, it has not responded therapeutically to traditional immunosuppressive agents. In addition, PBC may recur following liver transplantation, despite the absence of major histocompatibility complex (MHC) matching, in sharp contrast to the well-known paradigm of MHC restriction. We have suggested previously that invariant natural killer T (iNK T) cells are critical to the initiation of PBC. In this study we have taken advantage of our ability to induce autoimmune cholangitis with 2-octynoic acid, a common component of cosmetics, conjugated to bovine serum albumin (2-OA-BSA), and studied the natural history of pathology in mice genetically deleted for CD4 or CD8 following immunization with 2-OA-BSA in the presence or absence of α-galactosylceramide (α-GalCer). In particular, we address whether autoimmune cholangitis can be induced in the absence of traditional CD4 and CD8 responses. We report herein that CD4 and CD8 knock-out mice immunized with 2-OA-BSA/PBS or 2-OA-BSA/α-GalCer develop anti-mitochondrial antibodies (AMAs), portal infiltrates and fibrosis. Indeed, our data suggest that the innate immunity is critical for immunopathology and that the pathology is exacerbated in the presence of α-GalCer. In conclusion, these data provide not only an explanation for the recurrence of PBC following liver transplantation in the absence of MHC compatibility, but also suggest that effective therapies for PBC must include blocking of both innate and adaptive pathways.