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1.  Defective NKT Cell Activation by CD1d+ TRAMP Prostate Tumor Cells Is Corrected by Interleukin-12 with alpha-Galactosylceramide 
PLoS ONE  2010;5(6):e11311.
Numerical and functional defects of invariant natural killer T cells (iNKT) have been documented in human and mouse cancers, resulting in a defect in IFN production in several malignancies. iNKT cells recognize glycolipids presented on CD1d molecules by dendritic and related cells, leading to their activation and thereby regulating immune reactions. Activated iNKT cells cytokine secretion and cytotoxicity can inhibit existing and spontaneous tumor growth, progression, and metastasis. We have identified functional iNKT cell defects in the murine TRAMP prostate cancer model. We found that iNKT cells show the ability to migrate into TRAMP prostate tumors. This infiltration was mediated through CCL2: CCR5 chemokine: receptor interaction. Prostate tumor cells expressing CD1d partially activated iNKT cells, as appreciated by up-regulation of CD25, PD-1 and the IL-12R. However, despite inducing up-regulation of these activation markers and, hence, delivering positive signals, prostate tumor cells inhibited the IL-12-induced STAT4 phosphorylation in a cell-cell contact dependent but CD1d-independent manner. Consequently, tumor cells did not induce secretion of IFNγ by iNKT cells. Blocking the inhibitory Ly49 receptor on iNKT cells in the presence of α-GalCer restored their IFNγ production in vivo and in vitro. However, Ly49 blockade alone was not sufficient. Importantly, this defect could be also be reversed into vigorous secretion of IFNγ by the addition of both IL-12 and the exogenous CD1d ligand alpha-galactosylceramide, but not by IL-12 alone, both in vivo and in vitro. These data underscore the potential to optimize iNKT-based therapeutic approaches.
PMCID: PMC2892484  PMID: 20593019
2.  Development and Function of Invariant Natural Killer T Cells Producing TH2- and TH17-Cytokines 
PLoS Biology  2012;10(2):e1001255.
Four distinct subsets of invariant natural killer T (NKT) cells are shown to differentiate in the thymus, then migrate to peripheral tissues where they retain their phenotypic and functional characteristics.
There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in TH2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB+ iNKT cells are present in the thymic CD44+/− NK1.1− population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB− iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB+ and IL-17RB− subsets. The IL-17RB+ iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4+ IL-17RB+ iNKT cells produce TH2 (IL-13), TH9 (IL-9 and IL-10), and TH17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4− IL-17RB+ iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt+ subset producing TH17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB+ iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB+ iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases.
Author Summary
T cells are a diverse group of immune cells involved in cell-mediated acquired immunity. One subset of T cells is the innate-like invariant natural killer T (iNKT) cells that recognize glycolipid ligands on target cells instead of peptides. We know that functionally distinct subtypes of iNKT cells are involved in specific pathologies, yet their development, phenotypes, and functions are not well understood. Here, we determine the relationship between various mouse iNKT cell subsets, identify reliable molecular markers for these subsets, and show that these contribute to their functional differences. We identify four iNKT cell subsets that we show arise via different developmental pathways and exhibit different cytokine profiles. Importantly, we show that these subsets can be isolated from the thymus (the organ of all T cells), as well as from peripheral tissues such as spleen, liver, lung, and lymph nodes. Contrary to the general understanding that iNKT cells mature after their exit from the thymus and their migration into peripheral tissues, we conclude that distinct phenotypic and functional iNKT cell subsets can be distinguished in the thymus by virtue of the presence or absence of the cytokine receptor IL-17RB and another cell surface molecule called CD4, and these subsets then migrate to peripheral tissues where they retain their phenotypic and functional characteristics. Regarding functional significance, we show that those iNKT cell subsets that lead to airway hyper-responsiveness to respiratory viruses are different to those that lead to allergen-induced airway hyperreactivity, which will enable researchers to focus on specific subsets as potential targets for therapeutic intervention.
PMCID: PMC3274505  PMID: 22346732
3.  Innate-Like Control of Human iNKT Cell Autoreactivity via the Hypervariable CDR3β Loop 
PLoS Biology  2010;8(6):e1000402.
T-cell receptor variability gives rise to a functional hierarchy of human invariant Natural Killer T-cells through a powerful effect on CD1d binding affinity, which is independent of CD1d ligands.
Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3β, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3β loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3β in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist α-linked glycolipid antigen OCH and structurally different endogenous β-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3β sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3β for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3β dependent functional hierarchy of human iNKT cells.
Author Summary
Our immune system uses randomly modified T-cell receptors (TCRs) to adapt its discriminative capacity to rapidly changing pathogens. The T-cell receptor (TCR) has six flexible, variable peptide loops that make contact with antigens presented to them on the surface of other cells. Invariant Natural Killer T-cells (iNKT) are regulatory T-cells with a unique type of TCR (iNKT-TCR) that recognizes lipid antigens presented by specific MHC-like molecules known as CD1d. In human iNKT-TCRs, only one of the six loops, CDR3beta, is variable. By comparing how different human iNKT clones bind and react to different CD1d-lipid complexes we uncover the existence of a hierarchical order of the human iNKT cell repertoire in which strongly CD1d-binding clones are autoreactive while weak CD1d-binding clones are non-autoreactive. Direct measurements of iNKT-TCR binding to CD1d using surface plasmon resonance recapitulated this hierarchy at the protein level. The data show that variation in the CDR3beta loop conveys dramatic differences in human iNKT TCR affinity that are independent of the CD1d bound ligand. Thus the CDR3beta loop provides the structural basis for the functional hierarchy of the human iNKT repertoire. We postulate that during the life-course, CDR3beta-dependent asymmetrical activation of different human iNKT clones leads to a bias in the iNKT repertoire, and this could result in age-dependent defects of iNKT-mediated immune regulation in later life.
