Invariant natural killer T cells: innate-like T cells with potent immunomodulatory activities

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that react with glycolipid antigens presented by the major histocompatibility complex class I-related glycoprotein CD1d. Although iNKT cells express an antigen-specific receptor of the adaptive immune system, they behave more like cells of the innate immune system. A hallmark of iNKT cells is their capacity to produce copious amounts of immunoregulatory cytokines quickly after activation. The cytokines produced by iNKT cells can influence the level of activation of many cell types of the innate and adaptive immune systems as well as the quality of an adaptive immune response. As such, iNKT cells have emerged as important regulators of immune responses, playing a role in microbial immunity, autoimmunity, tumor immunity, and a variety of inflammatory conditions. Although several endogenous and exogenous glycolipid antigens of iNKT cells have been identified, how these glycolipids orchestrate iNKT-cell functions remains poorly understood. Nevertheless, iNKT cells hold substantial promise as targets for development of vaccine adjuvants and immunotherapies. These properties of iNKT cells have been investigated most extensively in mouse models of human disease using the marine sponge-derived agent α-galactosylceramide (α-GalCer) and related iNKT-cell antigens. While these preclinical studies have raised enthusiasm for developing iNKT-cell-based immunotherapies, they also showed potential health risks associated with iNKT cell activation. Although α-GalCer treatment in humans was shown to be safe in the short term, further studies are needed to develop safe and effective iNKT-cell-based therapies.     

自然杀伤T细胞在抗感染免疫中的作用研究进展

自然杀伤T细胞（NKT）是一类同时表达T细胞受体（TCR）dB和NK细胞表面标志的T细胞亚群,能识别由抗原提呈细胞（APC）表面CD1d分子提呈的脂类抗原。NKT细胞经抗原刺激后能迅速分泌大量的细胞因子（如IFN-1和IL-4）,调节固有免疫和适应性免疫,还能直接杀伤靶细胞,具有效应细胞的功能,在机体抗感染、肿瘤和自身免疫病方面发挥重要作用。     

Probiotic antigens stimulate hepatic natural killer T cells

Increasing evidence suggests that gut flora play an important role in the pathogenesis of non‐alcoholic fatty liver disease (NAFLD). Our previous studies show that hepatic natural killer T (NKT) cells play a significant role in the pathogenesis of NAFLD. In this study, we explore the mechanism by which modification of gut flora leads to the alteration of hepatic NKT cells and improvement of steatosis. Mice were fed a high‐fat (HF) diet to induce NAFLD. Some of them also received different doses of mixed‐strain probiotics (VSL#3); single‐strain probiotic (Bifidobacterium infantis) or antibiotics. Animal weight, glucose tolerance, liver steatosis and hepatic NKT cells were assessed. Lipid extracts from probiotics were tested for their ability to activate NKT cells. Toll‐like receptor 4 (TLR4) knockout mice were also evaluated for their responses to HF diet. High‐dose VSL#3 was more effective than low‐dose VSL#3 and B. infantis for the improvement of hepatic NKT cell depletion and steatosis. The lipids extracted from VSL#3 stimulated NKT cells both in vivo and in vitro. In contrast, lipids from B. infantis decreased α‐GalCer‐mediated NKT cell activation in vitro, but were able to stimulate NKT cells. TLR4 knockout mice have a similar response to HF‐diet‐induced NKT cell depletion and obesity. These results suggest that alterations in the gut flora have profound effects on hepatic NKT cells and steatosis, which are both strain‐specific and dose‐dependent, but not through TLR4 signalling. Furthermore, these data suggest that probiotics may contain bacterial glycolipid antigens that directly modulate the effector functions of hepatic NKT cells.     

Adipose invariant natural killer T cells

Adipose tissue is a dynamic organ that makes up a substantial proportion of the body; in severe obesity it can account for 50% of body mass. Details of the unique immune system resident in human and murine adipose tissue are only recently emerging, and so it has remained a largely unexplored and unappreciated immune site until now. Adipose tissue harbours a unique collection of immune cells, which often display unusual functions compared with their counterparts elsewhere in the body. These resident immune cells are key to maintaining tissue and immune homeostasis, yet in obesity their chronic aberrant stimulation can contribute to the inflammation and pathogenesis associated with obesity. Anti‐inflammatory adipose‐resident lymphocytes are often depleted in obesity, whereas pro‐inflammatory immune cells accumulate, leading to an overall inflammatory state, which is a key step in the development of obesity‐induced metabolic disease. A good example is invariant natural killer T (iNKT) cells, which make up a large proportion of lymphocytes in human and murine adipose tissue. Here, they are unusually poised to produce anti‐inflammatory or regulatory cytokines, however in obesity, iNKT cells are greatly reduced. As iNKT cells are potent transactivaors of other immune cells, and can act as a bridge between innate and adaptive immunity, their loss in obesity represents the loss of a major regulatory population. Restoring iNKT cells, or activating them in obese mice leads to improved glucose handling, insulin sensitivity, and even weight loss, and hence represents an exciting therapeutic avenue to be explored for restoring homeostasis in obese adipose tissue.     

