Hepatic iNKT Cells Research Articles

Abstract 1386: CD1d-restricted iNKT cells promote liver metastasis from colon cancer

Abstract The liver is a prominent site for metastasis, yet little is known about the role of liver immunity in antimetastatic defense. Compared to other organs, the liver harbors a unique immune environment characterized by the abundance of innate cell lineages, including CD1d-restricted invariant natural killer T (iNKT) cells. To investigate the influence of iNKT cells on metastasis, we established an orthotopic model of colorectal cancer (CRC) with spontaneous dissemination to the liver. We induce the primary tumor by implanting CRC organoids into the colon mucosa of syngeneic C57BL/6 mice, resulting in hepatic macrometastases within 5 weeks. We observed that liver metastasis hardly occurred in mouse strains lacking iNKT cells (Cd1d−/− and Traj18−/− mice), while the growth of the primary tumor was comparable to that in wild-type mice. These results indicate a tumor-promoting function for hepatic iNKT cells. We uncovered that hepatic iNKT cells respond to disseminating cancer by producing IL-4 and IL-13, two cytokines known to activate hepatic stellate cells (HSCs) into fibrogenic myofibroblasts. Indeed, we saw rapid activation of HSCs and deposition of extracellular matrix upon arrival of cancer cells in the liver of wild-type but not iNKT cell-deficient mice. We propose that hepatic iNKT cells contribute to the formation of a supportive niche for disseminated tumor cells by activating HSCs early in the development of hepatic metastasis. Taken together, our findings highlight an essential tumor-promoting role for iNKT cells during the development of liver metastasis and contribute to our understanding of immune regulation of metastasis. Citation Format: Marc Nater, Giulia Lucchiari, Michael Brügger, Tomas Valenta, Virginia Cecconi, Geo Forni, Stephan Benke, Maries van den Broek. CD1d-restricted iNKT cells promote liver metastasis from colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1386.

Invariant natural killer T cells drive hepatic homeostasis in Non-alcoholic fatty liver disease via sustained IL-10 expression in CD170+ Kupffer cells.

Kupffer cells (KCs) are liver-resident macrophages involved in hepatic inflammatory responses, including non-alcoholic fatty liver disease (NAFLD) development. However, the contribution of KC subsets to liver inflammation remains unclear. Here, using high-dimensional single-cell RNA sequencing, we characterised murine embryo-derived KCs (Em-KCs) and identified two KC populations with different gene expression profiles: KC-1 and KC-2. KC-1 expressed CD170, exhibiting immunoreactivity and immune-regulatory abilities, while KC-2 highly expressed lipid metabolism-associated genes. In a high-fat diet (HFD)-induced NAFLD model, KC-1 cells differentiated into pro-inflammatory phenotypes and initiated more frequent communications with invariant natural killer T (iNKT) cells. In KC-1, interleukin (IL)-10 expression was unaffected by the HFD but impaired by iNKT cell ablation and upregulated by iNKT cell adoptive transfer in vivo. Moreover, in a cellular co-culture system, primary hepatic iNKT cells promoted IL-10 expression in RAW264.7 and primary KC-1 cells. CD206 signal blocking in KC-1 or CD206 knockdown in RAW264.7 cells significantly reduced IL-10 expression. In conclusion, we identified two Em-KC subpopulations with distinct transcriptional profiles. The CD206-mediated crosstalk between iNKT and KC-1 cells maintains IL-10 expression in KC-1 cells, affecting hepatic immune balance. Therefore, KC-based therapeutic strategies must consider cellular heterogeneity and the local immune microenvironment for enhanced specificity and efficiency. This article is protected by copyright. All rights reserved.

Tissue-specific metabolic profile drives iNKT cell function during obesity and liver injury

Invariant natural killer T (iNKT) cells are a distinct population of lymphocytes characterized by their reactivity to glycolipids presented by CD1d. iNKT cells are found throughout the body, and little is known about their tissue-specific metabolic regulation. Here, we show that splenic and hepatic iNKT cells are metabolically comparable and rely on glycolytic metabolism to support their activation. Deletion of the pyruvate kinase M2 (Pkm2) gene in splenic and hepatic iNKT cells impairs their response to specific stimulation and their ability to mitigate acute liver injury. In contrast, adipose tissue (AT) iNKT cells exhibit a distinctive immunometabolic profile, with AMP-activated protein kinase (AMPK) being necessary for their function. AMPK deficiency impairs AT-iNKT physiology, blocking their capacity to maintain AT homeostasis and their ability to regulate AT inflammation during obesity. Our work deepens our understanding on the tissue-specific immunometabolic regulation of iNKT cells, which directly impacts the course of liver injury and obesity-induced inflammation.

