Sulfatide-activated type II NKT cells prevent allergic airway inflammation by inhibiting type I NKT cell function in a mouse model of asthma

Asthma is a common chronic inflammatory disease involving many different cell types. Recently, type I natural killer T (NKT) cells have been demonstrated to play a crucial role in the development of asthma. However, the roles of type II NKT cells in asthma have not been investigated before. Interestingly, type I and type II NKT cells have been shown to have opposing roles in antitumor immunity, antiparasite immunity, and autoimmunity. We hypothesized that sulfatide-activated type II NKT cells could prevent allergic airway inflammation by inhibiting type I NKT cell function in asthma. Strikingly, in our mouse model, activation of type II NKT cells by sulfatide administration and adoptive transfer of sulfatide-activated type II NKT cells result in reduced-inflammation cell infiltration in the lung and bronchoalveolar lavage fluid, decreased levels of IL-4 and IL-5 in the BALF; and decreased serum levels of ovalbumin-specific IgE and IgG1. Furthermore, it is found that the activation of sulfatide-reactive type II NKT cells leads to the functional inactivation of type I NKT cells, including the proliferation and cytokine secretion. Our data reveal that type II NKT cells activated by glycolipids, such as sulfatide, may serve as a novel approach to treat allergic diseases and other disorders characterized by inappropriate type I NKT cell activation.     

Cross-regulation between distinct natural killer T cell subsets influences immune response to self and foreign antigens

Natural killer T (NKT) cells generally recognize lipid-antigens presented in the context of the MHC class I-like molecule CD1d. CD1d-restricted NKT cells consist of two broad subsets: Type I, which express an invariant T cell receptor (TCR) and type II, which utilize diverse TCR gene segments. A major type II NKT subset has been shown to recognize a self-glycolipid, sulfatide. Both subsets play important roles in autoimmune diseases, tumor surveillance, and infectious diseases. While type I NKT cells protect from tumor growth by enhancing tumor surveillance, type II NKT cells may suppress anti-tumor immune responses. In a murine autoimmune hepatitis model, type I NKT cells contribute to pathogenesis, whereas activation of sulfatide-reactive type II NKT cells protects from disease. Sulfatide-mediated activation of type II NKT cells results in modification of dendritic cells and induction of anergy in type I NKT cells. Elucidation of this novel pathway of cross-regulation among NKT cell subsets will provide tools for intervention in autoimmune diseases and for designing strategies for effective anti-tumor immunity.     

The immunoregulatory role of type I and type II NKT cells in cancer and other diseases

NKT cells are CD1d-restricted T cells that recognize lipid antigens. They also have been shown to play critical roles in the regulation of immune responses. In the immune responses against tumors, two subsets of NKT cells, type I and type II, play opposing roles and cross-regulate each other. As members of both the innate and adaptive immune systems, which form a network of multiple components, they also interact with other immune components. Here, we discuss the function of NKT cells in tumor immunity and their interaction with other regulatory cells, especially CD4⁺CD25⁺Foxp3⁺ regulatory T cells.     

A novel immunoregulatory axis of NKT cell subsets regulating tumor immunity

There are many mechanisms that regulate and dampen the immune response to cancers, including several types of regulatory T cells. Besides the T reg cell, we have identified another immunoregulatory circuit initiated by NKT cells that produce IL-13 in response to tumor growth and this IL-13 then induces myeloid cells to make TGF-beta that inhibits cytotoxic T cell-mediated tumor immunosurveillance in several mouse tumor models. This finding created a paradox in the role of NKT cells in tumor immunity, in that they can also contribute to protection. We resolve this paradox by the finding that the suppressive NKT cell is a type II NKT cell that lacks the canonical invariant T cell receptor, whereas the protective cell is a type I NKT cell that expresses the invariant receptor. Further, we see that these two subsets of NKT cells counter-regulate each other, defining a new immunoregulatory axis. The balance along this axis may determine the outcome of tumor immunosurveillance as well as influence the efficacy of anti-cancer vaccines and immunotherapy.     