PMCID: PMC2889927  PMID: 20585371
4.  Invariant NKT Cell Response to Dengue Virus Infection in Human 
Dengue viral infection is a global health threat without vaccine or specific treatment. The clinical outcome varies from asymptomatic, mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF). While adaptive immune responses were found to be detrimental in the dengue pathogenesis, the roles of earlier innate events remain largely uninvestigated. Invariant natural killer T (iNKT) cells represent innate-like T cells that could dictate subsequent adaptive response but their role in human dengue virus infection is not known. We hypothesized that iNKT cells play a role in human dengue infection.
Blood samples from a well-characterized cohort of children with DF, DHF, in comparison to non-dengue febrile illness (OFI) and healthy controls at various time points were studied. iNKT cells activation were analyzed by the expression of CD69 by flow cytometry. Their cytokine production was then analyzed after α-GalCer stimulation. Further, the CD1d expression on monocytes, and CD69 expression on conventional T cells were measured.
iNKT cells were activated during acute dengue infection. The level of iNKT cell activation associates with the disease severity. Furthermore, these iNKT cells had altered functional response to subsequent ex vivo stimulation with α-GalCer. Moreover, during acute dengue infection, monocytic CD1d expression was also upregulated and conventional T cells also became activated.
iNKT cells might play an early and critical role in the pathogenesis of severe dengue viral infection in human. Targeting iNKT cells and CD1d serve as a potential therapeutic strategy for severe dengue infection in the future.
Author Summary
Almost half of the world population is at risk of dengue viral infection. The disease severity varies from mild to a deadly form-which is caused mainly by host overt immune reaction. Earlier studies focused on the disease-causing roles of adaptive immune cells - cells that are highly specific but require time and signal from rapidly activating immune cells to become active. Invariant Natural Killer T (iNKT) cells are unique T cells that get activated rapidly and can control later adaptive response by secreting cytokines. They can be activated by lipid loaded on CD1d, an antigen presenting molecule. However, their role in human dengue infection was not known. Here, we studied iNKT cells from dengue infected children and found that they were activated. Importantly, the more severe the disease, the higher level of iNKT cells activation. Their cytokine patterns also differ from those of healthy donors. Moreover, together with iNKT cells activation, the level of CD1d was higher and T cells became active. Therefore, iNKT cells likely play a role in the pathogenesis of human dengue infection. New drugs targeting iNKT cells might help dampen the disease severity before the adaptive immune cells become too active.
PMCID: PMC4063705  PMID: 24945350
5.  Amide Analogues of CD1d Agonists Modulate iNKT-Cell-Mediated Cytokine Production 
ACS Chemical Biology  2012;7(5):847-855.
Invariant natural killer T (iNKT) cells are restricted by the non-polymorphic MHC class I-like protein, CD1d, and activated following presentation of lipid antigens bound to CD1d molecules. The prototypical iNKT cell agonist is α-galactosyl ceramide (α-GalCer). CD1d-mediated activation of iNKT cells by this molecule results in the rapid secretion of a range of pro-inflammatory (Th1) and regulatory (Th2) cytokines. Polarization of the cytokine response can be achieved by modifying the structure of the glycolipid, which opens up the possibility of using CD1d agonists as therapeutic agents for a range of diseases. Analysis of crystal structures of the T-cell receptor−α-GalCer–CD1d complex led us to postulate that amide isosteres of known CD1d agonists should modulate the cytokine response profile upon iNKT-cell activation. To this end, we describe the synthesis and biological activity of amide analogues of α-GalCer and its non-glycosidic analogue threitol ceramide (ThrCer). All of the analogues were found to stimulate murine and human iNKT cells by CD1d-mediated presentation to varying degrees; however, the thioamide and carbamate analogues of ThrCer were of particular interest in that they elicited a strongly polarized cytokine response (more interferon-gamma (IFN-γ), no interleukin-4 (IL-4)) in mice. While the ThrCer-carbamate analogue was shown to transactivate natural killer (NK) cells, a mechanism that has been used to account for the preferential production of IFN-γ by other CD1d agonists, this pathway does not account for the polarized cytokine response observed for the thioamide analogue.
PMCID: PMC3409616  PMID: 22324848
6.  Contact-dependent interference with invariant NKT cell activation by HSV-infected cells 
Invariant CD1d-restricted natural killer T (iNKT) cells play important roles in generating protective immune responses against infections. Here, we have investigated the role of human iNKT cells in HSV-1 infection and their interaction with epidermal keratinocytes. These cells express CD1d and are the primary target of the virus. Keratinocytes loaded with α-GalCer could stimulate IFN-γ production and CD25 upregulation by iNKT cells. However, both α-GalCer- and cytokine-dependent activation of iNKT cells was impaired after coculture with HSV-1-infected cells. Interestingly, CD1d downregulation was not observed on infected keratinocytes, which were also found to inhibit TCR-independent iNKT cell activation. Further examination of the cytokine profile of iNKT/keratinocyte cocultures showed inhibition of IFN-γ, IL-5, IL-10, IL-13 and IL-17 secretion, but upregulation of IL-4 and TNF-α following the infection. Moreover, cell-to-cell contact between infected keratinocytes and iNKT cells was required for the inhibition of activation, as the cell-free supernatants containing virus did not affect activation. Productive infection of iNKT cells was however not required for the inhibitory effect. Following coculture with infected cells, iNKT cells were no longer responsive to further stimulation with α-GalCer-loaded CD1d. We found that exposure to HSV-1-infected cells resulted in impaired TCR signalling downstream of ZAP70. Additionally, infected cells upregulated the expression of the negative T cell regulator, galectin-9; however, blocking experiments indicated that the impairment of iNKT cell responses was independent of galectin-9. Thus, interference with activation of human iNKT cells by HSV-1 may represent a novel immunoevasive strategy utilized by the virus to avoid immune clearance.
