Sequential production of interferon-gamma by NK1.1(+) T cells and natural killer cells is essential for the antimetastatic effect of alpha-galactosylceramide

The antimetastatic effect of the CD1d-binding glycolipid, alpha-galactosylceramide (alpha-GalCer), is mediated by NK1.1(+)T (NKT) cells; however, the mechanisms behind this process are poorly defined. Although it has been shown to involve NK cells and interferon-gamma (IFN-gamma) production, the way these factors collaborate to mediate effective tumor rejection and the importance of other factors characteristic of NKT cell and NK cell activation are unknown. Using gene-targeted mice and antibody treatments, the critical need for interleukin 12 (IL-12), IFN-gamma, and NK cells has been shown in the antimetastatic activity of alpha-GalCer in the lungs and the liver. By contrast, in lung and liver metastasis models, cytotoxic molecules expressed by NK cells and NKT cells (perforin, Fas ligand, and tumor necrosis factor-related apoptosis-inducing ligand) and an NKT cell-secreted cytokine, IL-4, were not necessary for the antitumor activity of alpha-GalCer. Like IL-12, IL-18 was required for optimal serum IFN-gamma induction and control of lung metastases by alpha-GalCer. IL-18 was unnecessary for alpha-GalCer-related suppression of liver metastases. Most importantly, after adoptive transfer of alpha-GalCer-reactive NKT cells or NK cells into NKT cell-deficient, IFN-gamma-deficient, or RAG-1-deficient mice, it was demonstrated that the sequential production of IFN-gamma by NKT cells and NK cells was absolutely required to reconstitute the antimetastatic activity of alpha-GalCer.     

Neutralization of tumor necrosis factor abrogates hepatic failure induced by alpha-galactosylceramide without attenuating its antitumor effect in aged mice

BACKGROUND/AIMS: The functions of mouse liver NK1.1+ T (NKT) cells stimulated with alpha-galactosylceramide (alpha-GalCer) are enhanced age dependently, and the antitumor and anti-metastatic effect in the liver is dependent on IFN-gamma. However, hepatic injury is independent of IFN-gamma and Fas/Fas-ligand dependent. The aim of this study is to investigate how tumor necrosis factor is involved in the alpha-GalCer-mediated immune phenomena. METHODS: C57BL/6 mice were intraperitoneally treated with anti-TNF antibody 1 h before alpha-GalCer injection, and Fas-ligand expression of NKT cells, the serum ALT levels and histopathological findings of the liver, kidney and lung and mortality after alpha-GalCer injection were evaluated. IFN-gamma production and antitumor immunity in the liver after the intravenous injection of EL-4 cells were also assessed. RESULTS: Serum TNF levels after alpha-GalCer injection increased age dependently in mice. Anti-TNF Ab reduced Fas-ligand (Fas-L) expression of NKT cells while it completely inhibited organ injuries induced by alpha-GalCer and thereby reduced the mortality of old mice, whereas it did not affect the IFN-gamma production from NKT cells, the antitumor immunity in the liver nor the mouse survival after EL-4 injection. CONCLUSIONS: NKT cells activated by alpha-galactosylceramide participated in either antitumor immunity or hepatic injury using IFN-gamma and TNF/Fas-L, respectively.     

Concanavalin a injection activates intrahepatic innate immune cells to provoke an antitumor effect in murine liver

Concanavalin A (ConA), directly injected into mice, induces T cell-mediated liver injury. However, it remains unclear whether ConA injection can activate innate immune cells, including natural killer (NK) cells and natural killer T (NKT) cells, both of which exist abundantly in the liver. Here we report that ConA injection stimulated interferon (IFN)-gamma production from liver NKT cells as early as 2 hours after injection and augmented YAC-1 cytotoxicity of liver NK cells. ConA-induced NK cell activation required other types of immune cells and critically depended on IFN-gamma. Because a nonhepatotoxic low dose of ConA was capable of fully activating both NKT cells and NK cells, we next addressed the possibility of ConA injection displaying an antitumor effect in the liver without liver injury. A nonhepatotoxic low-dose ConA injection augmented the cytotoxicity of liver NK cells against Colon-26 colon cancer cells and suppressed hepatic metastasis of Colon-26 cells in a NK cell- and IFN-gamma-dependent manner. In conclusion, a nonhepatotoxic low dose of ConA might serve as an immunomodulator that can preferentially activate the innate immune cells to induce an antitumor effect against metastatic liver tumor.     