Invariant natural killer T cells: linking inflammation and neovascularization in human atherosclerosis

Atherosclerosis, a chronic inflammatory lipid storage disease of large arteries, is complicated by cardiovascular events usually precipitated by plaque rupture or erosion. Inflammation participates in lesion progression and plaque rupture. Identification of leukocyte populations involved in plaque destabilization is important for effective prevention of cardiovascular events. This study investigates CD1d-expressing cells and invariant NKT cells (iNKT) in human arterial tissue, their correlation with disease severity and symptoms, and potential mechanisms for their involvement in plaque formation and/or destabilization. CD1d-expressing cells were present in advanced plaques in patients who suffered from cardiovascular events in the past and were most abundant in plaques with ectopic neovascularization. Confocal microscopy detected iNKT cells in plaques, and plaque-derived iNKT cell lines promptly produced proinflammatory cytokines when stimulated by CD1d-expressing APC-presenting α-galactosylceramide lipid antigen. Furthermore, iNKT cells were diminished in the circulating blood of patients with symptomatic atherosclerosis. Activated iNKT cell-derived culture supernatants showed angiogenic activity in a human microvascular endothelial cell line HMEC-1-spheroid model of in vitro angiogenesis and strongly activated human microvascular endothelial cell line HMEC-1 migration. This functional activity was ascribed to IL-8 released by iNKT cells upon lipid recognition. These findings introduce iNKT cells as novel cellular candidates promoting plaque neovascularization and destabilization in human atherosclerosis.     

Molecular basis of lipid antigen presentation by CD1d and recognition by natural killer T cells

Together with peptides, T lymphocytes respond to hydrophobic molecules, mostly lipids, presented by the non-classical CD1 family (CD1a–e). These molecules have evolved complex and diverse binding grooves in order to survey different cellular compartments for self and exogenous antigens, which are then presented for recognition to T-cell receptors (TCRs) on the surface of T cells. In particular, most CD1d-presented antigens are recognized by a population of lymphocytes denominated natural killer T (NKT) cells, characterized by a strong immunomodulatory potential. Among NKT cells, two major subsets (type I and type II NKT cells) have been described, based on their TCR repertoire and antigen specificity. Here we review recent structural and biochemical studies that have shed light on the molecular details of CD1d-mediated antigen recognition by type I and II NKT cells, which are in many aspects distinct from what has been observed for peptide major histocompatibility complex-reactive TCRs.     

Contribution of Va24Vb11 natural killer T cells in Wilsonian hepatitis

Wilson disease (WD) is an autosomal recessive disorder of copper transport, resulting in copper accumulation and toxicity to the liver and brain. There is no evidence that the WD patient's immune system attacks copper accumulated hepatocytes. Here we describe that the frequency and absolute number of Valpha24+Vbeta11+ natural killer T (NKT) cells were significantly increased in 3 cases of WD, whereas those of CD3+CD161+ NKT cells were within the normal range. Patients no. 1 and 2 had a presymptomatic form of WD. Their tissue specimens showed pathological changes of mild degeneration of hepatocytes with a few infiltrating mononuclear cells and a low degree of fatty change. Patient no. 3 displayed fulminant hepatitis with Coombs-negative haemolytic anaemia. The tissue specimens of patient no. 3 showed macronodular cirrhosis with thick fibrosis, inflammatory infiltrates and spotty necrosis. Human Valpha24+Vbeta11+ NKT cells are almost equal to CD1d-restricted NKT cells. Therefore we investigated CD1d-restricted NKT cells in the LEC rat as an animal model of WD. In LEC rats before hepatitis onset, the number and phenotype of liver NKT cells were normal. At about 4 months of age all LEC rats developed acute hepatitis accompanied by acute jaundice, and CD161high NKT cells developed in their livers. CD161highalphabetaTCRbright NKT cells developed in some of them. Their hepatitis was severe. CD161highalphabetaTCRbright NKT cells expressed an invariant rat Valpha14-Jalpha281 chain, which is CD1d-restricted. Furthermore, liver lymphocytes in the acute jaundiced LEC rats with CD161highalphabetaTCRbright NKT cells had significant and CD1d-specific cytotoxic activity.     

