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

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

Negative regulation of TCR signaling by linker for activation of X cells via phosphotyrosine-dependent and -independent mechanisms

The activation of T cells and the initiation of an immune response is tightly controlled through the crosstalk of both positive and negative regulators. Two adaptors that function as negative regulators of T cell activation are adaptor in lymphocytes of unknown function X (ALX) and linker for activation of X cell (LAX). Previously, we showed that T cells from mice deficient in ALX and LAX display similar hyperresponsiveness, with increased IL-2 production and proliferation upon TCR/CD28 stimulation, and that these adaptors physically associate. In this study, we analyze the nature of the association between ALX and LAX. We demonstrate that this association occurs in the absence of TCR/CD28 signaling via a mechanism independent of both tyrosine phosphorylation of LAX and the SH2 domain of ALX. Cotransfection of ALX with LAX resulted in LAX tyrosine phosphorylation in the absence of TCR/CD28 stimulation. ALX-mediated LAX phosphorylation depends upon the ALX SH2 domain, which functions to recruit Lck to LAX. We also show that LAX, like ALX, can inhibit RE/AP reporter activation. However, in contrast to its inhibition of NFAT, the inhibition of RE/AP by LAX is independent of its tyrosine phosphorylation. Therefore, it can be concluded that inhibition of signaling events involved in T cell activation by LAX occurs through mechanisms both dependent on and independent of its tyrosine phosphorylation.     

Regulation of NKT cells by Ly49: analysis of primary NKT cells and generation of NKT cell line

TCRalphabeta(+)NK1.1(+) (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to alpha-galactosylceramide (alpha-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with alpha-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to alpha-GalCer. Moreover, alpha-GalCer presented by normal dendritic cells stimulated purified Ly49(-), but not Ly49(+), splenic NKT cells, whereas MHC class I-deficient dendritic cells presented alpha-GalCer to both Ly49(+) and Ly49(-) NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an alpha-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49(-)NK1.1(+)CD4(+) thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.     

Interplay of cytokines and microbial signals in regulation of CD1d expression and NKT cell activation

In this study we show that like MHC class I and class II molecules, cell surface CD1d expression on APC is regulated and affects T cell activation under physiological conditions. Although IFN-gamma alone is sufficient for optimum expression of MHC, CD1d requires two signals, one provided by IFN-gamma and a second mediated by microbial products or by the proinflammatory cytokine TNF. IFN-gamma-dependent CD1d up-regulation occurs on macrophages following infection with live bacteria or exposure to microbial products in vitro and in vivo. APC expressing higher CD1d levels more efficiently activate NKT cell hybridomas and primary NKT cells independently of whether the CD1d-restricted TCR recognizes foreign or self-lipid Ags. Our findings support a model in which CD1d induction regulates NKT cell activation.     

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.     

alpha-galactosylceramide induces early B-cell activation through IL-4 production by NKT cells

alpha-Galactosylceramide (alpha-GalCer), a glycolipid antigen, specifically activates natural killer T (NKT) cells by a CD1d-restricted mechanism. In this work, we found that in vivo administration of alpha-GalCer resulted in the activation of B cells in addition to NKT cells, namely, alpha-GalCer administration caused upregulation of the early activation marker, CD69, on both NKT and B cells. In addition, expression of B7.2 and I-A(b) on B cells was greatly upregulated by alpha-GalCer. However, serum levels of IgE, IgG1, and IgG2a were not significantly changed within 48 h. In the present experiments, it was also demonstrated that the upregulation of CD69 expression by alpha-GalCer was strongly blocked by anti-IL-4 monoclonal antibody. Moreover, B-cell activation by alpha-GalCer was not observed in NKT-deficient mice. These results suggested that antigen-stimulated NKT cells might play a critical role not only in early defense mechanisms but also in early B-cell activation through IL-4 production.     

NKT cells and viral immunity

Over the past 10 years a new population of cells has been the focus of much attention. The functions of these unique lymphocytes, characterized by the concomitant expression of T- and NK-cell markers and thus termed NKT cells, have been implicated in many diverse aspects of immunity, including regulation of autoimmune disorders, control of tumour growth and spread, and defence against a number of pathogens. Although much debate still remains as to the natural role of NKT cells, it is clear that these cells have the capacity, either constitutively or postactivation, to promote an amazing array of immunoregulatory responses. The involvement of NKT cells in viral immune-surveillance and their ability to induce protection against pathogens once activated make them an attractive clinical target.     