Involvement of decidual Valpha14 NKT cells in abortion

The immunological mechanisms that regulate abortion are largely unknown. Here, we found that a distinct subset of lymphocytes, Valpha14 NKT cells expressing an invariant antigen receptor encoded by Valpha14/Jalpha281 and Vbeta7 segments, accumulated in the decidua during pregnancy and provoked abortion upon stimulation with alpha-galactosylceramide (alpha-GalCer), a specific ligand for Valpha14 NKT cells. The alpha-GalCer-mediated abortion was not observed in Valpha14 NKT-, IFN-gamma-, tumor necrosis factor alpha-, or perforin-knock-out mice and appeared to be due to the degeneration of embryonic trophoblasts mediated by the activated Valpha14 NKT cells whose perforin-dependent killing and production of IFN-gamma and tumor necrosis factor alpha were essential. The possible role of the decidual Valpha14 NKT cells in the pathogenesis of abortion is discussed.     

alpha -galactosylceramide-activated Valpha 14 natural killer T cells mediate protection against murine malaria

Natural killer T (NKT) cells are a unique population of lymphocytes that coexpress a semiinvariant T cell and natural killer cell receptors, which are particularly abundant in the liver. To investigate the possible effect of these cells on the development of the liver stages of malaria parasites, a glycolipid, alpha-galactosylceramide (alpha-GalCer), known to selectively activate Valpha14 NKT cells in the context of CD1d molecules, was administered to sporozoite-inoculated mice. The administration of alpha-GalCer resulted in rapid, strong antimalaria activity, inhibiting the development of the intrahepatocytic stages of the rodent malaria parasites Plasmodium yoelii and Plasmodium berghei. The antimalaria activity mediated by alpha-GalCer is stage-specific, since the course of blood-stage-induced infection was not inhibited by administration of this glycolipid. Furthermore, it was determined that IFN-gamma is essential for the antimalaria activity mediated by the glycolipid. Taken together, our results provide the clear evidence that NKT cells can mediate protection against an intracellular microbial infection.     

Contribution of natural killer cells to inhibition of angiogenesis by interleukin-12

Interleukin-12 (IL-12) inhibits angiogenesis in vivo by inducing interferon-gamma (IFN-gamma) and other downstream mediators. Here, we report that neutralization of natural killer (NK) cell function with antibodies to either asialo GM1 or NK 1.1 reversed IL-12 inhibition of basic fibroblast growth factor (bFGF)-induced angiogenesis in athymic mice. By immunohistochemistry, those sites where bFGF-induced neovascularization was inhibited by IL-12 displayed accumulation of NK cells and the presence of IP-10-positive cells. Based on expression of the cytolytic mediators perforin and granzyme B, the NK cells were locally activated. Experimental Burkitt lymphomas treated locally with IL-12 displayed tumor tissue necrosis, vascular damage, and NK-cell infiltration surrounding small vessels. After activation in vitro with IL-12, NK cells from nude mice became strongly cytotoxic for primary cultures of syngeneic aortic endothelial cells. Cytotoxicity was neutralized by antibodies to IFN-gamma. These results document that NK cells are required mediators of angiogenesis inhibition by IL-12, and provide evidence that NK-cell cytotoxicity of endothelial cells is a potential mechanism by which IL-12 can suppress neovascularization.     

