Hyaluronan induces cell death in activated T cells through CD44

In the immune system, leukocyte activation induces CD44 to bind hyaluronan, a component of the extracellular matrix. Here we used gain and loss of hyaluronan-binding mutants of CD44 to examine the consequence of hyaluronan binding in T cells. Jurkat T cells transfected with CD44 mutated at S180, which prevented the addition of chondroitin sulfate, displayed constitutively high levels of hyaluronan binding. These cells were more susceptible to activation-induced cell death, whereas cells expressing a CD44 mutant unable to bind hyaluronan (R41A) were resistant to cell death. In TCR or PMA activated Jurkat T cells, hyaluronan induced rapid cell death. This depended on the level of hyaluronan binding by the cell, and the amount and size of hyaluronan. High molecular mass hyaluronan had the greatest effect and cell death occurred independently of Fas and caspase activation. In splenic T cells, high hyaluronan binding occurred in a subpopulation of cells undergoing activation-induced cell death. In addition, hyaluronan induced cell death in approximately 10% of reactivated splenic T cells when Fas-dependent apoptosis was prevented by Ab blocking or in Fas negative MRL/lpr T cells. This demonstrates that hyaluronan can induce cell death in activated, high hyaluronan binding T cells via a Fas-independent mechanism.     

DX5/CD49b-positive T cells are not synonymous with CD1d-dependent NKT cells

NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR alpha-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d-/- mice that are completely deficient for NKT cells. CD1d tetramer- CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer- CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.     

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.     

Efficient activation of Valpha14 invariant NKT cells by foreign lipid antigen is associated with concurrent dendritic cell-specific self recognition

A burst release of cytokines by Valpha14 invariant NKT (iNKT) cells upon their TCR engagement critically regulates innate and adaptive immune responses. However, it remains unclear in vivo why iNKT cells respond efficiently to microbial or intracellular lipid Ags that are at low levels or that possess suboptimal antigenicity. We found that dendritic cells (DCs) potentiated iNKT cells to respond to a minimal amount of ligand alpha-galactosylceramide (alphaGalCer) through CD1d-dependent autoreactive responses that require endosomal processing and CD1d trafficking. The ability of potentiation of NKT cells was DC specific and did not depend on costimulatory signals and IL-12 production by DCs. However, DCs that failed to synthesize a major endogenous lipid Ag isoglobotrihexosylceramide were unable to potentiate NKT cells for efficient activation. Further analysis showed that differences in the level and pattern of endogenous lipid Ag presentation differentiate DCs and B cells for effective potentiation and subsequent activation of iNKT cells in the presence of an exogenous Ag. Thus, CD1d-dependent potentiation by DCs may be crucial for iNKT cell-mediated immunity against infectious agents.     

Immunotherapy with ligands of natural killer T cells

Natural killer T (NKT) cells are innate lymphocytes that share receptor structures and functions with conventional T cells and natural killer cells. NKT cells are specific for glycolipid antigens bound by the major histocompatibility complex class I-like protein CD1d. One striking property of NKT cells is their capacity to rapidly produce large amounts of cytokines in response to T-cell receptor engagement, suggesting that activated NKT cells can modulate adaptive immune responses. Recent pre-clinical studies have revealed significant efficacy of NKT-cell ligands such as the glycolipid alpha-galactosylceramide for treatment of metastatic cancers and infections, and for prevention of autoimmune diseases. These findings suggest that appropriate stimulation of NKT cells could be exploited for prevention or treatment of human diseases.     

Direct regulatory role of NKT cells in allogeneic graft survival is dependent on the quantitative strength of antigenicity