PMCID: PMC3370127  PMID: 22581860
7.  Therapeutic Targeting the Diverse Immunologic Functions Expressed by Hepatic NKT cells 
NKT cells comprise approximately 30% of the hepatic lymphoid population in mice (~50% in humans). Most mouse hepatic NKT cells [invariant (i)NKT cells] express T cell receptors, composed of invariant Vα14Jα18 chains. Unlike conventional T cells, iNKT cells recognize glycolipid molecules presented in association with MHC class Ib (CD1d) molecules. Purportedly, iNKT cells serve a key function in a wide range of immunological events; the precise nature of this function is often unclear. Indeed, the consequences of hepatic iNKT cell activation can be beneficial or detrimental. α-Galactosylceramide, the prototypic glycolipid recognized by the iNKT cell receptor, stimulates the rapid production of both interferon-γ and interleukin-4. The reciprocal suppression exhibited by these cytokines limits the potential therapeutic value of α-galactosylceramide. An extensive research effort is ongoing to develop α-galactosylceramide analogs that modulate iNKT cell activity and selectively promote interferon-γ or interleukin-4.
Areas covered
This review provides a broad overview of hepatic iNKT cells and their purported role in liver disease. Efforts to develop therapeutic agents that promote their beneficial contributions are detailed.
Expert Opinion
While a growing body of literature documents the differential effects of α-GalCer analogs on IFN-γ and IL-4 production, the effects of these analogs on other iNKT cell activities, e.g., cytolysis and the production of other cytokines, remain to be determined. Similarly, an exhaustive examination of the effects of these analogs on inflammation and liver injury in animal models remains prior to considering their utility in clinical trials.
PMCID: PMC3133853  PMID: 21564001
8.  Invariant natural killer T cells from food allergic versus non-allergic children exhibit differential responsiveness to milk-derived sphingomyelin 
A key immunological feature of food allergy (FA) is the presence of a T-helper-2 (Th2)-type cytokine bias. Ligation of the invariant natural killer T cell (iNKT) T cell receptor (TCR) by sphingolipids (SL) presented via the CD1d molecule leads to copious secretion of Th2-type cytokines. Major food allergens (e.g. milk, egg) are the richest dietary source of SL (food-SL). Nonetheless, the role of iNKTs in FA is unknown.
To investigate the role of iNKTs in FA and to assess whether food-SL-CD1d complexes can engage the iNKT-TCR and induce iNKT cell functions.
Peripheral blood mononuclear cells from 15 children allergic to cow's milk (FA-MA), 12 children tolerant to cow's milk but with allergy to egg (FA-NMA) and 13 healthy controls were incubated with α-galactosylceramide (αGal), cow's milk-sphingomyelin-[SM] or hen's egg-ceramide-[CE]. iNKTs were quantified and their cytokine production and proliferation were assessed. Human CD1d tetramers loaded with milk-SM or egg-CE were used to determine food-SL binding to the iNKT-TCR.
Milk-SM, but not egg-CE, can engage the iNKT-TCR and induce iNKT-proliferation and Th2-type cytokine secretion. FA-children, especially those with MA, had significantly fewer peripheral blood (PB) iNKTs and their iNKTs exhibited a greater Th2-response to αGal and milk-SM compared to iNKTs of healthy controls.
iNKTs from FA-children, especially those with MA, are reduced in number and exhibit a Th2-bias in response to αGal and milk-SM. These data suggest a potential role for iNKTs in FA.
Clinical Implications
Milk-SM activate PB-iNKTs to produce Th2-cytokines and this effect is greater in FA-MA-children. Hence, SL contained in milk may promote an iNKT cell-mediated-Th2-type-cytokine bias that facilitates sensitization to food allergens.
PMCID: PMC3129401  PMID: 21458849
Food allergy; invariant natural killer T cells; sphingolipids
9.  iNKT Cell Production of GM-CSF Controls Mycobacterium tuberculosis 
PLoS Pathogens  2014;10(1):e1003805.
Invariant natural killer T (iNKT) cells are activated during infection, but how they limit microbial growth is unknown in most cases. We investigated how iNKT cells suppress intracellular Mycobacterium tuberculosis (Mtb) replication. When co-cultured with infected macrophages, iNKT cell activation, as measured by CD25 upregulation and IFNγ production, was primarily driven by IL-12 and IL-18. In contrast, iNKT cell control of Mtb growth was CD1d-dependent, and did not require IL-12, IL-18, or IFNγ. This demonstrated that conventional activation markers did not correlate with iNKT cell effector function during Mtb infection. iNKT cell control of Mtb replication was also independent of TNF and cell-mediated cytotoxicity. By dissociating cytokine-driven activation and CD1d-restricted effector function, we uncovered a novel mediator of iNKT cell antimicrobial activity: GM-CSF. iNKT cells produced GM-CSF in vitro and in vivo in a CD1d-dependent manner during Mtb infection, and GM-CSF was both necessary and sufficient to control Mtb growth. Here, we have identified GM-CSF production as a novel iNKT cell antimicrobial effector function and uncovered a potential role for GM-CSF in T cell immunity against Mtb.