Long-Chain Acylcarnitines Induce Senescence of Invariant Natural Killer T Cells in Hepatocellular Carcinoma.

Patients with HBV-related HCC exhibit a cell senescence-associated dysregulation of invariant natural killer cells that is related to altered lipid metabolism and accumulated LCACs in tumor tissue.

New insights into iNKT cells and their roles in liver diseases.

Natural killer T cells (NKTs) are an important part of the immune system. Since their discovery in the 1990s, researchers have gained deeper insights into the physiology and functions of these cells in many liver diseases. NKT cells are divided into two subsets, type I and type II. Type I NKT cells are also named iNKT cells as they express a semi-invariant T cell-receptor (TCR) α chain. As part of the innate immune system, hepatic iNKT cells interact with hepatocytes, macrophages (Kupffer cells), T cells, and dendritic cells through direct cell-to-cell contact and cytokine secretion, bridging the innate and adaptive immune systems. A better understanding of hepatic iNKT cells is necessary for finding new methods of treating liver disease including autoimmune liver diseases, alcoholic liver diseases (ALDs), non-alcoholic fatty liver diseases (NAFLDs), and liver tumors. Here we summarize how iNKT cells are activated, how they interact with other cells, and how they function in the presence of liver disease.

Hepatic iNKT cells produce type 2 cytokines and restrain antiviral T cells during acute hepacivirus infection.

Chronic hepatitis C virus (HCV) infection is a curable disease, but the absence of a vaccine remains a major problem in infection prevention. The lack of small animal models and limited access to human liver tissue impede the study of hepatic antiviral immunity and the development of new vaccine strategies. We recently developed an immune-competent mouse model using an HCV-related rodent hepacivirus which shares immunological features with human viral hepatitis. In this study, we used this new model to investigate the role of invariant natural killer T (iNKT) cells during hepacivirus infection in vivo. These cells are enriched in the liver, however their role in viral hepatitis is not well defined. Using high-dimensional flow cytometry and NKT cell deficient mice we analyzed a potential role of iNKT cells in mediating viral clearance, liver pathology or immune-regulation during hepacivirus infection. In addition, we identified new immune-dominant MHC class I restricted viral epitopes and analyzed the impact of iNKT cells on virus-specific CD8+ T cells. We found that rodent hepacivirus infection induced the activation of iNKT cell subsets with a mixed NKT1/NKT2 signature and significant production of type 2 cytokines (IL-4 and IL-13) during acute infection. While iNKT cells were dispensable for viral clearance, the lack of these cells caused higher levels of liver injury during infection. In addition, the absence of iNKT cells resulted in increased effector functions of hepatic antiviral T cells. In conclusion, our study reports a regulatory role of hepatic iNKT cells during hepacivirus infection in vivo. Specifically, our data suggest that iNKT cells skewed towards type 2 immunity limit liver injury during acute infection by mechanisms that include the regulation of effector functions of virus-specific T cells.

Identification of Adipose‐Specific iNKT Cell Subpopulation through Single‐Cell RNA Sequencing

Invariant natural killer T (iNKT) cells are innate T cells that recognize CD1d‐loaded lipid antigens. In adipose tissue, iNKT cells contribute to maintenance of adipose tissue homeostasis through active communication with CD1d‐positive adipocytes. By recognizing lipid antigens, adipose iNKT cells kill harmful cells, such as hypertrophic adipocytes and pro‐inflammatory macrophages, and promote stem cell proliferation. These characteristics seem not to be observed in hepatic or splenic iNKT cells. However, it has not been thoroughly understood how adipose iNKT cells control cell fates in adipose tissue. Here, we adopted single‐cell RNA sequencing to identify unique subpopulations of adipose iNKT cells involved in the clearance of deleterious cells or stem cell renewal. Adipose iNKT cells were largely categorized into 3 subpopulations; iNKT1A, iNKT1B, and iNKT17. Transcriptome analysis revealed that iNKT1A cells were the adipose‐specific subpopulation, while iNKT1B and iNKT17 cells were similar to splenic and thymic iNKT cells. Moreover, adoptive transfer experiments showed that iNKT1A cells were generated only in adipose tissue, but not in other organs, indicating that adipose tissue microenvironment plays crucial roles for generation of iNKT1A cells. Collectively, our data showed that iNKT1A cells would be the adipose‐specific iNKT subpopulation and could be generated by adipose tissue microenvironment.