The role of NKT cells during immune responses is diverse, ranging from antiviral and antitumor activity to the regulation of autoimmune diseases; however, the regulatory function of CD1d-dependent NKT cells in rejection responses against allogeneic graft is uncertain. In this study, we demonstrated the direct regulatory effects of CD1d-dependent NKT cells using an allogeneic skin transplantation model. H-Y-mismatched skin graft survival was shortened in CD1d-/- recipients compared with wild-type recipients. Adoptive transfer of syngeneic NKT cells via splenocytes or hepatic mononuclear cells into CD1d-/- recipients restored graft survival times to those of wild-type recipients. alpha-Galactosylceramide, a specific activator of NKT cells, further prolonged graft survival. Although CD1d-dependent NKT cells did not extend skin graft survival in either major or complete minor histocompatibility-mismatched models, these cells affected graft survival in minor Ag mismatch models according to the magnitude of the antigenic difference. The afferent arm of NKT cell activation during transplantation required CD1d molecules expressed on host APCs and the migration of CD1d-dependent NKT cells into grafts. Moreover, the regulatory effects of CD1d-dependent NKT cells against alloantigen were primarily IL-10 dependent. Taken together, we concluded that CD1d-dependent NKT cells may directly affect the outcome of allogeneic skin graft through an IL-10-dependent regulatory mechanism.     

Activation of invariant NKT cells by the helminth parasite schistosoma mansoni

Mouse CD1d-restricted NKT cells, including invariant (i)NKT cells, are innate cells activated by glycolipid Ags and play important roles in the initiation and regulation of immune responses. Through their ability to promptly produce large amounts of Th1 and/or Th2 cytokines upon TCR engagement, iNKT cells exert crucial functions in the immune/inflammatory system during bacterial, protozoan, fungal, and viral infections. However, their roles during metazoan parasite infection, which are generally associated with strong Th2 responses, still remain elusive. In this study, we show that during the course of murine schistosomiasis, iNKT cells exhibit an activated phenotype and that following schistosome egg encounter in the liver, hepatic iNKT cells produce both IFN-gamma and IL-4 in vivo. We also report that schistosome egg-sensitized dendritic cells (DCs) activate, in a CD1d-dependent manner, iNKT cells to secrete IFN-gamma and IL-4 in vitro. Interestingly, transfer of egg-sensitized DCs promotes a strong Th2 response in recipient wild-type mice, but not in mice that lack iNKT cells. Engagement of TLRs in DCs is not necessary for iNKT cell stimulation in response to egg-sensitized DCs, suggesting an alternative pathway of activation. Finally, we propose that self, rather than parasite-derived, CD1d-restricted ligands are implicated in iNKT cell stimulation. Taken together, our data show for the first time that helminths can activate iNKT cells to produce immunoregulatory cytokines in vivo, enabling them to influence the adaptive immune response.     

The interaction between regulatory T cells and NKT cells in the liver: a CD1d bridge links innate and adaptive immunity

Background/Aims

Regulatory T cells (Tregs) and natural killer T (NKT) cells are two distinct lymphocyte subsets that independently regulate hepatic adaptive and innate immunity, respectively. In the current study, we examine the interaction between Tregs and NKT cells to understand the mechanisms of cross immune regulation by these cells.

Methods

The frequency and function of Tregs were evaluated in wild type and NKT cell deficient (CD1dko) mice. In vitro lymphocyte proliferation and apoptosis assays were performed with NKT cells co-cultured with Tregs. The ability of Tregs to inhibit NKT cells in vivo was examined by adoptive transfer of Tregs in a model of NKT cell mediated hepatitis.

Results

CD1dko mice have a significant reduction in hepatic Tregs. Although, the Tregs from CD1dko mice remain functional and can suppress conventional T cells, their ability to suppress activation induced NKT cell proliferation and to promote NKT cell apoptosis is greatly diminished. These effects are CD1d dependent and require cell to cell contact. Adoptive transfer of Tregs inhibits NKT cell-mediated liver injury.

Conclusions

NKT cells promote Tregs, and Tregs inhibit NKT cells in a CD1d dependent manner requiring cell to cell contact. These cross-talk immune regulations provide a linkage between innate and adaptive immunity.     

The effect of intracellular trafficking of CD1d on the formation of TCR repertoire of NKT cells

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire. [BMB Reports 2014; 47(5): 241-248]     

Regulation of antitumour immunity by CD1d-restricted NKT cells

An understanding of the complex interactions occurring between tumours and the immune system is a prerequisite for the rational design of effective cancer immunotherapies. To date, attention has focused mainly on the role the adaptive immune system plays in controlling tumourigenesis, with conventional T cells, which recognize peptide antigens presented by classical MHC molecules, coming under close scrutiny. Accumulating reports now suggest that an additional T-cell subset, known as CD1d-restricted natural killer T (NKT) cells, also plays a pivotal role in modulating antitumour responses. Found in both humans and mice, CD1d-restricted NKT cells are a highly specialized cell type that, in contrast to conventional T cells, recognize lipid/glycolipid antigens presented by the non-classical MHC molecule CD1d. Several features of NKT cells, including their ability to rapidly produce large quantities of cytokines upon primary stimulation, make them ideal targets for developing anticancer immunotherapies. This intriguing cell type is the focus of this review.     