The glycolipid exoantigen derived from Chlamydia muridarum activates invariant natural killer T cells

The chlamydial glycolipid exoantigen (GLXA), a glycolipid antigen derived from Chlamydia muridarum, has been implicated in chlamydial–host cell interaction. Although glycolipid antigens from Sphingomonas and related bacteria have been shown to activate invariant natural killer T (iNKT) cells, it is not yet known whether GLXA can activate these cells. In this study, we have for the first time investigated the role of GLXA in iNKT cell activation using in vitro as well as in vivo settings. First, we examined the effect of GLXA on iNKT cell activation in a cell-free antigen-presentation assay, and found that GLXA specifically stimulated iNKT1.4 hybridoma cell produce enhanced amounts of IL-2. Next, we analyzed the effect of pharmacological activation of iNKT cells by GLXA using iNKT cell-deficient (iNKT knockout (KO)) mice and bone marrow-derived dendritic cell (BMDC)–liver mononuclear cell (LMC) coculture system. On stimulation with GLXA, iNKT cells produced higher quantities of cytokines in a CD1d-dependent fashion. More importantly, iNKT cells from GLXA-treated, but not from cell mock-treated, mice showed higher expression of activation marker, CD69, and enhanced production of interferon (IFN)-γ and IL-4 in vivo. Cumulatively, these data provide evidence on the pharmacological ability of GLXA in specifically activating iNKT cells.     

Invariant natural killer T cells are not affected by lysosomal storage in patients with Niemann-Pick disease type C

Invariant natural killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or NPC2, resulting in the storage of multiple lipids, including glycosphingolipids. In the NPC1 mouse model, iNKT cells are virtually undetectable, which is likely due to the inability of CD1d to be loaded with the selecting ligand due to defective lysosomal function and/or CD1d trafficking. However, in this study we have found that in NPC1 patients iNKT cells are present at normal frequencies, with no phenotypic or functional differences. In addi-tion, antigen-presenting cells derived from NPC1 patients are functionally competent to present several different CD1d/iNKT-cell ligands. This further supports the hypothesis that there are different trafficking requirements for the development of murine and human iNKT cells, and a functional lysosomal/late-endosomal compartment is not required for human iNKT-cell development.     

Regulation of immune responses by CD1d-restricted natural killer T cells

Natural killer T (NKT) cells are a unique subset of T lymphocytes that share receptor structures and properties with conventional T lymphocytes and natural killer (NK) cells. NKT cells are specific for glycolipid antigens such as the marine sponge-derived agent α-galactosylceramide (α-GalCer) presented by the major histocompatibility complex (MHC) class I-like molcule CD1d. My laboratory has evaluated the function of NKT cells by generating and analyzing CD1d-deficient mice. These studies showed that CD1d expression is required for NKT cell development, but not absolutely necessary for the generation of polarized T helper (Th) cell responses. Further, we have studied the in vivo response of NKT cells toα-GalCer stimulation and the capacity of α-GalCer to modulate innate and adaptive immune responses. Our results revealed that, quickly following administration of α-GalCer, NKT cells expand and produce cytokines, trans-activate a variety of innate and adaptive immune cells, and promote Th2 responses that are capable of suppressing Th1-dominant autoimmunity. Our findings indicate that NKT cells play a regulatory role in the immune response and that specific activation of these cells may be exploited for therapeutic purposes.     