Expansion of iNKT Cells Promotes Liver Repair Following Hepatic Ischemia Reperfusion Injury.

Invariant natural killer T (iNKT) cells are involved in the initiation and resolution of inflammatory responses. We previously reported that activated iNKT cells facilitate liver repair after hepatic ischemia reperfusion (I/R) injury by accelerating macrophage polarization during the early phase of hepatic I/R injury. Upon activation with α-galactosylceramide (α-GalCer), iNKT cell numbers transiently decrease before increasing within 72 h of stimulation. In the present study, we examined the role of expanded hepatic iNKT cells in the late phase of hepatic I/R injury. iNKT cells were activated by intraperitoneal injection of α-GalCer in male C57/BL6 mice at the induction of hepatic ischemia followed by reperfusion. Numbers of activated hepatic iNKT cells immediately diminished after hepatic I/R and reached minimal levels at 24 h and 48 h post-reperfusion. Numbers of hepatic iNKT cells then increased at 72 h and 96 h post-reperfusion to levels approximately 2-fold higher than in mice that underwent a sham operation. Liver repair as demonstrated by decreased necrotic area and increased expression of proliferating cell nuclear antigen (PCNA) was enhanced in α-GalCer-treated mice at 96 h post-reperfusion. Interleukin (IL)-13 production by proliferating iNKT cells was observed at 96 h post-reperfusion, which was associated with enhanced liver repair and increased numbers of reparative macrophages. Repopulation of hepatic iNKT cells promotes liver repair by stimulating macrophage phenotype switching in the late phase of hepatic I/R injury.

Chronic Stress Physically Spares But Functionally Impairs Innate-Like Invariant T Cells

Long-term stress disrupts the neuroimmune interface, often resulting in immunosuppression. The deleterious effects of stress on mainstream T cells are well-documented. However, innate-like invariant T cells have been overlooked. Chief among these are invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, which fulfill important functions in anticancer and antimicrobial immunity. We found stress to surprisingly abrogate both TH1- and TH2-type responses mediated by iNKT cells. This was not due to cell death since iNKT cells were unusually refractory to stress-inflicted apoptosis. Activated iNKT cells in stressed mice triggered an abnormal response characterized by a ‘split’ inflammatory signature along with a sudden and robust induction of IL-10, IL-23 and IL-27. Pharmacological inhibitors, conditional knockout systems and gene expression analyses revealed that dysregulated iNKT cell responses were driven by iNKT cell-intrinsic glucocorticoid receptor signaling. Accordingly, the inhibitory effects of chronic stress on iNKT cell functions were lost upon habituation to predictable stressors. Importantly, iNKT cells in stressed mice failed to potentiate cytotoxicity against lymphoma targets or promote immune surveillance against metastatic melanoma. Using a congenic mouse model in which MAIT cells are abundant, these cells remained resistant to stress-induced death but demonstrated hindered TH1- and TH2-type cytokine responses, reminiscent of their iNKT cell counterparts. This was corroborated in culture systems in which we interrogated human peripheral blood and hepatic iNKT and MAIT cells. Our findings uncover a previously unappreciated mechanism of stress-induced immunosuppression and implicate the stress response as a potential barrier to iNKT cell- and MAIT cell-based immunotherapies.

Activation of Liver X Receptor α Sensitizes Mice to T-Cell Mediated Hepatitis.

Autoimmune hepatitis (AIH) is an inflammatory disease of the liver. Liver X receptors (LXRs), including the α and β isoforms, are previously known for their anti‐inflammatory activities. The goal of this study is to determine whether and how LXR plays a role in AIH. LXRα gain‐of‐function and loss‐of‐function mouse models were used, in conjunction with the concanavalin A (ConA) model of T‐cell mediated hepatitis. We first showed that the hepatic expression of LXRα was decreased in the ConA model of hepatitis and in human patients with AIH. In the ConA model, we were surprised to find that activation of LXRα in the constitutively activated VP‐LXRα whole‐body knock‐in (LXRα‐KI) mice exacerbated ConA‐induced AIH, whereas the LXRα−/− mice showed attenuated ConA‐induced AIH. Interestingly, hepatocyte‐specific activation of LXRα in the fatty acid binding protein–VP‐LXRα transgenic mice did not exacerbate ConA‐induced hepatitis. Mechanistically, the sensitizing effect of the LXRα‐KI allele was invariant natural killer T (iNKT)–cell dependent, because the sensitizing effect was abolished when the LXRα‐KI allele was bred into the NKT‐deficient CD1d−/− background. In addition, LXRα‐enhanced ConA‐induced hepatitis was dependent on interferon gamma. In contrast, adoptive transfer of hepatic iNKT cells isolated from LXRα‐KI mice was sufficient to sensitize CD1d−/− mice to ConA‐induced AIH. Conclusion: Activation of LXRα sensitizes mice to ConA‐induced AIH in iNKT and interferon gamma–dependent manner. Our results suggest that LXRα plays an important role in the development of AIH.