CD1d-specific NK1.1+ T cells with a transgenic variant TCR

The majority of T lymphocytes carrying the NK cell marker NK1.1 (NKT cells) depend on the CD1d molecule for their development and are distinguished by their potent capacity to rapidly secrete cytokines upon activation. A substantial fraction of NKT cells express a restricted TCR repertiore using an invariant TCR Valpha14-Jalpha281 rearrangement and a limited set of TCR Vbeta segments, implying recognition of a limited set of CD1d-associated ligands. A second group of CD1d-reactive T cells use diverse TCR potentially recognizing a larger diversity of ligands presented on CD1d. In TCR-transgenic mice carrying rearranged TCR genes from a CD1d-reactive T cell with the diverse type receptor (using Valpha3. 2/Vbeta9 rearrangements), the majority of T cells expressing the transgenic TCR had the typical phenotype of NKT cells. They expressed NK1.1, CD122, intermediate TCR levels, and markers indicating previous activation and were CD4/CD8 double negative or CD4+. Upon activation in vitro, the cells secreted large amounts of IL-4 and IFN-gamma, a characteristic of NKT cells. In mice lacking CD1d, TCR-transgenic cells with the NKT phenotype were absent. This demonstrates that a CD1d-reactive TCR of the "non-Valpha 14" diverse type can, in a ligand-dependent way, direct development of NK1.1+ T cells expressing expected functional and cell-surface phenotype characteristics.     

Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids

NKT cells represent a unique subset of immunoregulatory T cells that recognize glycolipid Ags presented by the MHC class I-like molecule CD1d. Because of their immunoregulatory properties, NKT cells are attractive targets for the development of immunotherapies. The prototypical NKT cell ligand alpha-galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, has potent immunomodulatory activities in mice, demonstrating therapeutic efficacy against metastatic tumors, infections, and autoimmune diseases, but also has a number of adverse side effects. In vivo administration of alpha-GalCer to mice results in the rapid activation of NKT cells, which is characterized by cytokine secretion, surface receptor down-regulation, expansion, and secondary activation of a variety of innate and adaptive immune system cells. In this study, we have evaluated the in vivo immune response of mice to a set of structural analogues of alpha-GalCer. Our results show that, contrary to current thinking, beta-anomeric GalCer can induce CD1d-dependent biological activities in mice, albeit at lower potency than alpha-anomeric GalCer. In addition, we show that the response of NKT cells to distinct GalCer differs not only quantitatively, but also qualitatively. These findings indicate that NKT cells can fine-tune their immune responses to distinct glycolipid Ags in vivo, a property that may be exploited for the development of effective and safe NKT cell-based immunotherapies.     

自然杀伤T细胞抗胞内菌感染的研究进展

自然杀伤T细胞(NKT细胞)是免疫细胞中一类具有特定标记的T细胞亚群,能特异性识别南抗原呈递细胞表面CD1d分子所呈递的糖脂类抗原.活化后的NKT细胞能分泌多种细胞因子,参与机体的天然免疫和获得性免疫反应,还能直接杀伤靶细胞,具有效应细胞的功能.研究发现,NKT细胞在抗胞内菌感染中发挥着重要作用.     

Emerging role of bystander T cell activation in autoimmune diseases

Autoimmune disease is known to be caused by unregulated self-antigen-specific T cells, causing tissue damage. Although antigen specificity is an important mechanism of the adaptive immune system, antigen non-related T cells have been found in the inflamed tissues in various conditions. Bystander T cell activation refers to the activation of T cells without antigen recognition. During an immune response to a pathogen, bystander activation of self-reactive T cells via inflammatory mediators such as cytokines can trigger autoimmune diseases. Other antigen-specific T cells can also be bystander-activated to induce innate immune response resulting in autoimmune disease pathogenesis along with self-antigen-specific T cells. In this review, we summarize previous studies investigating bystander activation of various T cell types (NKT, γδ T cells, MAIT cells, conventional CD4⁺, and CD8⁺ T cells) and discuss the role of innate-like T cell response in autoimmune diseases. In addition, we also review previous findings of bystander T cell function in infection and cancer. A better understanding of bystander-activated T cells versus antigen-stimulated T cells provides a novel insight to control autoimmune disease pathogenesis.     