Gene Therapy against Murine Melanoma B16F10-Nex2 Using IL-13Ralpha2-Fc Chimera and Interleukin 12 in Association with a Cyclopalladated Drug

Interleukin 13 (IL-13) is immunoregulatory in many diseases, including cancer. The protective or suppressive role of CD1-restricted natural killer T cells (NKT cells) in tumor immunosurveillance and immunity is well documented. Interleukin 12 (IL-12) can activate type I NKT cells to produce interferon-gamma (IFN-gamma), whereas type II NKT cells may produce IL-13. The high-affinity chain of IL-13Ralpha2 may act as negative inhibitor, suppressing the action of IL-13 and helping to maintain tumor immunosurveillance. We constructed an mIL-13Ralpha2-Fc chimera in a eukaryotic expression vector and confirmed the identity of the recombinant protein by immunoblot analysis and binding to IL-13 in chemiluminescent ELISA. Such DNA vaccine was tested against syngeneic B16F10-Nex2 murine melanoma. In vivo experiments showed a protective effect mediated by high production of IFN-gamma and down-regulation of anti-inflammatory interleukins mainly by NKT 1.1(+) T cells. Biochemoterapy in vivo with plasmid encoding mIL-13Ralpha2-Fc in association with plasmid encoding IL-12 and the 7A cyclopalladated drug led to a significant reduction in the tumor evolution with 30% tumor-free mice. We conclude that IL-12 gene therapy, followed by continuous administration of IL-13Ralpha2-Fc gene along with 7A-drug has antitumor activity involving the high production of proinflammatory cytokines and low immune suppression, specifically by NK1.1(+)T cells producing IL-13 and IL-10.     

CD4(+) type II NKT cells mediate ICOS and programmed death-1-dependent regulation of type 1 diabetes

Type 1 diabetes (T1D) is a chronic autoimmune disease that results from T cell-mediated destruction of pancreatic β cells. CD1d-restricted NKT lymphocytes have the ability to regulate immunity, including autoimmunity. We previously demonstrated that CD1d-restricted type II NKT cells, which carry diverse TCRs, prevented T1D in the NOD mouse model for the human disease. In this study, we show that CD4(+) 24αβ type II NKT cells, but not CD4/CD8 double-negative NKT cells, were sufficient to downregulate diabetogenic CD4(+) BDC2.5 NOD T cells in adoptive transfer experiments. CD4(+) 24αβ NKT cells exhibited a memory phenotype including high ICOS expression, increased cytokine production, and limited display of NK cell markers, compared with double-negative 24αβ NKT cells. Blocking of ICOS or the programmed death-1/programmed death ligand 1 pathway was shown to abolish the regulation that occurred in the pancreas draining lymph nodes. To our knowledge, these results provide for the first time cellular and molecular information on how type II CD1d-restricted NKT cells regulate T1D.     

Involvement of secretory and endosomal compartments in presentation of an exogenous self-glycolipid to type II NKT cells

Natural Killer T (NKT) cells recognize both self and foreign lipid Ags presented by CD1 molecules. Although presentation of the marine sponge-derived lipid alphaGalCer to type I NKT cells has been well studied, little is known about self-glycolipid presentation to either type I or type II NKT cells. Here we have investigated presentation of the self-glycolipid sulfatide to a type II NKT cell that specifically recognizes a single species of sulfatide, namely lyso-sulfatide but not other sulfatides containing additional acyl chains. In comparison to other sulfatides or alphaGalCer, lyso-sulfatide binds with lower affinity to CD1d. Although plate-bound CD1d is inefficient in presenting lyso-sulfatide at neutral pH, it is efficiently presented at acidic pH and in the presence of saposin C. The lysosomal trafficking of mCD1d is required for alphaGalCer presentation to type I NKT cells, it is not important for presentation of lyso-sulfatide to type II NKT cells. Consistently, APCs deficient in a lysosomal lipid-transfer protein effectively present lyso-sulfatide. Presentation of lyso-sulfatide is inhibited in the presence of primaquine, concanamycin A, monensin, cycloheximide, and an inhibitor of microsomal triglyceride transfer protein but remains unchanged following treatment with brefeldin A. Wortmannin-mediated inhibition of lipid presentation indicates an important role for the PI-3kinase in mCD1d trafficking. Our data collectively suggest that weak CD1d-binding self-glycolipid ligands such as lyso-sulfatide can be presented via the secretory and endosomal compartments. Thus this study provides important insights into the exogenous self-glycolipid presentation to CD1d-restricted T cells.     