Differential Activation of Unconventional T Cells, Including iNKT Cells, in Alcohol-Related Liver Disease.

Liver is enriched in several innate-like unconventional T cells, but their role in alcohol-related liver disease (ALD) is not fully understood. Studies in several acute alcohol feeding models but not in chronic alcoholic steatohepatitis (ASH) model have shown that invariant natural killer T (iNKT) cells play a pathogenic role in ALD. Here, we investigated the activation of iNKT cells in an intragastric (iG) infusion model of chronic ASH as well as the frequency and cytokine phenotype of 3 different unconventional T cells: iNKT, mucosal-associated invariant T (MAIT), and CD8+ CD161hi Vα7.2- cells in peripheral blood of ALD patients. Hepatic iNKT cells were investigated using the iG model of chronic ASH that combines feeding of high-cholesterol/high-fat diet (HCFD) with intragastric feeding of ethanol diet (HCFD+iG Alc). Human iNKT, MAIT, and CD8+ CD161hi Vα7.2- cells were examined by flow cytometry in peripheral blood of patients with severe alcoholic hepatitis (SAH) and chronic alcoholics (ChA) and compared with healthy controls. In the iG model of chronic ASH, IFNγ+ iNKT cells accumulate in their livers compared with pair-fed control mice and activated hepatic iNKT cells show high expression of Fas and FasL. Notably, IFNγ+ iNKT cells are also significantly increased in peripheral blood of ChA patients compared with SAH patients. MAIT cells are significantly reduced in all ALD patients, but CD8+ CD161hi Vα7.2- cells are increased in SAH patients. Although MAIT and CD8+ CD161hi Vα7.2- cells displayed a similar cytokine production profile, the production of IFNγ and TNFα is significantly increased in SAH patients, while significant IL-17A production is found in ChA patients. We found that the 3 unconventional T cells are activated in ALD patients showing interesting differences in their frequency and cytokine production profile between SAH and ChA patients. In the iG murine model of chronic ASH, iNKT cells are also activated secreting proinflammatory cytokines suggesting their involvement in liver disease.

Activation of hepatic iNKT2 cells by α-GalCer ameliorates hepatic steatosis induced by high-fat diet in C57BL/6J mice

The existence of association between the subpopulation of iNKT cells with different functions and nonalcoholic fatty liver disease has not been confirmed. To investigative the role of iNKT cells in the pathogenesis of nonalcoholic fatty liver disease, we established a non-alcoholic fatty liver model by feeding C57BL/6J mice for 12 weeks with a high-fat diet and injecting α-GalCer through different routes to activate hepatic iNKT cells. The liver of the mice fed a high-fat diet (HFD) had severe hepatic steatosis appearance, elevated pro-inflammatory cytokines and reduced anti-inflammatory cytokines in the liver, and high serum levels of TC, LDL, HDL, and ALT. Our results showed that the percentage of iNKT cells in the liver of the HFD-fed mice was lower than that of the control mice. The expression levels of the related transcription factor of T-bet increased but that of GATA-3 decreased in the HFD-fed mice. The administration of α-GalCer by intraperitoneal injection resulted in increasing of hepatic iNKT and iNKT2 cells but decreasing of hepatic iNKT1 cells, and the expression of GATA-3 and anti-inflammatory cytokine (IL-4) was increased in the liver, and hepatic steatosis was ameliorated in the HFD-fed mice. The administration of α-GalCer by subcutaneous injection resulted in a decrease in hepatic iNKT and iNKT2 and an augmentation of hepatic iNKT1 cells. However, hepatic steatosis was not significantly improved. We concluded that the intraperitoneal injection with α-GalCer effectively improved hepatic steatosis, according to increasing the number of hepatic iNKT2 cells. The precise mechanism requires further exploration.

Stress-elicited glucocorticoid receptor signaling upregulates TIGIT in innate-like invariant T lymphocytes

Stress is known to impede certain host defense mechanisms, including those governed by conventional T lymphocytes. However, whether innate-like T lymphocytes, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, are impacted by stress is unclear. