Activated invariant NKT cells regulate osteoclast development and function

Invariant NKT (iNKT) cells modulate innate and adaptive immune responses through activation of myeloid dendritic cells and macrophages and via enhanced clonogenicity, differentiation, and egress of their shared myeloid progenitors. Because these same progenitors give rise to osteoclasts (OCs), which also mediate the egress of hematopoietic progenitors and orchestrate bone remodeling, we hypothesized that iNKT cells would extend their myeloid cell regulatory role to the development and function of OCs. In this study, we report that selective activation of iNKT cells by α-galactosylceramide causes myeloid cell egress, enhances OC progenitor and precursor development, modifies the intramedullary kinetics of mature OCs, and enhances their resorptive activity. OC progenitor activity is positively regulated by TNF-α and negatively regulated by IFN-γ, but is IL-4 and IL-17 independent. These data demonstrate a novel role of iNKT cells that couples osteoclastogenesis with myeloid cell egress in conditions of immune activation.     

Liver invariant NKT cells and listeriosis

The invariant (i) NKT cells represent unique T lymphocytes expressing TCRValpha14. Although iNKT cells have been regarded as T lymphocytes expressing NK1.1, they do not consistently express this marker. NK1.1 allows recognition of "missing-self" and thus controls inhibition/activation of iNKT cells. It is thus tempting to assume that iNKT cells participate in the regulation of host immune responses during microbial infection by controlling NK1.1 expression. These findings shed light on the unique role of iNKT cells in microbial infection and provide an evidence for unique aspects of the NK1.1 on these cells as a regulatory molecule.     

Activation of plasmacytoid dendritic cells with TLR9 agonists initiates invariant NKT cell-mediated cross-talk with myeloid dendritic cells

CD1d-restricted invariant NK T (iNKT) cells and dendritic cells (DCs) have been shown to play crucial roles in various types of immune responses, including TLR9-dependent antiviral responses initiated by plasmacytoid DCs (pDCs). However, the mechanism by which this occurs is enigmatic because TLRs are absent in iNKT cells and human pDCs do not express CD1d. To explore this process, pDCs were activated with CpG oligodeoxyribonucleotides, which stimulated the secretion of several cytokines such as type I and TNF-alpha. These cytokines and other soluble factors potently induced the expression of activation markers on iNKT cells, selectively enhanced double-negative iNKT cell survival, but did not induce their expansion or production of cytokines. Notably, pDC-derived factors licensed iNKT cells to respond to myeloid DCs: an important downstream cellular target of iNKT cell effector function and a critical contributor to the initiation of adaptive immune responses. This interaction supports the notion that iNKT cells can mediate cross-talk between DC subsets known to express mutually exclusive TLR and cytokine profiles.     

On/off TLR signaling decides proinflammatory or tolerogenic dendritic cell maturation upon CD1d-mediated interaction with invariant NKT cells

Invariant NKT (iNKT) cells play an effector/adjuvant function during antimicrobial and antitumoral immunity and a regulatory role to induce immune tolerance and prevent autoimmunity. iNKT cells that differentially modulate adaptive immunity do not bear a unique phenotype and/or specific cytokine secretion profile, thus opening questions on how a single T cell subset can exert opposite immunological tasks. In this study, we show that iNKT cells perform their dual roles through a single mechanism of action relying on the cognate interaction with myeloid dendritic cells (DCs) and leading to opposite effects depending on the presence of other maturation stimuli simultaneously acting on DCs. The contact of murine purified iNKT cells with immature autologous DCs directly triggers the tolerogenic maturation of DCs, rendering them able to induce regulatory T cell differentiation and prevent autoimmune diabetes in vivo. Conversely, the interaction of the same purified iNKT cells with DCs, in the presence of simultaneous TLR4 stimulation, significantly enhances proinflammatory DC maturation and IL-12 secretion. The different iNKT cell effects are mediated through distinct mechanisms and activation of different molecular pathways within the DC: CD1d signaling and activation of the ERK1/2 pathway for the tolerogenic action, and CD40-CD40L interaction and NF-κB activation for the adjuvant effect. Our data suggest that the DC decision to undergo proinflammatory or tolerogenic maturation results from the integration of different signals received at the time of iNKT cell contact and could have important therapeutic implications for exploiting iNKT cell adjuvant/regulatory properties in autoimmune diseases, infections, and cancer.     