Author Summary
Mycobacterium tuberculosis (Mtb) is the cause of tuberculosis, a leading cause of sickness and death worldwide. Although much is known about CD4+ and CD8+ T cell responses to Mtb, the role of other T cell subsets is poorly understood. Invariant natural killer T (iNKT) cells are innate lymphocytes that express a semi-invariant T cell receptor and recognize lipid antigens presented by CD1d. Although iNKT cells participate in the immune response to many different pathogens, little is known about how iNKT cells directly kill microbes. We previously showed that when co-cultured with Mtb-infected macrophages, iNKT cells inhibit intracellular Mtb replication. Now, we used this model to dissociate the signals that induce iNKT cell activation markers including IFNγ production, from the signals that activate iNKT cell antimicrobial activity. This allowed us to uncover a novel antimicrobial effector function produced by iNKT cells: GM-CSF. GM-CSF is essential for immunity to Mtb, but its role has never been defined. This study is the first report to demonstrate a protective function of GM-CSF production by any T cell subset during Mtb infection. T cell production of GM-CSF should be considered as a potential mechanism of antimicrobial immunity.
PMCID: PMC3879349  PMID: 24391492
10.  Identification of distinct human invariant natural killer T-cell response phenotypes to alpha-galactosylceramide 
BMC Immunology  2008;9:71.
Human CD1d-restricted, invariant natural killer T cells (iNKT) are a unique class of T lymphocytes that recognise glycolipid antigens such as α-galactosylceramide (αGalCer) and upon T cell receptor (TCR) activation produce both Th1 and Th2 cytokines. iNKT cells expand when cultured in-vitro with αGalCer and interleukin 2 (IL-2) in a CD1d-restricted manner. However, the expansion ratio of human iNKT cells varies between individuals and this has implications for attempts to manipulate this pathway therapeutically. We have studied a panel of twenty five healthy human donors to assess the variability in their in-vitro iNKT cell expansion responses to stimulation with CD1d ligands and investigated some of the factors that may influence this phenomenon.
Although all donors had comparable numbers of circulating iNKT cells their growth rates in-vitro over 14 days in response to a range of CD1d ligands and IL-2 were highly donor-dependent. Two reproducible donor response patterns of iNKT expansion were seen which we have called 'strong' or 'poor' iNKT responders. Donor response phenotype did not correlate with age, gender, frequency of circulating iNKT, or with the CD1d ligand utilised. Addition of exogenous recombinant human interleukin 4 (IL-4) to 'poor' responder donor cultures significantly increased their iNKT proliferative capacity, but not to levels equivalent to that of 'strong' responder donors. However in 'strong' responder donors, addition of IL-4 to their cultures did not significantly alter the frequency of iNKT cells in the expanded CD3+ population.
(i) in-vitro expansion of human iNKT cells in response to CD1d ligand activation is highly donor variable, (ii) two reproducible patterns of donor iNKT expansion were observed, which could be classified into 'strong' and 'poor' responder phenotypes, (iii) donor iNKT response phenotypes did not correlate with age, gender, frequency of circulating iNKT cells, or with the CD1d ligand utilised, (iv) addition of IL-4 to 'poor' but not 'strong' responder donor cultures significantly increased their in-vitro iNKT cell expansion to αGalCer.
PMCID: PMC2613383  PMID: 19055753
11.  Distinct and Overlapping Effector Functions of Expanded Human CD4+, CD8α+ and CD4-CD8α- Invariant Natural Killer T Cells 
PLoS ONE  2011;6(12):e28648.
CD1d-restricted invariant natural killer T (iNKT) cells have diverse immune stimulatory/regulatory activities through their ability to release cytokines and to kill or transactivate other cells. Activation of iNKT cells can protect against multiple diseases in mice but clinical trials in humans have had limited impact. Clinical studies to date have targeted polyclonal mixtures of iNKT cells and we proposed that their subset compositions will influence therapeutic outcomes. We sorted and expanded iNKT cells from healthy donors and compared the phenotypes, cytotoxic activities and cytokine profiles of the CD4+, CD8α+ and CD4−CD8α− double-negative (DN) subsets. CD4+ iNKT cells expanded more readily than CD8α+ and DN iNKT cells upon mitogen stimulation. CD8α+ and DN iNKT cells most frequently expressed CD56, CD161 and NKG2D and most potently killed CD1d+ cell lines and primary leukemia cells. All iNKT subsets released Th1 (IFN-γ and TNF-α) and Th2 (IL-4, IL-5 and IL-13) cytokines. Relative amounts followed a CD8α>DN>CD4 pattern for Th1 and CD4>DN>CD8α for Th2. All iNKT subsets could simultaneously produce IFN-γ and IL-4, but single-positivity for IFN-γ or IL-4 was strikingly rare in CD4+ and CD8α+ fractions, respectively. Only CD4+ iNKT cells produced IL-9 and IL-10; DN cells released IL-17; and none produced IL-22. All iNKT subsets upregulated CD40L upon glycolipid stimulation and induced IL-10 and IL-12 secretion by dendritic cells. Thus, subset composition of iNKT cells is a major determinant of function. Use of enriched CD8α+, DN or CD4+ iNKT cells may optimally harness the immunoregulatory properties of iNKT cells for treatment of disease.
PMCID: PMC3236218  PMID: 22174854
Invariant Natural Killer T 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). Here 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 antigen (Ag), IRBP. 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 α-C-GalCer, which induces a strong IFN-γ but only a weak IL-4 production by iNKT cells, in contrast to the ligands α-GalCer (both IFN-γ and IL-4) and OCH (primarily IL-4). The protective effect of α-C-Gal-Cer was associated with a reduction of adaptive Ag specific IFN-γ and IL-17 production and was negated by systemic neutralization of IFN-γ. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-γ and a consequent dampening of the Th1 as well as the Th17 effector responses.