Augmentation of lipopolysaccharide-induced nitric oxide production by alpha-galactosylceramide in mouse peritoneal cells

The effect of alpha-galactosylceramide (alpha-GalCer) on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in mouse peritoneal cells was studied. alpha-GalCer augmented LPS-induced NO production in mouse peritoneal cells, but not in RAW 264.7 macrophage cells. alpha-GalCer augmented NO production, but not tumor necrosis factor (TNF)-alpha production in LPS-stimulated peritoneal cells. Peritoneal cells produced a significant level of interferon (IFN)-gamma in response to alpha-GalCer and anti-IFN-gamma antibody abolished the augmentation of LPS-induced NO production by alpha-GalCer. Moreover, anti-IFN-gamma antibody prevented the enhanced expression of an inducible type of NO synthase mRNA by alpha-GalCer. alpha-GalCer did not augment LPS-induced NO production in peritoneal cells from natural killer T (NKT)-deficient mice. Therefore, it was suggested that alpha-GalCer might augment LPS-induced NO production in peritoneal cells through release of IFN-gamma from NKT cells.     

Glycolipid alpha-C-galactosylceramide is a distinct inducer of dendritic cell function during innate and adaptive immune responses of mice

alpha-Galactosylceramide (alpha-GalCer) is the prototype compound for studying the presentation of glycolipids on CD1d molecules to natural killer T (NKT) lymphocytes. A single i.v. dose of glycolipid triggers a cascade of events involving the production of several cytokines over the course of a day, a short-lived activation of NKT and natural killer (NK) cells, and a more prolonged adaptive T cell immune response if certain antigens are given together with alpha-GalCer. We find that a recently described analogue, alpha-C-galactosylceramide (alpha-C-GalCer), more potently induces these innate and adaptive immune responses in mice. alpha-C-GalCer acts as a more effective trigger for IL-12 and IFN-gamma production, although it minimally elicits IL-4 and TNF-alpha release into the serum. Also, alpha-C-GalCer better mobilizes NKT and natural killer cells to resist B16 melanoma. To help understand these effects, we find that alpha-C-GalCer binds more stably to dendritic cells than alpha-GalCer and that dendritic cells loaded with alpha-C-GalCer induce larger and more long lasting NKT cell responses in vivo. When glycolipid is targeted to dendritic cells in spleen together with antigens in dying cells, such as irradiated tumor cells, alpha-C-GalCer is active as an adjuvant for T cell-mediated immunity at lower doses, just 20 ng per mouse, where it is also able to up-regulate the required CD40L costimulatory molecule on NKT cells. Therefore, alpha-C-GalCer represents a glycolipid that binds more stably to dendritic cells and acts as a more effective link between innate and adaptive immunity in vivo.     

The T cell antigen receptor expressed by Valpha14i NKT cells has a unique mode of glycosphingolipid antigen recognition

Natural killer (NK) T cells with an invariant Valpha14 rearrangement (Valpha14i) are the largest population of lipid antigen-specific T lymphocytes identified in animals. They react to the glycolipid alpha-galactosyl ceramide (alpha-GalCer) presented by CD1d, and they may have important regulatory functions. It was previously shown that the Valpha14i T cell antigen receptor (TCR) has a high affinity for the alpha-GalCer/CD1d complex, driven by a long half-life (t(1/2)). Although this result could have reflected the unique attributes of alpha-GalCer, using several related glycolipid compounds, we show here that the threshold for full activation of Valpha14i NKT cells by these glycosphingolipids requires a relatively high-affinity TCR interaction with a long t(1/2). Furthermore, our data are consistent with the view that the mechanism of recognition of these compounds presented by CD1d to the Valpha14i NKT cell TCR is likely to fit a lock-and-key model. Overall, these findings emphasize the distinct properties of glycosphingolipid antigen recognition by Valpha14i NKT cells.     

Expansion of cytolytic CD8(+) natural killer T cells with limited capacity for graft-versus-host disease induction due to interferon gamma production

