Depletion Of Natural Killer T Cells Research Articles

A comparative study on roles of natural killer T cells in two diet-induced non-alcoholic steatohepatitis-related fibrosis in mice

Background Immune responses are important in the progression of non-alcoholic fatty liver disease (NAFLD). Natural killer T (NKT) cells are main components of the innate immune system that modulate immunity. However, the role of NKT cells in NAFLD remains controversial. Objective We aimed to investigate the role of NKT cells in non-alcoholic steatohepatitis (NASH)-related fibrosis in fast food diet (FFD)- and methionine choline-deficient (MCD) diet-induced mouse models. Methods Hepatic NKT cells were analysed in wild-type (WT) and CD1d-/- mice fed FFD or MCD diets. Hepatic pathology, cytokine profiles and liver fibrosis were evaluated. Furthermore, the effect of chronic administration of α-galactosylceramide (α-GalCer) on liver fibrosis was investigated in both FFD- and MCD-treated mice. Results FFD induced a significant depletion of hepatic NKT cells, thus leading to mild to moderate NASH and early-stage fibrosis, while mice fed MCD diets developed severe liver inflammation and progressive fibrosis without a significant change in hepatic NKT cell abundance. FFD induced a similar liver fibrogenic response in CD1d-/- and WT mice, while MCD induced a higher hepatic mRNA expression of Col1α1 and TIMP1 as well as relative fibrosis density in CD1d-/- mice than WT mice (31.8 vs. 16.3, p = .039; 40.0 vs. 22.6, p = .019; 2.24 vs. 1.59, p = .036). Chronic administration of α-GalCer induced a higher hepatic mRNA expression of TIMP1 in MCD-treated mice than controls (36.7 vs. 14.9, p = .005). Conclusion NKT cells have protective roles in NAFLD as the disease progresses. During diet-induced steatosis, mild to moderate NASH and the early stage of fibrosis, hepatic NKT cells are relatively depleted, leading to a proinflammatory status. In severe NASH and the advanced stage of liver fibrosis, NKT cells play a role in inhibiting the NASH-related fibrogenic response. Chronic administration of α-GalCer induces NKT cell anergy and tolerance, which may play a role in promoting the liver fibrogenic response.

Increased Expression of Tim-3 Is Associated With Depletion of NKT Cells In SARS-CoV-2 Infection.

In the ongoing coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), natural killer T (NKT) cells act as primary initiators of immune responses. However, a decrease of circulating NKT cells has been observed in COVID-19 different stages, of which the underlying mechanism remains to be elucidated. Here, by performing single-cell RNA sequencing analysis in three large cohorts of COVID-19 patients, we found that increased expression of Tim-3 promotes depletion of NKT cells during the progression stage of COVID-19, which is associated with disease severity and outcome of patients with COVID-19. Tim-3+ NKT cells also expressed high levels of CD147 and CD26, which are potential SARS-CoV-2 spike binding receptors. In the study, Tim-3+ NKT cells showed high enrichment of apoptosis, higher expression levels of mitochondrial genes and caspase genes, with a larger pseudo time value. In addition, Tim-3+ NKT cells in COVID-19 presented a stronger capacity to secrete IFN-γ, IL-4 and IL-10 compared with healthy individuals, they also demonstrated high expression of co-inhibitory receptors such as PD-1, CTLA-4, and LAG-3. Moreover, we found that IL-12 secreted by dendritic cells (DCs) was positively correlated with up-regulated expression of Tim-3 in NKT cells in COVID-19 patients. Overall, this study describes a novel mechanism by which up-regulated Tim-3 expression induced the depletion and dysfunction of NKT cells in COVID-19 patients. These findings not only have possible implications for the prediction of severity and prognosis in COVID-19 but also provide a link between NKT cells and future new therapeutic strategies in SARS-CoV-2 infection.

NK and NKT Cell Depletion Alters the Outcome of Experimental Pneumococcal Pneumonia: Relationship with Regulation of Interferon-γ Production.

