Activation Of Invariant NKT Cells Research Articles

α-Galatosylceramide and retinoic acid regulate splenic follicular B cell differentiation and antibody production in a CD1d-dependent manner

Abstract α-Galactosylceramide (αGalCer) and all-trans-retinoic acid (RA) are each potent immune regulators able to increase immune responses in various conditions. αGalCer is particularly known for its direct activation of invariant NKT cells and B cells. In the present study, we used wildtype (WT) and CD1d knockout (KO) mice to characterize the specificity of αGalCer and its interaction with RA on B cell activation. WT and CD1dKO spleen cells were cultured ± αGalCer (100 ng/ml), or LPS (100 ng/ml) as a direct B-cell stimulus, and RA (20 nM), and B cell proliferation and differentiation were determined by flow cytometry. αGalCer and LPS both markedly increased the CD19+ B cell division. Whereas αGalCer mainly enriched spleen follicular CD23+ B cells, LPS increased the marginal zone CD21+ B cell population. The effects of αGalCer were completely absent in CD1dKO mice. However, although spleen B cells of CD1dKO mice failed to respond to αGalCer, these cells had a higher rate of proliferation compared to cells of WT mice, suggesting that CD1d molecules may have immune tonic effects in addition to reacting to αGalCer. Moreover, RA worked together with αGalCer to increase the proportions of IgG+ and CD138+ cells among CD23+ B cells, suggesting enhanced B-cell differentiation in splenic follicles. An in vivo immunization study using Keyhole limpet hemocyanin (KLH, 50 μg, ip) further confirmed that administration of RA (37 μg, po) and αGalCer (2 μg, sc), given at the time of primary immunization, increased antibody production in both the primary and secondary responses, which was not observed in CD1dKO mice. Thus, immune enhancement by αGalCer requires CD1d, and, when combined with RA promotes follicular B cell differentiation and increased antibody production.

Antigen-dependent versus -independent activation of invariant NKT cells during infection.

CD1d-reactive invariant NKT cells (iNKT) play a vital role in determining the characteristics of immune responses to infectious agents. Previous reports suggest that iNKT cell activation during infection can be: 1) solely driven by cytokines from innate immune cells, 2) require microbial Ag, or 3) require self-Ag. In this study, we examined the role of Ag receptor stimulation in iNKT cells during several bacterial and viral infections. To test for Ag receptor signaling, Nur77(gfp) BAC transgenic mice, which upregulate GFP in response to Ag receptor but not inflammatory signals, were analyzed. iNKT cells in the reporter mice infected with mouse CMV produced IFN-γ but did not upregulate GFP, consistent with their reported CD1d-independent activation. However, two bacteria known to produce lipid Ags for iNKT cells induced GFP expression and cytokine production. In contrast, although Salmonella typhimurium was proposed to induce the presentation of a self-lipid, iNKT cells produced IFN-γ but did not upregulate GFP postinfection in vivo. Even in CD1d-deficient hosts, iNKT cells were still able to produce IFN-γ after S. typhimurium infection. Furthermore, although it has been proposed that endogenous lipid presentation is a result of TLR stimulation of APCs, injection of different TLR agonists led to iNKT cell IFN-γ but not increased GFP expression. These data indicate that robust iNKT cell responses to bacteria, as well as viruses, can be obtained in the absence of antigenic stimulation.

Activation of Invariant NKT Cells with Glycolipid Ligand α-Galactosylceramide Ameliorates Glucose-6-Phosphate Isomerase Peptide-Induced Arthritis