PMCID: PMC2597425  PMID: 18802082
NKT cells; autoimmune disease; cytokines
13.  Generation of Antitumor Invariant NKT Cell Lines in Multiple Myeloma and Promotion of Their Functions via Lenalidomide: A Strategy for Immunotherapy 
CD1d-restricted invariant NKT (iNKT) cells are important immunoregulatory cells in antitumor immune responses. However, the quantitative and qualitative defects of iNKT cells in advanced multiple myeloma (MM) hampered their antitumor effects. Therefore, the development of functional iNKT cells may provide a novel strategy for the immunotherapy in MM treatment.
Experimental Design
We activated and expanded iNKT cells from MM patients with α-galactosylceramide(α-GalCer)-pulsed-dendritic cells (DCs), characterized their antitumor effects by the cytokine production profile and cytotoxicity against MM cells, and explored the effects of immunomodulatory drug lenalidomide on these iNKT cells. We also investigated the expression of CD1d by primary MM cells and its function to activate iNKT cells.
We established highly purified functional iNKT cell lines from newly diagnosed and advanced MM patients. These CD1d-restricted iNKT cell lines produced high level of antitumor Th1 cytokine in response to α-GalCer-pulsed-primary MM cells, CD1d-transfected MM1S cell line or DCs. Moreover, MM iNKT cell lines displayed strong cytotoxicity against α-GalCer-pulsed-primary MM cells. Importantly, lenalidomide further augmented the Th1-polarization by iNKT cell lines via the increased Th1 cytokine production and the reduced Th2 cytokine production. We also demonstrated that CD1d was expressed in primary MM cells at mRNA and protein levels from the majority of MM patients, but not in normal plasma cells and MM cell lines, and CD1d+ primary MM cells presented antigens to activate iNKT cell lines.
Taken together, our results provide the pre-clinical evidence for the iNKT cells-mediated immunotherapy and a rationale for their use in combination with lenalidomide in MM treatment.
PMCID: PMC2967459  PMID: 18980990
iNKT cells; multiple myeloma; lenalidomide; immunotherapy
14.  Activated invariant NKT cells control central nervous system autoimmunity in a mechanism that involves myeloid-derived suppressor cells 
Invariant NKT (iNKT) cells are a subset of T lymphocytes that recognize glycolipid antigens presented by the MHC class I-related protein CD1d. Activation of iNKT cells with glycolipid antigens such as the marine sponge-derived reagent α-galactosylceramide (α-GalCer) results in the rapid production of a variety of cytokines and activation of many other immune cell types. These immunomodulatory properties of iNKT cells have been exploited for the development of immunotherapies against a variety of autoimmune and inflammatory diseases but mechanisms by which activated iNKT cells confer disease protection have remained incompletely understood. Here, we demonstrate that glycolipid-activated iNKT cells cooperate with myeloid-derived suppressor cells (MDSCs) in protecting mice against the development of experimental autoimmune encephalomyelitis (EAE) in mice, an animal model for multiple sclerosis (MS). We showed that α-GalCer induced the expansion and immunosuppressive activities of MDSCs in the spleen of mice induced for development of EAE. Disease protection in these animals also correlated with recruitment of MDSCs to the central nervous system. Depletion of MDSCs abrogated the protective effects of α-GalCer against EAE and, conversely, adoptive transfer of MDSCs from α-GalCer-treated mice ameliorated passive EAE induced in recipient animals. The cytokines GM-CSF, IL-4 and IFN-γ, produced by activated iNKT cells, and inducible nitric oxide synthase, arginase-1 and IL-10 produced by MDSCs, contributed to these effects. Taken together, our findings have revealed cooperative immunosuppressive interactions between iNKT cells and MDSCs that might be exploited for the development of improved immunotherapies for MS and other autoimmune and inflammatory diseases.
PMCID: PMC3577977  PMID: 23345328
15.  PD-1:PD-L blockade prevents anergy induction and enhances the anti-tumor activities of glycolipid-activated iNKT cells1 
Invariant natural killer T (iNKT) cells recognize glycolipid antigens such as the marine sponge-derived glycosphingolipid α-galactosylceramide (αGalCer) presented by the CD1d protein. In vivo activation of iNKT cells with αGalCer results in robust cytokine production followed by the acquisition of an anergic phenotype. Here, we have investigated mechanisms responsible for the establishment of αGalCer-induced iNKT cell anergy. We found that αGalCer-activated iNKT cells rapidly upregulated expression of the inhibitory co-stimulatory receptor programmed death (PD)-1 at their cell surface, and this increased expression was retained for at least one month. Blockade of the interaction between PD-1 and its ligands, PD-L1 and PD-L2, at the time of αGalCer treatment prevented the induction iNKT cell anergy, but was unable to reverse established iNKT cell anergy. Consistently, injection of αGalCer into PD-1-deficient mice failed to induce iNKT cell anergy. However, blockade of the PD-1:PD-L pathway failed to prevent bacterial- or sulfatide-induced iNKT cell anergy, suggesting additional mechanisms of iNKT cell tolerance. Finally, we showed that blockade of PD-1:PD-L interactions enhanced the antimetastatic activities of αGalCer. Collectively, our findings reveal a critical role for the PD-1:PD-L costimulatory pathway in the αGalCer-mediated induction of iNKT cell anergy that can be targeted for the development of immunotherapies.