Background. Natural killer (NK) and natural killer T (NKT) cells contribute to the innate host defense but their role in bacterial sepsis remains controversial. Methods. C57BL/6 mice were infected intratracheally with 5 × 105 cfu of Streptococcus pneumoniae. Animals were divided into sham group (Sham); pretreated with isotype control antibody (CON) group; pretreated with anti-asialo GM1 antibody (NKd) group; and pretreated with anti-CD1d monoclonal antibody (NKTd) group before bacterial challenge. Serum and tissue samples were analyzed for bacterial load, cytokine levels, splenocyte apoptosis rates, and cell characteristics by flow cytometry. Splenocyte miRNA expression was also analyzed and survival was assessed. Results. NK cell depletion prolonged survival. Upon inhibition of NKT cell activation, spleen NK (CD3−/NK1.1+) cells increased compared to all other groups. Inhibition of NKT cell activation led to higher bacterial loads and increased levels of serum and splenocyte IFN-γ. Splenocyte miRNA analysis showed that miR-200c and miR-29a were downregulated, while miR-125a-5p was upregulated, in anti-CD1d treated animals. These changes were moderate after NK cell depletion. Conclusions. NK cells appear to contribute to mortality in pneumococcal pneumonia. Inhibition of NKT cell activation resulted in an increase in spleen NK (CD3−/NK1.1+) cells and a higher IFN-γ production, while altering splenocyte miRNA expression.

NKT cell depletion in humans during early HIV infection.

Natural killer T (NKT) cells bridge across innate and adaptive immune responses and have an important role in chronic viral infections such as human immunodeficiency virus (HIV). NKT cells are depleted during chronic HIV infection, but the timing, drivers and implications of this NKT cell depletion are poorly understood. We studied human peripheral blood NKT cell levels, phenotype and function in 31 HIV-infected subjects not on antiretroviral treatment from a mean of 4 months to 2 years after HIV infection. We found that peripheral CD4(+) NKT cells were substantially depleted and dysfunctional by 4 months after HIV infection. The depletion of CD4(+) NKT cells was more marked than the depletion of total CD4(+) T cells. Further, the early depletion of NKT cells correlated with CD4(+) T-cell decline, but not HIV viral levels. Levels of activated CD4(+) T cells correlated with the loss of NKT cells. Our studies suggest that the early loss of NKT cells is associated with subsequent immune destruction during HIV infection.

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.

Clinical Grade Purification and Expansion of NK Cell Products for an Optimized Manufacturing Protocol

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, n = 18; D2.12depl, n = 13) or the faster depletion 3.1 (D3.1, n = 9) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12 days. The whole process resulted in a median number of 7.59 × 108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; p < 0.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

Blockade of IL-33 ameliorates Con A-induced hepatic injury by reducing NKT cell activation and IFN-γ production in mice

IL-33, a recently described member of the IL-1 family, has been identified as a cytokine endowed with pro-Th2 type functions. To date, there are only limited data on its role in physiological and pathological hepatic immune responses. In this study, we examined the role of IL-33 in immune-mediated liver injury by exploring the model of concanavalin A (Con A)-induced hepatitis. We observed that the level of IL-33 expression in the liver was dramatically increased at 12h after Con A injection. Meanwhile, ST2L, the receptor of IL-33, was significantly up-regulated in lymphocytes including T and natural killer T (NKT) cells, especially in NKT cells. Moreover, administration of recombinant IL-33 exacerbated Con A-induced hepatitis, while pretreatment of IL-33-blocking antibody or psST2-Fc plasmids showed a protective effect probably by inhibiting the activation of late stage of T cells and NKT cells and also decreasing the production of the cytokine IFN-γ. Furthermore, depletion of NKT cells abolished the protective effect of IL-33-blocking antibody, and IL-33 failed to exacerbate Con A-induced hepatitis in IFN-γ(-/-) mice. These data suggested the critical roles of NKT cells and IFN-γ in the involvement of IL-33 in Con A-induced hepatitis. Blockade of IL-33 may represent a novel therapeutic strategy through IL-33/ST2L signal to prevent immune-mediated liver injury.