ObjectiveInvariant natural killer T (iNKT) cells regulate collagen-induced arthritis (CIA) when activated by their potent glycolipid ligand, alpha-galactosylceramide (α-GalCer). Glucose-6-phosphate isomerase (GPI)-induced arthritis is a closer model of human rheumatoid arthritis based on its association with CD4+ T cells and cytokines such as TNF-α and IL-6 than CIA. Dominant T cell epitope peptide of GPI (GPI325-339) can induce arthritis similar to GPI-induced arthritis. In this study, we investigated the roles of activation of iNKT cells by α-GalCer in GPI peptide-induced arthritis.MethodsArthritis was induced in susceptible DBA1 mice with GPI peptide and its severity was assessed clinically. The arthritic mice were treated with either the vehicle (DMSO) or α-GalCer. iNKT cells were detected in draining lymph nodes (dLNs) by flow cytometry, while serum anti-GPI antibody levels were measured by enzyme-linked immunosorbent assay. To evaluate GPI peptide-specific cytokine production from CD4+ T cells, immunized mice were euthanized and dLN CD4+ cells were re-stimulated by GPI-peptide in the presence of antigen-presenting cells.Resultsα-GalCer induced iNKT cell expansion in dLNs and significantly decreased the severity of GPI peptide-induced arthritis. In α-GalCer-treated mice, anti-GPI antibody production (total IgG, IgG1, IgG2b) and IL-17, IFN-γ, IL-2, and TNF-α produced by GPI peptide-specific T cells were significantly suppressed at day 10. Moreover, GPI-reactive T cells from mice immunized with GPI and α-GalCer did not generate any cytokines even when these cells were co-cultured with APC from mice immunized with GPI alone. In vitro depletion of iNKT cells did not alter the suppressive effect of α-GalCer on CD4+ T cells.Conclusionα-GalCer significantly suppressed GPI peptide-induced arthritis through the suppression of GPI-specific CD4+ T cells.

Activation of Invariant NKT Cells in Early Phase of Experimental Autoimmune Encephalomyelitis Results in Differentiation of Ly6Chi Inflammatory Monocyte to M2 Macrophages and Improved Outcome

Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome.

The adenosine A2A receptor agonist regadenoson decreases activation of invariant NKT cells in human sickle cell subjects

Sickle cell disease (SCD) is associated with widely disseminated vaso‐occlusion caused by hyper‐adherent sickle red blood cells and leukocytes. Recurrent episodes of ischemia reperfusion‐injury in the vasculature of SCD individuals promote a pro‐inflammatory state. In a mouse model of SCD, adenosine A2A receptor (A2AR) activation significantly decreases pulmonary inflammation and dysfunction. Similar protective effects are produced by depleting invariant NKT (iNKT) cells or by blocking CD1d restricted activation of iNKT cells, indicating that A2AR agonists mediate their anti‐inflammatory effects primarily by inhibiting iNKT cell activation. We have initiated a clinical trial to investigate the safety and efficacy of a selective A2AR agonist, regadenoson, in human SCD subjects. Our findings indicate that iNKT cells in blood from normal subjects are fewer in number and less activated than from subjects with SCD. Furthermore, activated iNKT cells from SCD subjects are CD4+ and express elevated levels of phospho‐NFκB (P‐NFκB), CD69, and the A2AR. Most significantly, infused regadenoson was observed to diminish iNKT cell expression of P‐NFκB and other activation markers in a time dependent manner. These results imply that A2AR signaling and NFκB regulate iNKT cell activation and that selective A2AR agonists may be useful for treating acute vaso‐occlusion in SCD.

W1794 Activation of Invariant NKT Cells Prevents the Growth of Colitis-Associated Colorectal Cancers in Mice

W1794 Activation of Invariant NKT Cells Prevents the Growth of Colitis-Associated Colorectal Cancers in Mice

A CD1d-Dependent Antagonist Inhibits the Activation of Invariant NKT Cells and Prevents Development of Allergen-Induced Airway Hyperreactivity

The prevalence of asthma continues to increase in westernized countries, and optimal treatment remains a significant therapeutic challenge. Recently, CD1d-restricted invariant NKT (iNKT) cells were found to play a critical role in the induction of airway hyperreactivity (AHR) in animal models and are associated with asthma in humans. To test whether iNKT cell-targeted therapy could be used to treat allergen-induced airway disease, mice were sensitized with OVA and treated with di-palmitoyl-phosphatidyl-ethanolamine polyethylene glycol (DPPE-PEG), a CD1d-binding lipid antagonist. A single dose of DPPE-PEG prevented the development of AHR and pulmonary infiltration of lymphocytes upon OVA challenge, but had no effect on the development of OVA-specific Th2 responses. In addition, DPPE-PEG completely prevented the development of AHR after administration of alpha-galactosylceramide (alpha-GalCer) intranasally. Furthermore, we demonstrate that DPPE-PEG acts as antagonist to alpha-GalCer and competes with alpha-GalCer for binding to CD1d. Finally, we show that DPPE-PEG completely inhibits the alpha-GalCer-induced phosphorylation of ERK tyrosine kinase in iNKT cells, suggesting that DPPE-PEG specifically blocks TCR signaling and thus activation of iNKT cells. Because iNKT cells play a critical role in the development of AHR, the inhibition of iNKT activation by DPPE-PEG suggests a novel approach to treat iNKT cell-mediated diseases such as asthma.