Cutting edge: nonglycosidic CD1d lipid ligands activate human and murine invariant NKT cells

Invariant NKT cells (iNKT cells) recognize CD1d/glycolipid complexes. We demonstrate that the nonglycosidic compound threitolceramide efficiently activates iNKT cells, resulting in dendritic cell (DC) maturation and the priming of Ag-specific T and B cells. Threitolceramide-pulsed DCs are more resistant to iNKT cell-dependent lysis than alpha-galactosylceramide-pulsed DCs due to the weaker affinity of the human iNKT TCR for CD1d/ threitolceramide than CD1d/alpha-galactosylceramide complexes. iNKT cells stimulated with threitolceramide also recover more quickly from activation-induced anergy. Kinetic and functional experiments showed that shortening or lengthening the threitol moiety by one hydroxymethylene group modulates ligand recognition, as human and murine iNKT cells recognize glycerolceramide and arabinitolceramide differentially. Our data broaden the range of potential iNKT cell agonists. The ability of these compounds to assist the priming of Ag-specific immune responses while minimizing iNKT cell-dependent DC lysis makes them attractive adjuvants for vaccination strategies.     

IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand alpha-galactosylceramide (alphaGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using alphaGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-gamma production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8(+) CTL response, which was dependent on iNKT cell-derived IFN-gamma. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as alphaGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-gamma-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with alphaGC.     

Globosides but not isoglobosides can impact the development of invariant NKT cells and their interaction with dendritic cells

Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.     

Contact-dependent interference with invariant NKT cell activation by herpes simplex virus-infected cells

Invariant CD1d-restricted NKT (iNKT) cells play important roles in generating protective immune responses against infections. In this study, 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-galactosyl ceramide (α-GalCer) could stimulate IFN-γ production and CD25 upregulation by iNKT cells. However, both α-GalCer-dependent and cytokine-dependent activation of iNKT cells was impaired after coculture with HSV-1-infected cells. Notably, 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 after 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. After coculture with infected cells, iNKT cells were no longer responsive to further stimulation with α-GalCer-loaded CD1d-expressing cells. We found that exposure to HSV-1-infected cells resulted in impaired TCR signaling 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 used by the virus to avoid immune clearance.     

The TLR7 agonist R848 alleviates allergic inflammation by targeting invariant NKT cells to produce IFN-gamma

It has been documented that TLR7 stimulation triggers not only antiviral responses, but also alleviates experimental asthma. Considering the implication of invariant NKT (iNKT) cells in both situations, we postulated that they might contribute to the anti-inflammatory effect of TLR7 ligands. We show in this study that spleen cells activated by the TLR7 agonist resiquimod (R848) attenuate allergic inflammation upon adoptive transfer when they are recovered from wild-type, but not from iNKT cell-deficient Jα18(-/-) mice, which proves the specific involvement of this regulatory population. Furthermore, we provide evidence that IFN-γ is critical for the protective effect, which is lost when transferred iNKT cells are sorted from IFN-γ-deficient mice. In support of a direct activation of iNKT cells through TLR7 signaling in vivo, we observed a prompt increase of serum IFN-γ levels, associated with upregulation of CD69 expression on iNKT cells. Moreover, we demonstrate that iNKT cells effectively express TLR7 and respond to R848 in vitro by producing high levels of IFN-γ in the presence of IL-12, consistent with the conclusion that their contribution to the alleviation of allergic inflammation upon treatment with TLR7 ligands is mediated through IFN-γ.     