T cells with natural killer cell phenotype and function (NKT cells) have been described in both human and murine tissues. In this study, culture conditions were developed that resulted in the expansion of CD8(+) NKT cells from bone marrow, thymus, and spleen by the timed addition of interferon-gamma (IFN-gamma), interleukin 2 (IL-2), and anti-CD3 monoclonal antibody. After 14 to 21 days in culture, dramatic expansion of CD3(+), CD8(+), alphabetaT-cell receptor(+) T cells resulted with approximately 20% to 50% of the cells also expressing the NK markers NK1.1 and DX5. The CD8(+) NKT cells demonstrated lytic activity against several tumor target cells with more than 90% lysis by day 14 to day 21 of culture. Cytotoxicity was observed against both syngeneic and allogeneic tumor cell targets with the greatest lytic activity by the cells expressing either NK1.1 or DX5. The expanded CD8(+) NKT cells produce T(H)1-type cytokines with high levels of IFN-gamma and tumor necrosis factor alpha. Expansion of the CD8(+) NKT cells was independent of CD1d. Ly49 molecules were expressed on only a minority of cells. A single injection of expanded CD8(+) NKT cells was capable of protecting syngeneic animals from an otherwise lethal dose of Bcl1 leukemia cells. Expanded CD8(+) NKT cells produced far less graft-versus-host disease (GVHD) than splenocytes across major histocompatibility barriers, even when 10 times the number of CD8(+) NKT cells as compared to splenocytes were injected. This reduction in GVHD was related to IFN-gamma production since cells expanded from IFN-gamma knock-out animals caused acute lethal GVHD, whereas cells expanded from animals defective in fas ligand, fas, IL-2, and perforin did not. These data indicate that CD8(+) NKT cells expanded in this fashion could be useful for preserving graft-versus-leukemia activity without causing GVHD.     

Therapeutic activation of Valpha24+Vbeta11+ NKT cells in human subjects results in highly coordinated secondary activation of acquired and innate immunity

Human Valpha24+Vbeta11+ natural killer T (NKT) cells are a distinct CD1d-restricted lymphoid subset specifically and potently activated by alpha-galactosylceramide (alpha-GalCer) (KRN7000) presented by CD1d on antigen-presenting cells. Preclinical models show that activation of Valpha24+Vbeta11+ NKT cells induces effective antitumor immune responses and potentially important secondary immune effects, including activation of conventional T cells and NK cells. We describe the first clinical trial of cancer immune therapy with alpha-GalCer-pulsed CD1d-expressing dendritic cells. The results show that this therapy has substantial, rapid, and highly reproducible specific effects on Valpha24+Vbeta11+ NKT cells and provide the first human in vivo evidence that Valpha24+Vbeta11+ NKT cell stimulation leads to activation of both innate and acquired immunity, resulting in modulation of NK, T-, and B-cell numbers and increased serum interferon-gamma. We present the first clinical evidence that Valpha24+Vbeta11+ NKT cell memory produces faster, more vigorous secondary immune responses by innate and acquired immunity upon restimulation.     

Functional alterations of liver innate immunity of mice with aging in response to CpG-oligodeoxynucleotide

Immune functions of liver natural killer T (NKT) cells induced by the synthetic ligand alpha-galactosylceramide enhanced age-dependently; hepatic injury and multiorgan dysfunction syndrome (MODS) induced by ligand-activated NKT cells were also enhanced. This study investigated how aging affects liver innate immunity after common bacteria DNA stimulation. Young (6 weeks) and old (50-60 weeks) C57BL/6 mice were injected with CpG oligodeoxynucleotides (CpG-ODN), and the functions of liver leukocytes were assessed. A CpG-ODN injection into the old mice remarkably increased tumor necrosis factor (TNF) production in Kupffer cells, and MODS and lethal shock were induced, both of which are rarely seen in young mice. Old Kupffer cells showed increased Toll-like receptor-9 expression, and CpG-ODN challenge augmented TNF receptor and Fas-L expression in liver NKT cells. Experiments using mice depleted of natural killer (NK) cells by anti-asialoGM1 antibody (Ab), perforin knockout mice, and mice pretreated with neutralizing interferon (IFN)-gamma Ab demonstrated the important role of liver NK cells in antitumor immunity. The production capacities of old mice for IFN-gamma, IFN-alpha, and perforin were much lower than those of young mice, and the CpG-induced antitumor cytotoxicity of liver NK cells lessened. Lethal shock and MODS greatly decreased in old mice depleted/deficient in TNF, FasL, or NKT cells. However, depletion of NK cells also decreased serum TNF levels and FasL expression of NKT cells, which resulted in improved hepatic injury and survival, suggesting that NK cells are indirectly involved in MODS/lethal shock induced by NKT cells. Neutralization of TNF did not reduce the CpG-induced antitumor effect in the liver. CONCLUSION: Hepatic injury and MODS mediated by NKT cells via the TNF and FasL-mediated pathway after CpG injection increased, but the antitumor activity of liver NK cells decreased with aging.     