NK细胞和NKT细胞发育的转录调控

自然杀伤（natural killer,NK）细胞和自然杀伤T（natural killer T,NKT）细胞是参与机体抗病毒免疫和肿瘤免疫的两群淋巴细胞亚群,是介导先天性免疫（innate immunity）应答和调节适应性免疫（adaptive immunity）应答的重要效应细胞。近年来,随着对NK细胞和NKT细胞及其转录调控因子研究的不断深入,NK细胞和NKT细胞的发育机制逐步被阐明,这将为提高NK细胞和NKT细胞的抗病毒和肿瘤免疫疗效提供新的策略。     

CD1d-expressing breast cancer cells modulate NKT cell-mediated antitumor immunity in a murine model of breast cancer metastasis

Background

Tumor tolerance and immune suppression remain formidable obstacles to the efficacy of immunotherapies that harness the immune system to eradicate breast cancer. A novel syngeneic mouse model of breast cancer metastasis was developed in our lab to investigate mechanisms of immune regulation of breast cancer. Comparative analysis of low-metastatic vs. highly metastatic tumor cells isolated from these mice revealed several important genetic alterations related to immune control of cancer, including a significant downregulation of cd1d1 in the highly metastatic tumor cells. The cd1d1 gene in mice encodes the MHC class I-like molecule CD1d, which presents glycolipid antigens to a specialized subset of T cells known as natural killer T (NKT) cells. We hypothesize that breast cancer cells, through downregulation of CD1d and subsequent evasion of NKT-mediated antitumor immunity, gain increased potential for metastatic tumor progression.

Methodology/Principal Findings

In this study, we demonstrate in a mouse model of breast cancer metastasis that tumor downregulation of CD1d inhibits iNKT-mediated antitumor immunity and promotes metastatic breast cancer progression in a CD1d-dependent manner in vitro and in vivo. Using NKT-deficient transgenic mouse models, we demonstrate important differences between type I and type II NKT cells in their ability to regulate antitumor immunity of CD1d-expressing breast tumors.

Conclusions/Significance

The results of this study emphasize the importance of determining the CD1d expression status of the tumor when tailoring NKT-based immunotherapies for the prevention and treatment of metastatic breast cancer.     

Role of natural killer T cells in host defence against cryptococcal infection

Cryptococcosis is an opportunistic fungal infectious disease that often occurs in severely immunocompromised patients. Host defence against the causative microorganism is largely mediated by cellular immunity, and Th1 cytokines, such as IFN-gamma, play central roles in the host protective responses. IL-12 and IL-18 activate the synthesis of IFN-gamma by innate immune cells, including NK, NKT and gamma delta T cells and promote the differentiation of Th1-type acquired immune responses. Recently, NKT cells, which are involved in the recognition of glycolipid antigens, have attracted much attention based on their potent immunomodulating activities. Several studies have reported the role of this particular component of innate immune responses in tumor immunity and pathogenesis of autoimmune diseases. In this review, I outline the recent findings on the role of NKT cells in host defence against infectious microorganisms, with a special focus on our data emphasizing the importance of this subset of immunocytes in the development of acquired as well as early host protection against cryptococcal infection.     

Cutting edge: inhibition of experimental tumor metastasis by dendritic cells pulsed with alpha-galactosylceramide.

A unique lymphoid lineage, Valpha14 NKT cells, bearing an invariant Ag receptor encoded by Valpha14 and Jalpha281 gene segments, play crucial roles in various immune responses, including protective immunity against malignant tumors. A specific ligand of Valpha14 NKT cells is determined to be alpha-galactosylceramide (alpha-GalCer) which is presented by the CD1d molecule. Here, we report that dendritic cells (DCs) pulsed with alpha-GalCer effectively induce potent antitumor cytotoxic activity by specific activation of Valpha14 NKT cells, resulting in the inhibition of tumor metastasis in vivo. Moreover, a complete inhibition of B16 melanoma metastasis in the liver was observed when alpha-GalCer-pulsed DCs were injected even 7 days after transfer of tumor cells to syngeneic mice where small but multiple metastatic nodules were already formed. The potential utility of DCs pulsed with alpha-GalCer for tumor immunotherapy is discussed.     

Natural killer T cells: rapid responders controlling immunity and disease

Natural killer T (NKT) cells are a subset of T cells that share properties of natural killer cells and conventional T cells. They are involved in immediate immune responses, tumor rejection, immune surveillance and control of autoimmune diseases. Most NKT cells express both an invariant T cell antigen receptor and the NK cell receptor NK1.1, and are referred to as invariant NKT cells. This invariant T cell receptor is restricted to interactions with glycolipids presented by the non-classical MHC, CD1d. These NKT cells rapidly produce high levels of interleukin (IL)-2, IFN-gamma, TNF-alpha, and IL-4 upon stimulation through their TCR. Most also have cytotoxic activity similar to NK cells. NKT cells are involved in a number of pathological conditions, and have been shown to regulate viral infections in vivo, and control tumor growth. They may also play both protective and harmful roles in the progression of certain autoimmune diseases, such as diabetes, lupus, atherosclerosis, and allergen-induced asthma.     