Mice Lacking NKT Cells but with a Complete Complement of CD8+ T-Cells Are Not Protected against the Metabolic Abnormalities of Diet-Induced Obesity

The contribution of natural killer T (NKT) cells to the pathogenesis of metabolic abnormalities of obesity is controversial. While the combined genetic deletion of NKT and CD8+ T-cells improves glucose tolerance and reduces inflammation, interpretation of these data have been complicated by the recent observation that the deletion of CD8+ T-cells alone reduces obesity-induced inflammation and metabolic dysregulation, leaving the issue of the metabolic effects of NKT cell depletion unresolved. To address this question, CD1d null mice (CD1d−/−), which lack NKT cells but have a full complement of CD8+ T-cells, and littermate wild type controls (WT) on a pure C57BL/6J background were exposed to a high fat diet, and glucose intolerance, insulin resistance, dyslipidemia, inflammation, and obesity were assessed. Food intake (15.5±4.3 vs 15.3±1.8 kcal/mouse/day), weight gain (21.8±1.8 vs 22.8±1.4 g) and fat mass (18.6±1.9 vs 19.5±2.1 g) were similar in CD1d−/− and WT, respectively. As would be expected from these data, metabolic rate (3.0±0.1 vs 2.9±0.2 ml O2/g/h) and activity (21.6±4.3 vs 18.5±2.6 beam breaks/min) were unchanged by NKT cell depletion. Furthermore, the degree of insulin resistance, glucose intolerance, liver steatosis, and adipose and liver inflammatory marker expression (TNFα, IL-6, IL-10, IFN-γ, MCP-1, MIP1α) induced by high fat feeding in CD1d−/− were not different from WT. We conclude that deletion of NKT cells, in the absence of alterations in the CD8+ T-cell population, is insufficient to protect against the development of the metabolic abnormalities of diet-induced obesity.

Accelerated liver fibrosis in hepatitis B virus transgenic mice: Involvement of natural killer T cells

The immunopathogenic process from hepatitis B virus (HBV) infection to liver fibrosis is incompletely understood because it lacks an animal model. In this study we observed the development of liver fibrosis in HBV transgenic (HBV-tg) mice and found the roles of natural killer T (NKT) cells in HBV-related liver fibrosis. We found liver fibrosis spontaneously developed in HBV-tg mice with the elevated transcription of col1a1, matrix metalloproteinase (MMP)2, and tissue inhibitor of metalloproteinase (TIMP)1. Mice were then injected with repetitive hepatotoxin carbon tetrachloride (CCl(4) ) to induce prominent liver fibrosis. After chronic CCl(4) treatment, the serum alanine aminotransferase (ALT) was higher, the liver regenerative nodules became more and bigger, and the fibrosis area was remarkably increased in HBV-tg mice than in C57BL/6 mice. Moreover, the increase in col1a1 and MMP2 transcription was greater, with a sustaining high level of TIMP1 and a greater activation of hepatic stellate cells (HSCs) in the livers of CCl(4) -treated HBV-tg mice. Our data also showed that there were more liver mononuclear cells (MNCs) in HBV-tg mice after CCl(4) injection, and Rag1(-/-) mice adoptive transferred lymphocytes from HBV-tg mice displayed increased collagen deposition. Further study demonstrated the number of liver NKT cells increased after CCl(4) treatment and NKT cells were overactivated in HBV-tg mice in the long term. It was further confirmed that NKT cells were critical for HSCs activation by depletion of NKT cells of HBV-tg mice and adoptive transfer of purified NKT cells from HBV-tg mice into recipient Rag1(-/-) mice. The inflammatory cytokines IL-4 and IL-13 produced by NKT cells played a pivotal role in HSCs activation in an in vitro coculture experiment. These data suggest that NKT cells from HBV-tg mice induce the HSC activation in liver fibrogenesis.