Application of Natural Killer T-Cells to Posttransplantation Immunotherapy

Graft-versus-host disease (GVHD) and graft-versus leukemia (GVL) effects are closely related to each other after allogeneic stem cell transplantation. This association exists because of the extensive and complicated interaction between cellular donor components and recipient components concomitant with cytokine storms. It has been demonstrated that part of this interaction may be related to the induction of a variety of regulatory cells, such as regulatory T-cells and natural killer T (NKT) cells. A lower number of NKT cells may be found in patients with autoimmune diseases, cancer, viral infection, and severe GVHD. When activated, NKT cells rapidly release suppressive cytokines, such as interleukin 4 (IL-4), IL-10, and IL-13, as well as inflammatory cytokines, such as interferon gamma and tumor necrosis factor alpha. NKT cells therefore act as a double-edged sword in their progressive or suppressive effects on diseases. Such contradictory phenomena may be related to the function or types of antigen-presenting cells (APCs) in response to their ligand. A single-dose injection of a ligand for NKT cells, alpha-galactosylceramide (alpha-GalCer), can induce immunity through fully mature dendritic cells in an antigen-specific manner. By contrast, multiple injections of alpha-GalCer would induce tolerance, which may be caused by immature APCs. This response suggests that the function of NKT cells can be determined by alpha-GalCer for controlling the immune response. Furthermore, activation of NKT cells followed by activation of APCs and IL-12 production may lead to activation of NK cells and suppress GVHD in mismatched major histocompatibility complex combinations or may induce GVL effects. Control and modification of NKT cell function may play an important role in regulating GVHD/GVL effects.     

Potent immune-modulating and anticancer effects of NKT cell stimulatory glycolipids

Alpha-galactosylceramide (alpha-GalCer), a glycolipid that stimulates natural killer T (NKT) cells to produce both T helper (Th)1 and Th2 cytokines, has shown antitumor effects in mice but failed in clinical trials. We evaluated 16 analogs of alpha-GalCer for their CD1-mediated T cell receptor (TCR) activation of na?ve human NKT cells and their anticancer efficacy. In vitro, glycolipids containing an aromatic ring in their acyl tail or sphingosine tail were more effective than alpha-GalCer in inducing Th1 cytokines/chemokines, TCR activation, and human NKT cell expansion. None of these glycolipids could directly stimulate human dendritic cell maturation, except for a glycolipid with an aromatic ring on the sphingosine tail. Here, we show that glycolipids activated the TCR of NKT cells with phosphorylation of CD3epsilon, ERK1/2, or CREB, which correlated with their induction of Th1 cytokines. Notably, the extent of NKT cell activation when glycolipid was presented by antigen-presenting cells was greater than when glycolipid was presented by non-antigen-presenting cells. In vivo, in mice bearing breast or lung cancers, the glycolipids that induced more Th1-biased cytokines and CD8/CD4 T cells displayed significantly greater anticancer potency than alpha-GalCer. These findings indicate that alpha-GalCer analogs can be designed to favor Th1-biased immunity, with greater anticancer efficacy and other immune-enhancing activities than alpha-GalCer itself.     

Superoxide produced by Kupffer cells is an essential effector in concanavalin A-induced hepatitis in mice

Although concanavalin A (Con-A)-induced experimental hepatitis is thought to be induced by activated T cells, natural killer T (NKT) cells, and cytokines, precise mechanisms are still unknown. In the current study, we investigated the roles of Kupffer cells, NKT cells, FasL, tumor necrosis factor (TNF), and superoxide in Con-A hepatitis in C57BL/6 mice. Removal of Kupffer cells using gadolinium chloride (GdCl(3)) from the liver completely inhibited Con-A hepatitis, whereas increased serum TNF and IFN-gamma levels were not inhibited at all. Unexpectedly, anti-FasL antibody pretreatment did not inhibit Con-A hepatitis, whereas it inhibited hepatic injury induced by a synthetic ligand of NKT cells, alpha-galactosylceramide. Furthermore, GdCl(3) pretreatment changed neither the activation-induced down-regulation of NK1.1 antigens as well as T cell receptors of NKT cells nor the increased expression of the CD69 activation antigen of hepatic T cells. CD68(+) Kupffer cells greatly increased in proportion in the early phase after Con-A injection; this increase was abrogated by GdCl(3) pretreatment. Anti-TNF antibody (Ab) pretreatment did not inhibit the increase of Kupffer cells, but it effectively suppressed superoxide/reactive oxygen production from Kupffer cells and the resulting hepatic injury. Conversely, depletion of NKT cells in mice by NK1.1 Ab pretreatment did suppress both the increase of CD68(+) Kupffer cells and Con-A hepatitis. Consistently, the diminution of oxygen radicals produced by Kupffer cells by use of free radical scavengers greatly inhibited Con-A hepatitis without suppressing cytokine production. However, adoptive transfer experiments also indicate that a close interaction/cooperation of Kupffer cells with NKT cells is essential for Con-A hepatitis. Conclusion: Superoxide produced by Kupffer cells may be the essential effector in Con-A hepatitis, and TNF and NKT cells support their activation and superoxide production.     

Bacterial glycolipids and analogs as antigens for CD1d-restricted NKT cells

The CD1 family of proteins binds self and foreign glycolipids for presentation to CD1-restricted T cells. To identify previously uncharacterized active CD1 ligands, especially those of microbial origin, numerous glycolipids were synthesized and tested for their ability to stimulate mouse and human natural killer T (NKT) cells. They included analogs of the well known NKT cell agonist alpha-galactosyl ceramide (alpha-GalCer), bacterial glycolipids, and variations of the self-glycolipid, sulfatide. Bacterial glycolipids, alpha-galacturonosyl-ceramides from Sphingomonas wittichii, although structurally similar to alpha-GalCer, have significant differences in the sugar head group as well as the ceramide portion. The Sphingomonas glycosphingolipids (GSLs) and sulfatide variants were shown to activate human NKT cells as measured by IL-4 and IFN-gamma secretion. Moreover, CD1d-dimer staining revealed human NKT cell reactivity toward these GSLs and to the sulfatides in a fashion comparable with alpha-GalCer. Because alpha-GalCer is a marine-sponge-derived ligand, our study here shows that bacterium-derived antigens are also able to stimulate mouse and human NKT cells.     

Mouse V alpha 14i natural killer T cells are resistant to cytokine polarization in vivo

Under different circumstances, natural killer T (NKT) cells can cause a T helper (Th) 1 or a Th2 polarization of immune responses. We show here, however, that mouse NKT cells with an invariant V alpha 14 rearrangement (V alpha 14i NKT cells) rapidly produce both IL-4 and IFN-gamma, and this pattern could not be altered by methods that polarize naive CD4+ T cells. Surprisingly, although cytokine protein was detected only after activation, resting V alpha 14i NKT cells contained IL-4 and IFN-gamma mRNAs. Despite this finding, in vivo priming of mice with the glycolipid antigen recognized by V alpha 14i NKT cells resulted in a more Th2-oriented response upon antigen re-exposure. The V alpha 14i NKT cells from primed mice retain the ability to produce IL-4 and IFN-gamma, but they are less effective at activating NK cells to produce IFN-gamma. Our data therefore indicate that V alpha 14i NKT cells have a relatively inflexible immediate cytokine response, but that changes in their ability to induce IFN-gamma secretion by NK cells may determine the extent to which they promote Th1 responses.     