NKT cells stimulated by long fatty acyl chain sulfatides significantly reduces the incidence of type 1 diabetes in nonobese diabetic mice

Sulfatide-reactive type II NKT cells have been shown to regulate autoimmunity and anti-tumor immunity. Although, two major isoforms of sulfatide, C16:0 and C24:0, are enriched in the pancreas, their relative role in autoimmune diabetes is not known. Here, we report that sulfatide/CD1d-tetramer(+) cells accumulate in the draining pancreatic lymph nodes, and that treatment of NOD mice with sulfatide or C24:0 was more efficient than C16:0 in stimulating the NKT cell-mediated transfer of a delay in onset from T1D into NOD.Scid recipients. Using NOD.CD1d(-/-) mice, we show that this delay of T1D is CD1d-dependent. Interestingly, the latter delay or protection from T1D is associated with the enhanced secretion of IL-10 rather than IFN-g by C24:0-treated CD4(+) T cells and the deviation of the islet-reactive diabetogenic T cell response. Both C16:0 and C24:0 sulfatide isoforms are unable to activate and expand type I iNKT cells. Collectively, these data suggest that C24:0 stimulated type II NKT cells may regulate protection from T1D by activating DCs to secrete IL-10 and suppress the activation and expansion of type I iNKT cells and diabetogenic T cells. Our results raise the possibility that C24:0 may be used therapeutically to delay the onset and protect from T1D in humans.     

Dynamic roles of type I and type II IFNs in early infection with Mycobacterium tuberculosis

Although the protective role of type II IFN, or IFN-γ, against Mycobacterium tuberculosis has been established, the effects of type I IFNs are still unclear. One potential confounding factor is the overlap of function between the two signaling pathways. We used mice carrying null mutations in the type I IFNR, type II IFNR, or both and compared their immune responses to those of wild-type mice following aerosol infection with M. tuberculosis. We discovered that, in the absence of a response to IFN-γ, type I IFNs play a nonredundant protective role against tuberculosis. Mice unable to respond to both types of IFNs had more severe lung histopathology for similar bacterial loads and died significantly earlier than did mice with impaired IFN-γ signaling alone. We excluded a role for type I IFN in T cell recruitment, which was IFN-γ dependent, whereas both types of IFNs were required for optimal NK cell recruitment to the lungs. Type I IFN had a time-dependent influence on the composition of lung myeloid cell populations, in particular by limiting the abundance of M. tuberculosis-infected recruited macrophages after the onset of adaptive immunity. We confirmed that response to IFN-γ was essential to control intracellular mycobacterial growth, without any additional effect of type I IFN. Together, our results imply a model in which type I IFN limit the number of target cells that M. tuberculosis can infect in the lungs, whereas IFN-γ enhances their ability to restrict bacterial growth.     

NKT cells in tumor immunity: opposing subsets define a new immunoregulatory axis

NKT cells are true Ag-specific T cells that also have innate properties and form a bridge between the innate and adaptive immune systems. Distinct NKT cell subsets play positive and negative regulatory roles and define a new immunoregulatory axis with broad implications for tumor immunity and other immunological and disease settings.     

Donor bone marrow type II (non-Valpha14Jalpha18 CD1d-restricted) NKT cells suppress graft-versus-host disease by producing IFN-gamma and IL-4

NKT cells in donor bone marrow (BM) have been demonstrated to protect against graft-vs-host disease (GVHD) following BM transplantation. Murine NKT cells are divided into two distinct subsets based on the invariant Valpha14Jalpha18 TCR expression. However, details of the subset and mechanisms of the BM NKT cells involved in suppressing GVHD have not been clarified. Irradiated BALB/c or C3H/HeN mice administered B6 or Jalpha18(-/-) BM cells show attenuation of GVHD, whereas recipients given CD1d(-/-) BM cells did not show attenuation. Moreover, coinjection of BM non-Valpha14Jalpha18 CD1d-restricted (type II) NKT cells and CD1d(-/-) BM cells suppressed GVHD, whereas coinjection of BM Valpha14Jalpha18 TCR (type I) NKT cells did not. These protective effects on GVHD depended upon IFN-gamma-producing type II NKT cells, which induced the apoptosis of donor T cells. The splenocytes of mice administered BM cells from B6.IL-4(-/-) or Jalpha18(-/-)IL-4(-/-) mice produced lower levels of IL-4 and IL-10 than the splenocytes of mice transplanted with BM cells from B6, B6.IFN-gamma(-/-), Jalpha18(-/-), or Jalpha18(-/-)IFN-gamma(-/-) mice. Taken together, our results show that IFN-gamma-producing BM type II NKT cells suppress GVHD by inducing the apoptosis of donor T cells, while IL-4-producing BM type II NKT cells protect against GVHD by deviating the immune system toward a Th2-type response.     