Involvement of natural killer T cells in halothane-induced liver injury in mice

Drug-induced liver injury (DILI) causes significant patient morbidity and mortality, and is the most common reason for drug withdrawals. It is imperative to gain a thorough understanding of the underlying mechanisms of DILI to effectively predict and prevent these reactions. We have recently developed a murine model of halothane-induced liver injury (HILI). The aim of the present study was to investigate the role of hepatic natural killer T (NKT) cells in the pathogenesis of HILI. The degrees of HILI were compared between WT and CD1d −/− mice, which are deficient in NKT cells. The data revealed that CD1d −/− mice were resistant in developing HILI. This resistance appeared to be a direct result of NKT cell depletion rather than an indirect one due to the absence of cross-talk between NKT cells and other hepatic innate immune cells. Compared with WT mice, CD1d −/− mice exhibited a significantly lower number of hepatic infiltrating neutrophils upon halothane challenge (470,000 ± 100,000/liver in WT vs. 120,000 ± 31,500/liver in CD1d −/− mice). This result in conjunction with our previous finding of an indispensable role of neutrophils in HILI strongly suggests that NKT cells play a critical role in regulating neutrophil recruitment, thereby contributing to the development of HILI. Collectively, the current study and published reports indicate that this murine model of HILI provides an experimental system for the investigation of the underlying mechanisms of DILI. In addition, this model may yield the discovery of susceptibility factors that may control the development of liver injury in patients treated with halothane and potentially other drugs.

Paucity of CD4+ Natural Killer T (NKT) Lymphocytes in Sooty Mangabeys Is Associated with Lack of NKT Cell Depletion after SIV Infection

Lack of chronic immune activation in the presence of persistent viremia is a key feature that distinguishes nonpathogenic simian immunodeficiency virus (SIV) infection in natural hosts from pathogenic SIV and HIV infection. To elucidate novel mechanisms downmodulating immune activation in natural hosts of SIV infection, we investigated natural killer T (NKT) lymphocytes in sooty mangabeys. NKT lymphocytes are a potent immunoregulatory arm of the innate immune system that recognize glycolipid antigens presented on the nonpolymorphic MHC-class I-like CD1d molecules. In a cross-sectional analysis of 50 SIV-negative and 50 naturally SIV-infected sooty mangabeys, ligand α-galactosylceramide loaded CD1d tetramers co-staining with Vα24-positive invariant NKT lymphocytes were detected at frequencies ≥0.002% of circulating T lymphocytes in approximately half of the animals. In contrast to published reports in Asian macaques, sooty mangabey NKT lymphocytes consisted of CD8+ and CD4/CD8 double-negative T lymphocytes that were CXCR3-positive and CCR5-negative suggesting that they trafficked to sites of inflammation without being susceptible to SIV infection. Consistent with these findings, there was no difference in the frequency or phenotype of NKT lymphocytes between SIV-negative and SIV-infected sooty mangabeys. On stimulation with α-galactosylceramide loaded on human CD1d molecules, sooty mangabey NKT lymphocytes underwent degranulation and secreted IFN-γ, TNF-α, IL-2, IL-13, and IL-10, indicating the presence of both effector and immunoregulatory functional capabilities. The unique absence of CD4+ NKT lymphocytes in sooty mangabeys, combined with their IL-10 cytokine-secreting ability and preservation following SIV infection, raises the possibility that NKT lymphocytes might play a role in downmodulating immune activation in SIV-infected sooty mangabeys.