PMCID: PMC2709814  PMID: 19234176
16.  β-mannosylceramide activates type I natural killer T cells to induce tumor immunity without inducing long-term functional anergy 
Most studies characterizing antitumor properties of iNKT cells have used the agonist, α-galactosylceramide (α-GalCer). However, α-GalCer induces strong, long-lasting anergy of iNKT cells, which could be a major detriment for clinical therapy. A novel iNKT cell agonist, β-mannosylceramide (β-ManCer), induces strong antitumor immunity through a mechanism distinct from that of α-GalCer. The objective of this study was to determine whether β-ManCer induces anergy of iNKT cells.
Experimental design
Induction of anergy was determined by ex vivo analysis of splenocytes from mice pre-treated with iNKT cell agonists as well as in the CT26 lung metastasis in vivo tumor model.
β-ManCer activated iNKT cells without inducing long-term anergy. The transience of anergy induction correlated with a shortened duration of PD-1 upregulation on iNKT cells activated with β-ManCer, compared with α-GalCer. Moreover, while mice pretreated with α-GalCer were unable to respond to a second glycolipid stimulation to induce tumor protection for up to two months, mice pretreated with β-ManCer were protected from tumors by a second stimulation equivalently to vehicle-treated mice.
The lack of long-term functional anergy induced by β-ManCer, which allows for a second dose to still give therapeutic benefit, suggests the strong potential for this iNKT cell agonist to succeed in settings where α-GalCer has failed.
Translational Relevance
Activation of iNKT cells with α-galactosylceramide was very successful in preclinical mouse models of cancer; however, its success in clinical trials has been very limited. It has been very well-documented, that once iNKT cells are activated with α-galactosylceramide, they remain unresponsive to restimulation for months. This functional anergy could be a contributing factor to the failure of α-galactosylceramide clinically, as most therapeutics require multiple dosing to achieve maximum benefit. Here, we report that a different iNKT cell agonist, β-mannosylceramide, which is capable of inducing tumor immunity similarly to α-galactosylceramide but by a different mechanism, does not induce anergy. This suggests that β-mannosylceramide has the potential to work well clinically since it can be given in multiple doses without inducing anergy.
PMCID: PMC3819527  PMID: 23804426
NKT; CD1d; tumor immunology; immunotherapeutics; anergy
17.  α-Galactosylceramide Analogs with Weak Agonist Activity for Human iNKT Cells Define New Candidate Anti-Inflammatory Agents 
PLoS ONE  2010;5(12):e14374.
CD1d-restricted natural killer T cells with invariant T cell receptor α chains (iNKT cells) are a unique lymphocyte subset that responds to recognition of specific lipid and glycolipid antigens. They are conserved between mice and humans and exert various immunoregulatory functions through their rapid secretion of a variety of cytokines and secondary activation of dendritic cells, B cells and NK cells. In the current study, we analyzed the range of functional activation states of human iNKT cells using a library of novel analogs of α-galactosylceramide (αGalCer), the prototypical iNKT cell antigen. Measurement of cytokines secreted by human iNKT cell clones over a wide range of glycolipid concentrations revealed that iNKT cell ligands could be classified into functional groups, correlating with weak versus strong agonistic activity. The findings established a hierarchy for induction of different cytokines, with thresholds for secretion being consistently lowest for IL-13, higher for interferon-γ (IFNγ), and even higher for IL-4. These findings suggested that human iNKT cells can be intrinsically polarized to selective production of IL-13 by maintaining a low level of activation using weak agonists, whereas selective polarization to IL-4 production cannot be achieved through modulating the strength of the activating ligand. In addition, using a newly designed in vitro system to assess the ability of human iNKT cells to transactivate NK cells, we found that robust secondary induction of interferon-γ secretion by NK cells was associated with strong but not weak agonist ligands of iNKT cells. These results indicate that polarization of human iNKT cell responses to Th2-like or anti-inflammatory effects may best be achieved through selective induction of IL-13 and suggest potential discrepancies with findings from mouse models that may be important in designing iNKT cell-based therapies in humans.
PMCID: PMC3003687  PMID: 21179412
18.  Peripheral Blood Invariant Natural Killer T Cells of Pig-Tailed Macaques 
PLoS ONE  2012;7(10):e48166.
In humans, invariant natural killer T (iNKT) cells represent a small but significant population of peripheral blood mononuclear cells (PBMCs) with a high degree of variability. In this study, pursuant to our goal of identifying an appropriate non-human primate model suitable for pre-clinical glycolipid testing, we evaluated the percentage and function of iNKT cells in the peripheral blood of pig-tailed macaques. First, using a human CD1d-tetramer loaded with α-GalCer (α-GalCer-CD1d-Tet), we found that α-GalCer-CD1d-Tet+ CD3+ iNKT cells make up 0.13% to 0.4% of pig-tailed macaque PBMCs, which are comparable to the percentage of iNKT cells found in human PBMCs. Second, we observed that a large proportion of Vα24+CD3+ cells are α-GalCer-CD1d-Tet+CD3+ iNKT cells, which primarily consist of either the CD4+ or CD8+ subpopulation. Third, we found that pig-tailed macaque iNKT cells produce IFN-γ in response to α-GalCer, as shown by ELISpot assay and intracellular cytokine staining (ICCS), as well as TNF-α, as shown by ICCS, indicating that these iNKT cells are fully functional. Interestingly, the majority of pig-tailed macaque iNKT cells that secrete IFN-γ are CD8+ iNKT cells. Based on these findings, we conclude that the pig-tailed macaques exhibit potential as a non-human animal model for the pre-clinical testing of iNKT-stimulating glycolipids.