Fine specificity of natural killer T cells against GD3 ganglioside and identification of GM3 as an inhibitory natural killer T-cell ligand

GD3, a ganglioside expressed on melanoma, is the only tumour-associated glycolipid described to date that can induce a CD1d-restricted natural killer T (NKT)-cell response. We analysed the fine specificity of GD3-reactive NKT cells and discovered that immunization with GD3 induced two populations of GD3-reactive NKT cells. One population was CD4+ CD8- and was specific for GD3; the other population was CD4- CD8- and cross-reacted with GM3 in a CD1d-restricted manner, but did not cross-react with GM2, GD2, or lactosylceramide. This indicated that the T-cell receptors reacting with GD3 recognize glucose-galactose linked to at least one N-acetyl-neuraminic acid but will not accommodate a terminal N-acetylgalactosamine. Immunization with GM2, GM3, GD2, or lactosylceramide did not induce an NKT-cell response. Coimmunization of GM3-loaded antigen-presenting cells (APCs) with GD3-loaded APCs suppressed the NKT-cell response to GD3 in a CD1d-restricted manner. This suppressive effect was specific for GM3 and was a local effect lasting 2-4 days. In vitro, GM3-loaded APCs also suppressed the interleukin-4 response, but not the interferon-gamma response, of NKT cells to alpha-galactosylceramide. However, there was no effect on the T helper type 2 responses of conventional T cells. We found that this suppression was not mediated by soluble factors. We hypothesize that GM3 induces changes to the APC that lead to suppression of T helper type 2-like NKT-cell responses.     

Hepatic expression of CCL2 in alcoholic liver disease is associated with disease severity and neutrophil infiltrates

Serum levels and liver expression of CCL2 are increased in patients with alcoholic hepatitis (AH). In an experimental model of alcoholic liver disease (ALD), CCL2 was implicated in proinflammatory cytokines activation and hepatic lipid metabolism, but its role in human disease is currently unknown. In a large cohort of ALD patients, we analysed plasma levels and liver expression of CCL2 and their association with liver disease severity and histological lesions. We also studied the relationship between −2518 A > G CCL2 and CCR2 190 A/G polymorphisms and severity of ALD. We show that CCL2 plasma levels are increased in ALD patients compared with healthy subjects. AH patients had significantly higher plasma levels and hepatic expression of CCL2 than patients without AH. Plasma levels and hepatic expression of CCL2 were associated with disease severity. CCL2 liver expression was correlated with neutrophil infiltrate and interleukin (IL)-8 expression, but not with steatosis. Moreover, there were more G-allele carriers of −2518 A > G CCL2 polymorphism in severe AH patients than in other ALD patients. Our results demonstrate that CCL2 is increased in ALD, particularly in severe forms, and suggest a role for CCL2 in the pathogenesis of ALD via neutrophil recruitment.     

In vivo activation of invariant V alpha 14 natural killer T cells by alpha-galactosylceramide sequentially induces Fas-dependent and -independent cytotoxicity

The present study was designed to clarify the cytotoxic capacities of invariant V alpha 14 natural killer T (iNKT) cells activated in vivo. We found that as early as 2 h after a single injection of alpha-galactosylceramide (alpha-GalCer), sorted iNKT splenocytes from treated mice kill Fas-transfected target cells. The implication of the Fas pathway in this lysis was strengthened by both the blockage of cytotoxicity in the presence of anti-Fas ligand (FasL) monoclonal antibody (mAb) and the up-regulation of FasL expression on iNKT cells. Sorted NK cells did not participate in the lytic activity at this time point. Yet, they became cytotoxic later on, 24 h post-treatment, when target cell lysis was mainly independent of the Fas pathway. This type of cell killing was predominant at this later time point, even though iNKT cells conserved a slight Fas-dependent cytotoxicity. NK cells failed to acquire the ability to kill target cells when IFN-gamma production in alpha-GalCer-injected mice was blocked by anti-IFN-gamma mAb, underscoring the major role of this cytokine. In conclusion, our findings provide the first direct evidence that iNKT cells can exert Fas-dependent cytotoxicity very shortly after in vivo alpha-GalCer activation and later, through IFN-gamma secretion, enable NK cells to kill target cells in a Fas-independent pathway.     

Antigen presentation by human uterine epithelial cells to autologous T cells

PROBLEM: Epithelial cells, as sentinels of immune protection in the endometrium, use innate immune mechanisms to protect against infection from pathogenic microbes. Our goal in this study was to assess the ability of human uterine epithelial cells to present antigen to cells of the adaptive immune system. METHOD OF STUDY: Highly purified preparations of uterine epithelial cells from 11 patients were assessed for their ability to present tetanus toxoid (TT) to autologous T cells. Leukocyte contamination in the epithelial cell preparations was numerically and functionally determined. Using confocal microscopy, epithelial cells were tested for the expression of CD40 and CD1d. RESULTS: Purified preparations of endometrial epithelial cells isolated from every patient presented TT recall antigen to autologous T cells. Leukocyte contamination of epithelial cell preparations was insignificant. Uterine epithelial cells express CD40 and CD1d. CONCLUSION: Antigen presentation is an additional aspect of uterine epithelial cell function in maintaining women's health.     