Reply

We would like to thank Dr. Alisi and colleagues for their interest in our recent manuscript detailing the relationship between Kupffer cell-derived interleukin 12 (IL12) production and loss of hepatic natural killer T (NKT) cells in the steatotic liver.1 It is clear from our study and several others that NKT cells are highly sensitive to hepatic lipid accumulation, a process which we can link to IL12 production by Kupffer cells.1-4 In response to their first comment, we expected that if NKT cells were restored, hepatic lipid accumulation would also be reduced; we were very surprised to see otherwise. Indeed, these results in this model of nonalcoholic fatty liver suggest that NKT cell depletion is a consequence of lipid accumulation and Kupffer cell activation, which challenges the notion that they function to suppress hepatocellular lipid accumulation in nonalcoholic fatty liver disease. As for the mechanism(s) governing IL12 production by Kupffer cells in the nonalcoholic fatty liver, a number of potential possibilities exist with gut-liver interactions being a strong candidate. There is no doubt that gut-derived factors, including gut-derived lipopolysaccharide, contribute to Kupffer cell activation and IL12 production. Recent studies by Ma and colleagues have demonstrated the ability of probiotics, which likely alter gut microbial populations, to protect the hepatic NKT cells within the steatotic liver.5 In addition, it has been shown that Toll-like receptor-9 engagement can promote Kupffer cell-dependent and IL12-dependent NKT cell recruitment to the nonsteatotic liver and exacerbate concanavalin A-mediated hepatitis.6 This highlights the potential impact of gut-derived factors, including bacterial DNA, as key regulators of NKT cell function, although their direct influence on NKT cells within the steatotic liver remains to be determined. It is very likely that a combination of hepatocellular lipid accumulation and Kupffer cell activation promote this response. This complex association is evidenced by data from our study where isolated Kupffer cells from the steatotic liver do not produce more IL12 than those Kupffer cells isolated from a normal liver when activated in vitro, despite showing large and significant increases in its production when activated by lipopolysaccharide in vivo.1 Thus, multiple levels of regulation likely exist and gut-derived antigens no doubt contribute at least in part to this process. Current studies are directed at better understanding the mechanism(s) of Kupffer cell activation and IL12 production, as well as the potential contribution of gut-derived factors in this model of nonalcoholic fatty liver disease. Ian N. Hines Ph.D.*, * Department of Medicine, Division of Gastroenterology and Hepatology, University of North Carolina, Chapel Hill, NC.

The link between hepatosteatosis and cells of the immune system

We read with great interest the article by Kremer et al., 1 on the role of interleukin-12 (IL-12) production by Kupffer cells in fatty liver, and its possible impact in the reduction of hepatic resident natural killer T (NKT) cells using an animal model of hepatosteatosis (mice fed choline-deficient diet for 0-20 weeks). As previously demonstrated, 2 in this study the authors found that hepatosteatosis in mice that were fed a chronic choline-deficient diet is characterized at the hepatic level by the induction of interferon-gamma and tumor necrosis factor-alpha, elevated content of IL-12, and by a remarkable reduction in the number and activity of NKT cells. Interestingly, all these effects are undetectable in IL-12–depleted mice fed a choline-deficient diet, even if fatty liver is equally present. Several studies 2- 4 conducted on different metabolic animal models of hepatic steatosis have reported the relationship between lipid accumulation, increased Th1 cytokine production (i.e., tumor necrosis factor, IL-12, and interferon-gamma), and hepatic NKT cell depletion. IL-12 is well known as a NKT cell inductor able to stimulate the production/release of large amounts of interferon-gamma and to activate specific transcription factors, including signal transducer and activator of transcription 4 (STAT4). 5 However, the IL-12 increase may not be the sole factor involved in death-dependent NKT cell depletion: others factors, such as dietary factors, might interfere, for example, with mechanisms that mediate the hepatic homing and apoptosis of NKT cells. 6 All these findings noticeably demonstrated that NKT cell reduction is an effect of a combination between intrahepatic fat accumulation and IL-12 increase rather than a cause of hepatosteatosis. However, the real unresolved question is the connection between intrahepatic fat accumulation and up-regulated hepatic IL-12 messenger RNA levels. Once again, as demonstrated by Kremer et al., 1 and as anticipated by other studies, 7 the activation of Kupffer cells by an endotoxin-mediated mechanism could be the link between fatty liver, inflammatory response, and a reduction of NKT cell population. Noteworthy, this phenomenon could be either an effect of fatty liver or an early signal for the development of fibrosis. 8 Therefore, it might be very interesting to investigate whether the number of NKT cells may be a predictive marker of liver fibrosis. Furthermore, research favors the hypothesis of the role of endotoxin and the toll like receptor-4 in diet-induced steatohepatitis. 9 Yet, we would emphasize how many points are still obscure on the molecular mechanisms regulating this intricate network of interactions between cells of the immune system and hepatocellular damage. Anna Alisi Ph.D.*, Nadia Panera*, Valerio Nobili M.D.*, * Liver Unit, Bambino Gesù Children's Hospital and Research Institute, Rome, Italy.