PMCID: PMC3479117  PMID: 23110202
19.  Impaired selection of invariant natural killer T cells in diverse mouse models of glycosphingolipid lysosomal storage diseases 
The Journal of Experimental Medicine  2006;203(10):2293-2303.
Glycolipid ligands for invariant natural killer T cells (iNKT cells) are loaded onto CD1d molecules in the late endosome/lysosome. Accumulation of glycosphingolipids (GSLs) in lysosomal storage diseases could potentially influence endogenous and exogenous lipid loading and/or presentation and, thus, affect iNKT cell selection or function. The percentages and frequency of iNKT cells were reduced in multiple mouse models of lysosomal GSL storage disease, irrespective of the specific genetic defect or lipid species stored. Reduced numbers of iNKT cells resulted in the absence of cytokine production in response to α-galactosylceramide (α-GalCer) and reduced iNKT cell–mediated lysis of wild-type targets loaded with α-GalCer. The reduction in iNKT cells did not result from defective expression of CD1d or a lack of antigen-presenting cells. Although H-2 restricted CD4+ T cell responses were generally unaffected, processing of a lysosome-dependent analogue of α-GalCer was impaired in all the strains of mice tested. These data suggest that GSL storage may result in alterations in thymic selection of iNKT cells caused by impaired presentation of selecting ligands.
PMCID: PMC2118110  PMID: 16982810
20.  NF-κB Is Activated in CD4+ iNKT Cells by Sickle Cell Disease and Mediates Rapid Induction of Adenosine A2A Receptors 
PLoS ONE  2013;8(10):e74664.
Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11–7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists.
PMCID: PMC3790763  PMID: 24124453
21.  Brief Treatment with iNKT Cell Ligand α-Galactosylceramide Confers a Long-term Protection Against Lupus 
Journal of clinical immunology  2011;32(1):106-113.
CD1d presents glycolipid antigens such as α-galactosylceramide (αGalCer) to invariant natural killer T cells (iNKT). We have reported that activated iNKTs inhibit IL-10-producing autoreactive B cells, while promoting or leaving intact the normal B cell responses, making iNKT modulation an attractive therapeutic modality. Here, we report that a brief treatment of young lupus-prone (NZB/NZW)F1 (BWF1) mice with two injections of αGalCer conferred a long-term protection against lupus. Long-term repeated administrations of αGalCer, however, afforded no clinical benefit. These disparate clinical effects correlated with iNKT responsiveness. While a brief treatment with αGalCer enhanced iNKT responses upon in vitro recall, the long-term αGalCer treatment resulted in reduced iNKT responses in BWF1 mice. The improvement in disease with αGalCer treatment was associated with the reduced IL-10 production. Furthermore, iNKTs directly inhibited IL-10-secreting cells in vivo in reconstituted SCID mice and inhibited IL-10-secreting B cells in vitro in co-cultures. Thus, a brief treatment with a CD1d-binding glycolipid enhances iNKT responses, reduces IL-10 production, and delays the onset of lupus, whereas long-term repeated treatments induce marked iNKT hyporesponsiveness and do not affect disease outcome in BWF1 mice. Identifying glycolipid regimens that can modulate iNKT responsiveness will have important implications for developing iNKT-based therapies for autoimmune diseases.
PMCID: PMC3432318  PMID: 22002593
Rodent; T cells; autoimmunity; systemic lupus erythematosus
22.  Examining the Role of CD1d and Natural Killer T Cells in the Development of Nephritis in a Genetically Susceptible Lupus Model 
Arthritis and rheumatism  2007;56(4):1219-1233.
CD1d-reactive invariant natural killer T (iNKT) cells secrete multiple cytokines upon T cell receptor (TCR) engagement and modulate many immune-mediated conditions. The purpose of this study was to examine the role of these cells in the development of autoimmune disease in genetically lupus-prone (NZB × NZW)F1 (BWF1) mice.
The CD1d1-null genotype was crossed onto the NZB and NZW backgrounds to establish CD1d1-knockout (CD1d0) BWF1 mice. CD1d0 mice and their wild-type littermates were monitored for the development of nephritis and assessed for cytokine responses to CD1d-restricted glycolipid α-galactosylceramide (αGalCer), anti-CD3 antibody, and con-canavalin A (Con A). Thymus and spleen cells were stained with CD1d tetramers that had been loaded with αGalCer or its analog PBS-57 to detect iNKT cells, and the cells were compared between BWF1 mice and class II major histocompatibility complex–matched nonautoimmune strains, including BALB/c, (BALB/c × NZW)F1 (CWF1), and NZW.
CD1d0 BWF1 mice had more severe nephritis than did their wild-type littermates. Although iNKT cells and iNKT cell responses were absent in CD1d0 BWF1 mice, the CD1d0 mice continued to have significant numbers of interferon-γ–producing NKT-like (CD1d-independent TCRβ+,NK1.1+ and/or DX5+) cells. CD1d deficiency also influenced cytokine responses by conventional T cells: upon in vitro stimulation of splenocytes with Con A or anti-CD3, type 2 cytokine levels were reduced, whereas type 1 cytokine levels were increased or unchanged in CD1d0 mice as compared with their wild-type littermates. Additionally, numbers of thymic iNKT cells were lower in young wild-type BWF1 mice than in nonautoimmune strains.
Germline deletion of CD1d exacerbates lupus in BWF1 mice. This finding, together with reduced thymic iNKT cells in young BWF1 mice as compared with nonautoimmune strains, implies a regulatory role of CD1d and iNKT cells during the development of lupus.