Role of Valpha 14 NKT cells in the development of impaired liver regeneration in vivo

Although we have previously demonstrated that IL-12 stimulation increases the number of hepatic natural killer (NK) T (NKT) cells and enhances liver injury during the early phase of liver regeneration, the role of NKT cells has remained unknown. We therefore evaluated the influence of NKT cells activated by IL-12 or by alpha-galactosylceramide (alpha-GalCer) on murine liver regeneration using Valpha 14 NKT knockout (Jalpha 281(-/-)) mice. Levels of serum alanine aminotransferase (sALT) 24 hours after partial hepatectomy were enhanced in Jalpha 281(+/+) but not in Jalpha 281(-/-) mice by both procedures. Hepatic NKT cells expressed considerably more interferon (IFN) gamma and tumor necrosis factor alpha (TNF-alpha) messenger RNA (mRNA) after stimulation with both factors in Jalpha 281(+/+) mice. Either anti-IFN-gamma or TNF-alpha antibody inhibited the enhancement of liver injury. Furthermore, recombinant TNF-alpha injection similarly caused injury in hepatectomized livers of both Jalpha 281(+/+) and Jalpha 281(-/-) mice; indeed, adoptively transferred TNF-alpha(+/+) NKT cells enhanced liver injury after hepatectomy in TNF-alpha knockout mice. TNF receptor expressions on hepatocytes increased and peaked 24 hours after partial hepatectomy. In conclusion, simultaneous TNF-alpha synthesis and high levels of TNF receptor expression on hepatocytes cause severe liver damage by activated NKT cells during liver regeneration.     

Pathogenic role of natural killer T and natural killer cells in acetaminophen-induced liver injury in mice is dependent on the presence of dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) is commonly used in biological studies to dissolve drugs and enzyme inhibitors with low solubility. Although DMSO is generally thought of as being relatively inert, it can induce biological effects that are often overlooked. An example that highlights this potential problem is found in a recent report demonstrating a pathogenic role for natural killer T (NKT) and natural killer (NK) cells in acetaminophen-induced liver injury (AILI) in C57Bl/6 mice in which DMSO was used to facilitate acetaminophen (APAP) dissolution. We report that NKT and NK cells do not play a pathologic role in AILI in C57Bl/6 mice in the absence of DMSO. Although AILI was significantly attenuated in mice depleted of NKT and NK cells prior to APAP treatment in the presence of DMSO, no such effect was observed when APAP was dissolved in saline. Because of this unexpected finding, the effects of DMSO on hepatic NKT and NK cells were subsequently investigated. When given alone, DMSO activated hepatic NKT and NK cells in vivo as evidenced by increased NKT cell numbers and higher intracellular levels of the cytotoxic effector molecules interferon-gamma (IFN-gamma) and granzyme B in both cell types. Similarly, when used as a solvent for APAP, DMSO again increased NKT cell numbers and induced IFN-gamma and granzyme B expression in both cell types. CONCLUSION: These data demonstrate a previously unappreciated effect of DMSO on hepatic NKT and NK cells, suggesting that DMSO should be used cautiously in experiments involving these cells.     

Natural killer-like nonspecific tumor cell lysis mediated by specific ligand-activated Vα14 NKT cells

We have recently identified α-galactosylceramide (α-GalCer) as a specific ligand for an invariant Vα14/Vβ8.2 T cell receptor exclusively expressed on the majority of Vα14 NKT cells, a novel subset of lymphocytes. Here, we report that α-GalCer selectively activates Vα14 NKT cells resulting in prevention of tumor metastasis. The effector mechanisms of the ligand-activated Vα14 NKT cells seem to be mediated by natural killer (NK)-like nonspecific cytotoxicity. Indeed, the cytotoxic index obtained by α-GalCer-activated Vα14 NKT cells was reduced by the addition of cold target tumor cells or by treatment with concanamycin A, which inhibits activation and secretion of perforin, but not by mAbs against molecules involved in the NKT cell recognition and conventional cytotoxicity, such as CD1d, Vβ8, NK1.1, Ly49C, Fas, or Fas ligand. These results suggest that the ligand-activated Vα14 NKT cells kill tumor cells directly through a CD1d/Vα14 T cell receptor-independent, NK-like mechanism.