In Vivo Characterization of Rabbit Anti-Mouse Thymocyte Globulin: A Surrogate for Rabbit Anti-Human Thymocyte Globulin

Polyclonal rabbit anti-human thymocyte globulin (Thymoglobulin) is used clinically for immunosuppression in solid organ transplantation; however, it is difficult to fully characterize the effects of this agent in humans. A surrogate rabbit anti-murine thymocyte globulin (mATG) was generated analogously to the commercial product Thymoglobulin and in vivo activities were evaluated, including pharmacokinetics, T-cell depletion, dose response and kinetics, depletion/sparing of T-cell subsets or other leukocyte populations, and depletion in different lymphoid organs. Within 1 day, T cells are depleted by mATG in the blood, spleen, lymph node, and bone marrow down to doses of 1 mg/kg. Although mATG binds and depletes thymocytes in vitro, there is no thymocyte depletion in vivo at any dose level, suggesting decreased antibody accessibility to the thymus. After two doses of mATG given 3 days apart, T-cell reconstitution begins as early as day 9 and returns to basal levels by day 21 and 29 for CD4 and CD8 T cells, respectively. There is also preferential depletion of naïve T cells that results in increased ratios of regulatory and memory T cells within 1 day after mATG administration. Depletion of natural killer-T cells, natural killer cells, plasma cells, and plasmablasts occurs, but is modest and more transient compared with T cells. B cells, macrophages, dendritic cells, hematopoetic stem cells, and bone marrow stromal cells seem resistant to mATG depletion. These studies characterize the depletive effects of mATG in normal mice and provide insight into mechanisms of action of Thymoglobulin.

Distinct contributions of CD4+T cell subsets in hepatic ischemia/reperfusion injury

Helper T cells are known to mediate hepatic ischemia/reperfusion (I/R) injury. However, the precise mechanisms and subsets of CD4(+) T cells that contribute to this injury are still controversial. Therefore, we sought to determine the contributions of different CD4(+) T cell subsets during hepatic I/R injury. Wild-type, OT-II, or T cell receptor (TCR)-delta-deficient mice were subjected to 90 min of partial hepatic ischemia followed by 8 h of reperfusion. Additionally, wild-type mice were pretreated with anti-CD1d, -NK1.1, or -IL-2R-alpha antibodies before I/R injury. OT-II mice had diminished liver injury compared with wild-type mice, implicating that antigen-dependent activation of CD4(+) T cells through TCRs is involved in hepatic I/R injury. TCR-delta knockout mice had decreased hepatic neutrophil accumulation, suggesting that gammadelta T cells regulate neutrophil recruitment. We found that natural killer T (NKT) cells, but not NK cells, contribute to hepatic I/R injury via CD1d-dependent activation of their TCRs, as depletion of NKT cells by anti-CD1d antibody or depletion of both NKT cells and NK cells by anti-NK1.1 attenuated liver injury. Although regulatory T cells (Treg) are known to suppress T cell-dependent inflammation, depletion of Treg cells had little effect on hepatic I/R injury. The data suggest that antigen-dependent activation of CD4(+) T cells contributes to hepatic I/R injury. Among the subsets of CD4(+) T cells, it appears that gammadelta T cells contribute to neutrophil recruitment and that NKT cells directly injure the liver. In contrast, NK cells and Treg have little effects on hepatic I/R injury.

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. 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.