PMCID: PMC2291538  PMID: 17393451
23.  TLR4, 5, and 9 Agonists Inhibit Murine Airway Invariant Natural Killer T Cells in an IL-12-Dependent Manner 
Invariant natural killer T (iNKT) cells may play an important role in the pathogenesis of asthma in mice and humans. Thus, an agent that modulates the function of iNKT cells may have therapeutic potential to control asthma. We hypothesized that lipopolysaccharide (LPS)-, flagellin-, or CpG-induced changes in the cytokine milieu may modify and even inhibit the function of airway iNKT cells in asthma.
Because increased α-galactosylceramide (GalCer)-induced airway hyperreactivity (AHR) reflects the presence of airway iNKT cells, α-GalCer-induced AHR, as well as inflammatory cells and cytokines in bronchoalveolar lavage (BAL) fluid, were determined 24 hours after in vivo treatment with LPS, flagellin, or CpG in naïve BALB/c mice. Intracellular IL-4 and IFN-γ were measured in spleen iNKT cells after in vitro treatment with LPS, flagellin, or CpG. A role for IL-12 following the treatments was determined.
Intranasal administration of LPS, flagellin, or CpG reduced development of α-GalCer-induced AHR, eosinophilic airway inflammation, and Th1 and Th2 cytokine responses in BAL fluid, while producing IL-12 in BAL fluid. Intraperitoneal administration of IL-12 mAb blocked the suppressive effect of LPS, flagellin, or CpG. In vitro treatment with LPS, flagellin, or CpG reduced production of IL-4 and IFN-γ from α-GalCer-stimulated spleen iNKT cells; these effects were ameliorated by addition of anti-IL-12 mAb.
TLR4, 5, and 9 agonists may suppress the function of airway and spleen iNKT cells via IL-12-dependent mechanisms. Anergy of iNKT cells by IL-12 might play a role in suppression by these TLR agonists.
PMCID: PMC3423604  PMID: 22950036
CpG; flagellin; lipopolysaccharide; natural killer T cells
24.  Invariant and Noninvariant Natural Killer T Cells Exert Opposite Regulatory Functions on the Immune Response during Murine Schistosomiasis▿  
Infection and Immunity  2007;75(5):2171-2180.
CD1d-restricted natural killer T (NKT) cells represent a heterogeneous population of innate memory immune cells expressing both NK and T-cell markers distributed into two major subsets, i.e., invariant NKT (iNKT) cells, which express exclusively an invariant T-cell receptor (TCR) α chain (Vα14Jα18 in mice), and non-iNKT cells, which express more diverse TCRs. NKT cells quickly produce Th1- and/or Th2-type cytokines following stimulation with glycolipid antigen (Ag) and, through this property, play potent immunoregulatory roles in autoimmune diseases, cancer, and infection. No study has addressed the role of NKT cells in metazoan parasite infections so far. We show that during murine schistosomiasis, the apparent frequency of both iNKT cells and non-iNKT cells decreased in the spleen as early as 3 weeks postinfection (p.i.) and that both populations expressed a greater amount of the activation marker CD69 at 6 weeks p.i., suggesting an activated phenotype. Two different NKT-cell-deficient mouse models, namely, TCR Jα18−/− (exclusively deficient in iNKT cells) and CD1d−/− (deficient in both iNKT and non-iNKT cells) mice, were used to explore the implication of these subsets in infection. We show that whereas both iNKT and non-iNKT cells do not have a major impact on the immune response during the early phase (1 and 4 weeks) of infection, they exert important, although opposite, effects on the immune response during the acute phase of the disease (7 and 12 weeks), after schistosome egg production. Indeed, iNKT cells contribute to Th1 cell differentiation whereas non-iNKT cells might be mostly implicated in Th2 cell differentiation in response to parasite Ag. Our findings suggest, for the first time, that helminths activate both iNKT and non-iNKT cells in vivo, enabling them to differentially influence the Th1/Th2 balance of the immune response.
PMCID: PMC1865739  PMID: 17353286
25.  Human invariant natural killer T cells acquire transient innate responsiveness via histone H4 acetylation induced by weak TCR stimulation 
The Journal of Experimental Medicine  2012;209(5):987-1000.
Weak TCR stimulation of iNKT cells, such as that resulting from self-antigen recognition, induces histone modifications at the IFNG locus that allow the iNKT cells to subsequently produce IFN-γ in response to proinflammatory cytokines alone.
Invariant NKT cells (iNKT cells) are innate T lymphocytes that are thought to play an important role in producing an early burst of IFN-γ that promotes successful tumor immunosurveillance and antimicrobial immunity. The cellular activation processes underlying innate IFN-γ production remain poorly understood. We show here that weak T cell receptor (TCR) stimulation that does not directly activate iNKT cell IFN-γ messenger RNA transcription nevertheless induces histone H4 acetylation at specific regions near the IFNG gene locus. This renders the iNKT cells able to produce IFN-γ in an innate manner (i.e., not requiring concurrent TCR stimulation) upon exposure to IL-12 and IL-18. The iNKT cells retain the capacity for innate activation for hours to days after the initial weak TCR stimulation, although their innate responsiveness gradually declines as a function of histone deacetylation. These results explain how iNKT cells are able to mediate rapid innate IFN-γ secretion in a manner that does not require them to undergo permanent TH1 differentiation. Moreover, our results also indicate that iNKT cell motility is maintained during activation by IL-12 and IL-18. Therefore, iNKT cells activated through this pathway can continue to migrate and may thus disseminate the IFN-γ that they produce, which may amplify its impact.
PMCID: PMC3348100  PMID: 22508835

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