Activation of invariant NKT cells confers protection against Chlamydia trachomatis-induced arthritis

The role of invariant NKT (iNKT) cells in reactive arthritis is unknown. We explored the functional role of NKT cells in reactive arthritis using an established murine model of Chlamydia trachomatis-induced arthritis (CtIA). CtIA in wild-type and CD1d knockout (KO) mice was induced by intra-articular injection of C. trachomatis. The effect of alpha-galactosylceramide (alpha-GalCer) activation of iNKT cells was investigated by intra-peritoneal administration of alpha-GalCer. Histopathological and phenotypic changes, chlamydial clearance and cytokine and chemokine production in synovial tissue of the knee joint were investigated after onset of the arthritis. The severity of CtIA was significantly increased in CD1d KO mice, which was associated with decrease in bactericidal cytokine IFN-gamma, regulatory cytokines IL-4 and IL-10 and increase in pro-inflammatory chemokines macrophage inflammatory protein-2 (MIP-2) and IFN-gamma-inducible protein-10 (IP-10). Local clearance of the pathogen from the joint was also decreased. Prior treatment of mice with alpha-GalCer, a potent activator of iNKT cells, significantly reduced the severity of CtIA in mice. The amelioration of CtIA was associated with decrease in chlamydial load and induction of cytokines IFN-gamma, IL-4 and IL-10 and significant suppression of MIP-2 and IP-10. Treatment of established CtIA with alpha-GalCer also demonstrated modulation of CtIA and decrease in chlamydial load. These results suggest that iNKT cells are protective against CtIA and alpha-GalCer-activated iNKT cells have an immunoregulatory role not only in preventing the induction of reactive arthritis but also in modulating established disease.

TNF driven activation of invariant NKT cells favours regulation of combined gut and joint inflammation. (134.22)

Abstract Invariant natural killer T (iNKT) cells are a subset of glycolipid reactive T cells, implicated in the protection against microbial pathogens and immune regulation. While the pathways leading to iNKT cell activation in the context of infectious diseases have been uncovered, much less is known how iNKT cells become activated in the context of chronic inflammatory diseases. Here, we unveil a novel mode of iNKT cell activation which occurs during chronic TNF driven inflammation through generation of inflammatory dendritic cells. This in turn, leads to a spontaneous activation of iNKT cells by promoting CD1d mediated presentation of endogenous glycolipids. This mode of activation is capable of attenuating combined Crohn's like ileitis and joint inflammation occurring in TNFΔARE mice, and thus may represent a natural counter regulatory mechanism to dampen TNF driven inflammation.

Activation of Invariant NKT Cells Ameliorates Experimental Ocular Autoimmunity by A Mechanism Involving Innate IFN-γ Production and Dampening of the Adaptive Th1 and Th17 Responses

Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with alpha-C-GalCer, which induces a strong IFN-gamma but only a weak IL-4 production by iNKT cells, in contrast to the ligands alpha-GalCer (both IFN-gamma and IL-4) and OCH (primarily IL-4). The protective effect of alpha-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-gamma and IL-17 production and was negated by systemic neutralization of IFN-gamma. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-gamma and a consequent dampening of the Th1 as well as the Th17 effector responses.

Activation of invariant NKT cells enhances the innate immune response and improves the disease course in influenza A virus infection

Invariant NKT (iNKT) cells have an indubitable role in antiviral immunity, although the mechanisms by which these cells exert their functions are not fully elucidated. With the emerging importance of high-pathogenicity influenza A virus infections in humans, we questioned whether iNKT cells contribute to immune defence against influenza A virus and whether activation of these cells influences outcome. We show that activation of iNKT cells with alpha-galactosylceramide (alpha-GC) during influenza virus infection transiently enhanced early innate immune response without affecting T cell immunity, and reduced early viral titres in lungs of C57BL/6 mice. This is accompanied by a better disease course with improved weight loss profile. Temporal changes in iNKT cells in the liver, blood and lungs suggest activation and migration of iNKT cells from the liver to the lungs in mice that were administered alpha-GC. Improvement in viral titres appears dependent on activation of iNKT cells via the intraperitoneal route since intranasal administration of alpha-GC did not have the same effect. We conclude that activation of iNKT cells enhances early innate immune response in the lungs and contribute to antiviral immunity and improved disease course in influenza A virus infection.