Invariant natural killer T cells balance B cell immunity

Invariant natural killer T (iNKT) cells mediate rapid immune responses which bridge the gap between innate and adaptive responses to pathogens while also providing key regulation to maintain immune homeostasis. Both types of important iNKT immune responses are mediated through interactions with innate and adaptive B cells. As such, iNKT cells sit at the decision‐making fulcrum between regulating inflammatory or autoreactive B cells and supporting protective or regulatory B cell populations. iNKT cells interpret the signals in their environment to set the tone for subsequent adaptive responses, with outcomes ranging from getting licensed to maintain homeostasis as an iNKT regulatory cell (iNKT_reg) or being activated to become an iNKT follicular helper (iNKT_FH) cell supporting pathogen‐specific effector B cells. Here we review iNKT and B cell cooperation across the spectrum of immune outcomes, including during allergy and autoimmune disease, tumor surveillance and immunotherapy, or pathogen defense and vaccine responses. Because of their key role as influencers, iNKT cells provide a valuable target for therapeutic interventions. Understanding the nature of the interactions between iNKT and B cells will enable the development of clinical interventions to strategically target regulatory iNKT and B cell populations or inflammatory ones, depending on the circumstance.     

Endogenous lipid antigens for invariant natural killer T cells hold the reins in adipose tissue homeostasis

The global obesity epidemic and its associated co‐morbidities, including type 2 diabetes, cardiovascular disease and certain types of cancers, have drawn attention to the pivotal role of adipocytes in health and disease. Besides their ‘classical’ function in energy storage and release, adipocytes interact with adipose‐tissue‐resident immune cells, among which are lipid‐responsive invariant natural killer T (iNKT) cells. The iNKT cells are activated by lipid antigens presented by antigen‐presenting cells as CD1d/lipid complexes. Upon activation, iNKT cells can rapidly secrete soluble mediators that either promote or oppose inflammation. In lean adipose tissue, iNKT cells elicit a predominantly anti‐inflammatory immune response, whereas obesity is associated with declining iNKT cell numbers. Recent work showed that adipocytes act as non‐professional antigen‐presenting cells for lipid antigens. Here, we discuss endogenous lipid antigen processing and presentation by adipocytes, and speculate on how these lipid antigens, together with ‘environmental factors’ such as tissue/organ environment and co‐stimulatory signals, are able to influence the fate of adipose‐tissue‐resident iNKT cells, and thereby the role of these cells in obesity and its associated pathologies.     

Focal hepatocellular necrosis and portal lymphocytic infiltration of the liver in chronic alcoholics: Histopathological study of 40 liver biopsies

Forty liver biopsies of hepatitis B surface antigen negative chronic alcoholics were histologically studied to assess the influence of hepatitis C virus (HCV)-infection. A moderate degree of focal hepatocellular necrosis and/or portal lymphocytic infiltration (FHN-PLI) was observed in 28% of the specimens, being especially prevalent in advanced cases of fibrosis and cirrhosis, and significantly correlated with HCV-infection. Eleven of these cases were examined in detail: HCV-infection was detected in eight and lymphocytic infiltration was apparent in the portal area, accompanied by formation of lymph follicles. The FHN-PLI was ascribed to alcoholic hepatitis in two HCV-negative cases and was concluded to be of unknown etiology In the remaining one HCV-negative case. This study thus indicated that over two-thirds of cases of chronic hepatitis in alcoholics can be attributed to HCV-infection, with the remainder being at least partly related to alcoholic hepatitis. The prevalence of alcohol-induced chronic hepatitis based on immunopathological findings was unclear, but was probably less frequent than previously reported.     

自然杀伤T细胞在抗感染免疫中的作用研究进展

Natural killer T cell(NKT) are a unique T cell lineage expressing T cell receptor(TCR)and natural killer cell(NK) lineage receptors, and can recognize glycolipids presented by CD1d molecules expressed on antigen presenting cell through cell recognition pathway. After activated, NKT cells can release large amounts of cytokines, such as IL-4 and IFN-γ, and play a regulatory role in both innate and adaptive immune response. NKT cells can also act as direct effector cells via cytolysis or granulysin. NKT cells play an important role in infectious diseases, autoimmune disease,and cancer.