Natural killer T cells as targets for immunotherapy of autoimmune diseases

CD1d-restricted natural killer T (NKT) cells are innate lymphocytes that play a regulatory role during an immune response. The identification of alpha-galactosylceramide (alpha-GalCer), a marine sponge-derived glycosphingolipid, as a potent stimulator of NKT cells led many laboratories to investigate the effects of NKT cell activation on the regulation of immune responses. These studies revealed that alpha-GalCer induces rapid and robust cytokine production by NKT cells, secondary activation of a variety of innate and adaptive immune cells, and modulation of Th cell responses. Further, alpha-GalCer influences disease progression in a variety of experimental models of autoimmunity and inflammation in mice, including models for type 1 diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, and atherosclerosis. While these studies have raised significant enthusiasm for manipulation of NKT cells as a means of preventing autoimmunity in the clinical setting, there are significant concerns regarding the safety of repeated alpha-GalCer injections in human subjects.     

Cutting edge: NKT cell development is selectively impaired in Fyn- deficient mice.

Most NK1.1+ T (NKT) cells express a biased TCRalphabeta repertoire that is positively selected by the monomorphic MHC class I-like molecule CD1d. The development of CD1d-dependent NKT cells is thymus dependent but, in contrast to conventional T cells, requires positive selection by cells of hemopoietic origin. Here, we show that the Src protein tyrosine kinase Fyn is required for development of CD1d-dependent NKT cells but not for the development of conventional T cells. In contrast, another Src kinase, Lck, is required for the development of both NKT and T cells. Impaired NKT cell development in Fyn-deficient mice cannot be rescued by transgenic expression of CD8, which is believed to increase the avidity of CD1d recognition by NKT cells. Taken together, our data reveal a selective and nonredundant role for Fyn in NKT cell development.     

Lack of chemokine receptor CCR5 promotes murine fulminant liver failure by preventing the apoptosis of activated CD1d-restricted NKT cells

Fulminant liver failure (FLF) consists of a cascade of events beginning with a presumed uncontrolled systemic activation of the immune system. The etiology of FLF remains undefined. In this study, we demonstrate that CCR5 deficiency promotes the development of acute FLF in mice following Con A administration by preventing activated hepatic CD1d-restricted NKT cells (but not conventional T cells) from dying from activation-induced apoptosis. The resistance of CCR5-deficient NKT cells from activation-induced apoptosis following Con A administration is not due to a defective Fas-driven death pathway. Moreover, FLF in CCR5-deficient mice also correlated with hepatic CCR5-deficient NKT cells, producing more IL-4, but not IFN-gamma, relative to wild-type NKT cells. Furthermore, FLF in these mice was abolished by IL-4 mAb or NK1.1 mAb treatment. We propose that CCR5 deficiency may predispose individuals to the development of FLF by preventing hepatic NKT cell apoptosis and by regulating NKT cell function, establishing a novel role for CCR5 in the development of this catastrophic liver disease that is independent of leukocyte recruitment.     

Cutting edge: activation by innate cytokines or microbial antigens can cause arrest of natural killer T cell patrolling of liver sinusoids

Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of alpha-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-gamma, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-gamma production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, alpha-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response.     

Role of natural killer T lymphocytes during helminthic infection

Natural killer (NK)T cells are innate lymphocytes that release important amount of immunoregulatory cytokines (IFN-gamma and/or IL-4) shortly after T cell receptor engagement by (glyco)lipid antigens presented by the CD1d molecules. Through this property, NKT cells play pivotal role in many physiopathologic situations. Here, we review the current knowledge of the functions and mechanisms of activation of NKT cells during infection, with a particular emphasis on helminthic infections. Recent findings suggest that, although dispensable for host resistance, NKT cells play part in the development of the acquired immune response and in the control of the pathology during murine schistosomiasis.