A cell-type specific CD1d expression program modulates invariant NKT cell development and function

Invariant NK T (iNKT) cells are a distinct subset of T cells that rapidly produce an array of immunoregulatory cytokines upon activation. Cytokines produced by iNKT cells subsequently transactivate other leukocytes and elicit their respective effector functions. In this way, iNKT cells play a central role in coordinating the development of immune responses in a variety of settings. However, the mechanisms governing the quality of the iNKT cell response elicited remain poorly defined. To address whether changes in the CD1d expression pattern could regulate iNKT cell function, we generated a transgenic (Tg) mouse model in which thymocytes and peripheral T cells express high levels of CD1d (Lck-CD1d Tg+ mice). The expression of CD1d by T cells was sufficient to rescue development of iNKT cells in mice deficient of endogenous CD1d. However, the relative proportions of iNKT cell subsets in Lck-CD1d Tg+ mice were distinctly different from those in wild-type mice, suggesting an altered developmental program. Additionally, iNKT cells were hyporesponsive to antigenic stimulation in vivo. Interestingly, Lck-CD1d Tg+ mice develop liver pathology in the absence of any exogenous manipulation. The results of these studies suggest that changes to the CD1d expression program modulate iNKT cell development and function.     

Evaluation of the function of human invariant NKT cells from cancer patients using alpha-galactosylceramide-loaded murine dendritic cells

NKT cells play a role in immunological regulation of certain diseases, and their frequency and/or function may be related to disease prognosis. However, it is often difficult to evaluate NKT cell function in patients with malignancies due to reduced numbers of NKT cells as well as the dysfunction of the APCs used as stimulators. We found that NKT cell function could not be evaluated by conventional ELISPOT assays, confirming the impaired function of APCs in chronic myelogenous leukemia (CML)-chronic phase patients. To overcome this problem, we have established a sensitive assay using murine dendritic cells to evaluate the function of small numbers of human NKT cells independent of autologous APCs. We found that imatinib-treated CML-chronic phase patients showing a complete cytogenetic response had NKT cells capable of producing IFN-gamma, whereas NKT cells from patients who were only partially responsive to imatinib treatment did not produce IFN-gamma. Functional NKT cells found in imatinib-treated, CML-complete cytogenetic response patients may offer the promise of effective immunotherapy with ex vivo-generated alpha-galactosylceramide-pulsed dendritic cells. This new approach should be available for evaluating the functions of NKT cells and APCs in cancer patients.     

Dendritic cells and NK cells stimulate bystander T cell activation in response to TLR agonists through secretion of IFN-alpha beta and IFN-gamma

Recognition of conserved features of infectious agents by innate pathogen receptors plays an important role in initiating the adaptive immune response. We have investigated early changes occurring among T cells after injection of TLR agonists into mice. Widespread, transient phenotypic activation of both naive and memory T cells was observed rapidly after injection of molecules acting through TLR3, -4, -7, and -9, but not TLR2. T cell activation was shown to be mediated by a combination of IFN-alphabeta, secreted by dendritic cells (DCs), and IFN-gamma, secreted by NK cells; notably, IFN-gamma-secreting NK cells expressed CD11c and copurified with DCs. Production of IFN-gamma by NK cells could be stimulated by DCs from TLR agonist-injected mice, and although soluble factors secreted by LPS-stimulated DCs were sufficient to induce IFN-gamma, maximal IFN-gamma production required both direct contact of NK cells with DCs and DC-secreted cytokines. In vitro, IFN-alphabeta, IL-18, and IL-12 all contributed to DC stimulation of NK cell IFN-gamma, whereas IFN-alphabeta was shown to be important for induction of T cell bystander activation and NK cell IFN-gamma production in vivo. The results delineate a pathway involving innate immune mediators through which TLR agonists trigger bystander activation of T cells.     

Activation of invariant NKT cells exacerbates experimental visceral leishmaniasis

We report that natural killer T (NKT) cells play only a minor physiological role in protection from Leishmania donovani infection in C57BL/6 mice. Furthermore, attempts at therapeutic activation of invariant NKT (iNKT) cells with α-galactosylceramide (α-GalCer) during L. donovani infection exacerbated, rather than ameliorated, experimental visceral leishmaniasis. The inability of α-GalCer to promote anti-parasitic immunity did not result from inefficient antigen presentation caused by infection because α-GalCer–loaded bone marrow–derived dendritic cells were also unable to improve disease resolution. The immune-dampening affect of α-GalCer correlated with a bias towards increased IL-4 production by iNKT cells following α-GalCer stimulation in infected mice compared to naïve controls. However, studies in IL-4–deficient mice, and IL-4 neutralisation in cytokine-sufficient mice revealed that α-GalCer–induced IL-4 production during infection had only a minor role in impaired parasite control. Analysis of liver cell composition following α-GalCer stimulation during an established L. donovani infection revealed important differences, predominantly a decrease in IFNγ⁺ CD8⁺ T cells, compared with control-treated mice. Our data clearly illustrate the double-edged sword of NKT cell–based therapy, showing that in some circumstances, such as when sub-clinical or chronic infections exist, iNKT cell activation can have adverse outcomes.     