Characterization of the immature dendritic cells and cytotoxic cells both expanded after activation of invariant NKT cells with α-galactosylceramide in vivo

Invariant natural killer T (iNKT) cells can perform multiple functions characteristic of both innate and acquired immunity. Activation of iNKT cells in vivo by repeated α-GalCer injections can induce immune tolerance, but the mechanisms responsible for such immunoregulation remain unclear. We prepared α-GalCer-liposomes, a single injection of which into mice resulted in the expansion of splenic CD11c lowCD45RB high cells, which consists of two populations, CD180 + and CD49b +. Expansion of these cells was not observed in α-GalCer-liposome-treated mice deficient in IL-10 or iNKT cells. MHC and co-stimulatory molecules were down-regulated in CD11c lowCD180 + cells compared with conventional dendritic cells (cDCs), suggesting that the former possess characteristics of immature DCs. Meanwhile, the CD11c lowCD49b + cells expressed IL-10 and Ctla4, and possessed greater lytic activity than resting NK cells. These observations suggest that both immature DCs (CD11c lowCD180 +) and cytotoxic cells (CD11c lowCD49b +) might be expanded by α-GalCer-activated iNKT cells and could therefore be involved in immune tolerance.

Activation of Invariant NKT Cells by Toll-like Receptor 9-Stimulated Dendritic Cells Requires Type I Interferon and Charged Glycosphingolipids

Invariant natural killer T (iNKT) cells are a subset of innate lymphocytes that recognize lipid antigens in the context of CD1d and mediate potent immune regulatory functions via the rapid production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). We investigated whether diverse Toll-like receptor (TLR) signals in myeloid dendritic cells (DCs) could differentially stimulate iNKT cells. Together with the lipopolysaccharide-detecting receptor TLR4, activation of the nucleic acid sensors TLR7 and TLR9 in DCs were particularly potent in stimulating iNKT cells to produce IFN-gamma, but not IL-4. iNKT cell activation in response to TLR9 stimulation required combined synthesis of type I interferon and de novo production of charged beta-linked glycosphingolipid(s) by DCs. In addition, DCs stimulated via TLR9 activated both iNKT cells and NK cells in vivo and protected mice against B16F10-induced melanoma metastases. These data underline the role of TLR9 in iNKT cell activation and might have relevance to infectious diseases and cancer.

Activation of invariant NKT cells by alphaGalCer administration protects mice from MOG35-55-induced EAE: critical roles for administration route and IFN-gamma.

Invariant NKT (inv. NKT) cells co-express an invariant alpha beta T cell receptor and the NK receptor NK1.1 and, upon CD1d-restricted recognition of the glycosphingolipid antigen alpha-galactosyl ceramide (alphaGalCer), secrete large amounts of regulatory cytokines. We investigated whether alphaGalCer-dependent activation of inv. NKT cells protects from experimental autoimmune encephalomyelitis (EAE), an immune-mediated disease of the central nervous system mimicking multiple sclerosis, induced in C57BL/6 mice by the myelin oligodendrocyte glycoprotein (MOG) encephalitogenic peptide aa 35-55. alphaGalCer was administered at the time of immunization s.c., mixed with complete Freund's adjuvant and MOG35-55 peptide, or administered i.p., diluted in PBS. EAE onset was delayed and disease severity was decreased only when alphaGalCer was s.c. administered. The protective effect of s.c. administration of alphaGalCer was associated with a markedly enhanced IFN-gamma production by liver-confined inv. NKT cells which, in turn, suppressed Th1-cytokine production and fostered secretion of IL-10 from MOG35-55-specific T cells. In vivo neutralization of IFN-gamma, but notIL-4, reversed the protective effect induced by s.c. administration of alphaGalCer, further confirming the critical regulatory role exerted by IFN-gamma-producing inv. NKT cells. Our results indicate that alphaGalCer, properly administered, may elicit an inv. NKT-cell-mediated suppressive effect on the effector function of encephalitogenic T cells; this effect is able to ameliorate autoimmunedemyelination.