NKT cells and HIV infection

Natural killer T (NKT) cells are a subset of lymphocytes that express a semi-invariant T cell receptor (TCR) that recognizes glycolipids presented by the non-polymorphic MHC class I-like molecule CD1d. NKT cells regulate a wide variety of immune functions against autoantigens and pathogens. Recently, it was shown that NKT cells are targeted by HIV-1 and selectively lost in HIV-infected individuals. This review will focus on the mechanisms, consequences and therapeutic implications of these findings.     

Preferential activation of an IL-2 regulatory sequence transgene in TCR gamma delta and NKT cells: subset-specific differences in IL-2 regulation

A transgene with 8.4-kb of regulatory sequence from the murine IL-2 gene drives consistent expression of a green fluorescent protein (GFP) reporter gene in all cell types that normally express IL-2. However, quantitative analysis of this expression shows that different T cell subsets within the same mouse show divergent abilities to express the transgene as compared with endogenous IL-2 genes. TCR gamma delta cells, as well as alpha beta TCR-NKT cells, exhibit higher in vivo transgene expression levels than TCR alpha beta cells. This deviates from patterns of normal IL-2 expression and from expression of an IL-2-GFP knock-in. Peripheral TCR gamma delta cells accumulate GFP RNA faster than endogenous IL-2 RNA upon stimulation, whereas TCR alpha beta cells express more IL-2 than GFP RNA. In TCR gamma delta cells, IL-2-producing cells are a subset of the GFP-expressing cells, whereas in TCR alpha beta cells, endogenous IL-2 is more likely to be expressed without GFP. These results are seen in multiple independent transgenic lines and thus reflect functional properties of the transgene sequences, rather than copy number or integration site effects. The high ratio of GFP: endogenous IL-2 gene expression in transgenic TCR gamma delta cells may be explained by subset-specific IL-2 gene regulatory elements mapping outside of the 8.4-kb transgene regulatory sequence, as well as accelerated kinetics of endogenous IL-2 RNA degradation in TCR gamma delta cells. The high levels and percentages of transgene expression in thymic and splenic TCR gamma delta and NKT cells, as well as skin TCR gamma delta-dendritic epidermal T cells, indicate that the IL-2-GFP-transgenic mice may provide valuable tracers for detecting developmental and activation events in these lineages.     

The location of splenic NKT cells favours their rapid activation by blood-borne antigen

Natural killer T (NKT) cells play an important role in mounting protective responses to blood-borne infections. However, though the spleen is the largest blood filter in the body, the distribution and dynamics of NKT cells within this organ are not well characterized. Here we show that the majority of NKT cells patrol around the marginal zone (MZ) and red pulp (RP) of the spleen. In response to lipid antigen, these NKT cells become arrested and rapidly produce cytokines, while the small proportion of NKT cells located in the white pulp (WP) exhibit limited activation. Importantly, disruption of the splenic MZ by chemical or genetic approaches results in a severe reduction in NKT cell activation indicating the need of cooperation between both MZ macrophages and dendritic cells for efficient NKT cell responses. Thus, the location of splenic NKT cells in the MZ and RP facilitates their access to blood-borne antigen and enables the rapid initiation of protective immune responses.     

Role of NKT cells in the digestive system. II. NKT cells and diabetes

Natural killer T (NKT) cells are a subset of regulatory T lymphocytes that recognize glycolipid antigens presented by the major histocompatibility complex class I-related glycoprotein CD1d. NKT cells have been implicated in regulating the progression of Type 1 diabetes (T1D) in human patients and in an animal model for T1D. In addition, glycolipid agonists of NKT cells have been successful in preventing diabetes in mice, raising enthusiasm for the development of NKT cell-based therapies for T1D.     

Caspase-8 and Apaf-1-independent caspase-9 activation in Sendai virus-infected cells

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome c from mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.     

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.     

Characterization of the immature dendritic cells and cytotoxic cells both expanded after activation of invariant NKT cells with alpha-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.