Forskolin regulates retinal endothelial cell permeability through TLR4 actions in vitro

To investigate whether forskolin, a protein kinase A agonist, regulates toll-like receptor 4 actions on retinal endothelial cell permeability in vitro. We also evaluated whether PKA could regulate TLR4 signaling independent of exchange protein activated by cAMP in REC in culture. REC were grown in normal (5 mM) or high (25 mM) glucose. Cells were treated with forskolin to increase PKA levels, siRNA against TLR4, siRNA against myeloid differentiation primary response 88, siRNA against translocating chain associated membrane protein 1, siRNA against epac1, or scrambled siRNA, or a combination of these treatments. Western blotting was done for zonula occludens 1 and occludin protein levels, as well as TLR4 signaling cascade proteins. Permeability measurements were done for REC in culture following inhibition of TLR4 or its signaling cascades. Forskolin restored high glucose-associated decreases in ZO-1 and occludin, which was associated with improved in vitro permeability levels. Both forskolin and TLR4 inhibition reduced high glucose-induced increases in REC permeability, but the actions were not cooperative. Forskolin regulated both MyD88-dependent and -independent signaling pathways, independent of Epac1. Finally, blockade of MyD88 or TRAM1 reduced permeability in REC grown in high glucose. A PKA agonist regulated TLR4 signaling independent of Epac1. PKA agonism or TLR4 inhibition is effective at reducing high glucose-induced permeability in REC in vitro. These studies offer new avenues for therapeutic development.

     

Critical roles of RasGRP1 for invariant NKT cell development

The invariant NKT (iNKT) cell lineage contains CD4(+) and CD4(-) subsets. The mechanisms that control such subset differentiation and iNKT cell maturation in general have not been fully understood. RasGRP1, a guanine nucleotide exchange factor for TCR-induced activation of the Ras-ERK1/2 pathway, is critical for conventional αβ T cell development but dispensable for generating regulatory T cells. Its role in iNKT cells has been unknown. In this study, we report severe decreases of iNKT cells in RasGRP1(-/-) mice through cell intrinsic mechanisms. In the remaining iNKT cells in RasGRP1(-/-) mice, there is a selective absence of the CD4(+) subset. Furthermore, RasGRP1(-/-) iNKT cells are defective in TCR-induced proliferation in vitro. These observations establish that RasGRP1 is not only important for early iNKT cell development but also for the generation/maintenance of the CD4(+) iNKT cells. Our data provide genetic evidence that the CD4(+) and CD4(-) iNKT cells are distinct sublineages with differential signaling requirements for their development.     

TLR ligands can activate dendritic cells to provide a MyD88-dependent negative signal for Th2 cell development

During infection, CD4(+) Th cell responses polarize to become primarily Th1 or Th2. Th1 cells, which make IFN-gamma, are crucial for immunity to many bacterial and protozoal infections, whereas Th2 cells, which make IL-4, IL-5, and IL-13, are important for resistance to helminth infections. Polarized Th1 responses are induced by dendritic cells (DCs), which respond to pathogen-derived TLR ligands to produce IL-12 and related cytokines that are instrumental in Th1 cell outgrowth, and coordinately process and present Ag in the context of MHC class II to activate naive Th cells. In this study we show that in addition to providing positive signals for Th1 cell development, mouse DCs activated by TLR engagement can also provide a potent negative signal that prevents the development of Th2 cells. Production of this signal, which is not IL-12, IL-18, IL-23, IL-27, or IFN-gamma and is not provided via Th1 cells, is dependent upon a MyD88-dependent, TNF receptor-associated factor-6-independent signaling pathway in DCs. The signal is released from DCs in response to activation via TLR ligands and exerts an effect directly on Th cells rather than through a third-party cell. Our findings indicate that DCs can provide potent negative as well as positive instruction for Th response polarization, and that these instructional signals are distinct and independent.