Impairment of Vα24-Jα18+Vβ11+ natural killer T cells in adult acute lymphoblastic leukemia patients

Type I natural killer T (NKT) cells are attractive candidates for cancer immunotherapy. In this study, we examined the characteristics of type I NKT cells in patients with adult B-cell acute lymphoblastic leukemia (ALL). We first identified type I NKT cells as Vα24-Jα18 and Vβ11 double-positive CD3⁺ lymphocytes. Using this method, we found that the adult B-cell ALL patients presented significantly lower level of type I NKT cells than the age- and sex-matching control subjects. The expression of IL-21 by type I NKT cells was then examined using intracellular flow cytometry, which showed that with α-GalCer stimulation, the adult B-cell ALL patients presented significantly lower level of IL-21⁺ type I NKT cells than control subjects. By both flow cytometry and ELISA, we found that the vast majority of IL-21-expressing type I NKT cells expressed IL-21R, which was also reduced in adult B-cell ALL patients. Using an in vitro co-culture system, we demonstrated that IL-21R⁺, but not IL-21R^-, type I NKT cells could promote the IFN-γ, granzyme B, and perforin expression by CD8 T cells in an IL-21-dependent fashion. This type I NKT cell-mediated stimulatory effect was reduced in adult B-cell ALL patients than in control subjects. In addition, we observed a positive correlation between the frequency of IL-21R⁺ type I NKT cells and the frequencies of IFN-γ-, granzyme B-, and perforin-expressing circulating CD8 T cells in adult B-cell ALL patients directly ex vivo. Overall, this study identified an IL-21-related impairment in type I NKT cells from adult B-cell ALL patients.     

Cross-talk between NKT and regulatory T cells (Tregs) in modulation of immune response in patients with colorectal cancer following different pain management techniques

Natural killer T (NKT) and regulatory T cells (Tregs) play an important role in innate immune response. Natural killer (NK) and NKT cells are indispensable factors in the body's ongoing defense against tumor development, as well as viral infection. NKT cells are a subset of T cells that shares properties of natural killer cells and conventional T cells. They are involved in innate immune responses, tumor rejection, post transplantation immunotherapy, immune surveillance and control of autoimmune diseases. They may also play both protective and harmful roles in the progression of certain autoimmune diseases, such as diabetes, lupus, atherosclerosis, and allergen-induced asthma. Immune surveillance involves the process whereby precancerous and malignant cells are recognized by the host immune system as damaged and are consequently targeted for elimination. The pharmacological management of postoperative pain in patients with malignancies uses very different techniques whose possible cytotoxic functions we still known very poor. The present study compared effects of two different postoperative pain management techniques in patients undergoing colorectal cancer surgery on the innate immunity. Our data indicate that the patients with colorectal cancer have significantly increased the percentage of Tregs and NKT cells. The values were statistically higher during epidural analgesia in comparison with intravenous analgesia, indicating that epidural pain management technique ameliorate the immune suppression after surgery.     

IL-21 is produced by NKT cells and modulates NKT cell activation and cytokine production

The common gamma-chain cytokine, IL-21, is produced by CD4(+) T cells and mediates potent effects on a variety of immune cells including NK, T, and B cells. NKT cells express the receptor for IL-21; however, the effect of this cytokine on NKT cell function has not been studied. We show that IL-21 on its own enhances survival of NKT cells in vitro, and IL-21 increases the proliferation of NKT cells in combination with IL-2 or IL-15, and particularly with the CD1d-restricted glycosphingolipid Ag alpha-galactosylceramide. Similar to its effects on NK cells, IL-21 enhances NKT cell granular morphology, including granzyme B expression, and some inhibitory NK receptors, including Ly49C/I and CD94. IL-21 also enhanced NKT cell cytokine production in response to anti-CD3/CD28 in vitro. Furthermore, NKT cells may be subject to autocrine IL-21-mediated stimulation because they are potent producers of this cytokine following in vitro stimulation via CD3 and CD28, particularly in conjunction with IL-12 or following in vivo stimulation with alpha-galactosylceramide. Indeed, NKT cells produced much higher levels of IL-21 than conventional CD4 T cells in this assay. This study demonstrates that NKT cells are potentially a major source of IL-21, and that IL-21 may be an important factor in NKT cell-mediated immune regulation, both in its effects on NK, T, and B cells, as well as direct effects on NKT cells themselves. The influence of IL-21 in NKT cell-dependent models of tumor rejection, microbial clearance, autoimmunity, and allergy should be the subject of future investigations.