Type I Natural Killer T (iNKT) Cells Regulate Inflammatory Immune Response in Mice Liver Injury Induced by CCl4

Natural killer T (NKT) cells are the predominant lymphocyte population (up to 30 %) in the mouse liver and play an important role in Concanavalin A (Con A) and α‐Galactosylceramide (α‐GalCer) induced liver injury by producing inflammatory cytokines, such as IFN‐γ and TNF‐α and activating other cell populations including NK and T cells. In this study, we examined the role of NKT cells in carbon tetrachloride (CCl4) liver injury using type I, or invariant NKT (iNKT) KO mice (Ja18−/−) and WT C57BL/6 mice. Liver injury in mice was induced by intraperitoneal single injection of 1 ml/kg body weight of 10% CCl4 and samples were collected at different time points. The administration of CCl4 to mice induced liver injury with depletion of NKT cells and elevation of NK cells in the liver in both mice groups. Compared to WT mice, Jα 18−/− mice showed severe liver damage and increased neutrophil infiltration at early time point. Serum levels of inflammatory cytokines, such as TNF‐α, MCP‐1, IL‐4 and IL‐6, as well as mRNA expression were remarkably increased in Jα18−/− mice after CCl4 administration compared to WT mice. These findings suggest that type I NKT cells may protect against inflammatory immune response via suppressing the production of proinflammatory cytokines in early stage liver injury.

Deficiency of invariant Vα14 natural killer T cells decreases atherosclerosis in LDL receptor null mice

AimsCD1d-restricted natural killer T (NKT) cells function by regulating numerous immune responses during innate and adaptive immunity. Depletion of all populations of CD1d-dependent NKT cells has been shown by several groups to reduce atherosclerosis in two different mouse models of the disease. In this study, we determined if removal of a single (Vα14) NKT cell population protects mice from the disease.Methods and resultsTargeted deletion of the Jα18 gene results in selective depletion of CD1d-dependent Vα14 NKT cells in C57BL/6 mice without affecting the population of other NKT, NK, and conventional T cells. Therefore, to study the effect of Vα14 NKT cell depletion on the progression of atherosclerosis, we examined the extent of lesion formation using paired littermate LDL receptor null mice that were either +/+ or −/− for the Jα18 gene following the feeding of these mice a cholesterol- and fat-enriched diet for 8 weeks. At the end of the study, we found no difference in either serum total- or lipoprotein-cholesterol distributions between groups. However, quantification of atherosclerosis revealed that Vα14 NKT cell deficiency significantly decreased lesion size in the aortic root (20–28%) and arch (28–38%) in both genders of mice. By coupling the techniques of laser capture microdissection with quantitative real-time RT–PCR, we found that expression of the proatherogenic cytokine interferon (IFN)-γ was significantly reduced in lesions from Jα18−/− mice.ConclusionThis study is the first to identify a specific subpopulation of NKT cells that promotes atherosclerosis via a mechanism appearing to involve IFN-γ expression.

The Immunoregulatory Roles of Natural Killer T Cells in Cyclophosphamide-Induced Tolerance

Recent studies have indicated that natural killer T (NKT) cells are essential for the establishment of transplantation tolerance. In the present study, we have elucidated the role of recipient and donor NKT cells in cyclophosphamide (CP)-induced tolerance. DBA/2 (DBA; H-2) mice were used as donors and BALB/c (BALB; H-2) wild-type (WT) or Valpha14 NKT-knockout (KO, BALB/c background) mice were used as recipients. Recipients were treated with CP-induced tolerance regimen, which consists of donor spleen cells (SC) on day 0 and CP on day 2. In some experiments, NKT KO mice, which received NKT cells from either WT, inferon-gamma KO, or interleukin-4 KO mice, were treated with tolerant regimen. To deplete Ly49 inhibitory receptors on NKT cells in the recipient mice, anti-Ly49 monoclonal antibody cocktails were injected on day -1 when indicated. Donor skin graft was permanently accepted in recipient BALB WT mice with induction of donor mixed chimerism. On the contrary, donor DBA skin allografts were chronically rejected in NKT KO recipient. Lower levels of mixed chimerism were observed in NKT KO recipients comparing to the WT recipients. The production of interferon-gamma or interleukin-4 from NKT cells did not affect the induction of tolerance. Depletion of Ly49 positive NKT cells abrogated the induction of skin graft tolerance. Recipient NKT cells, but not donor NKT cells, were dominantly required for the induction of allograft tolerance. Our results indicated that the single cytokine produced by NKT cells did not mediate the regulatory function in the induction of allograft tolerance.

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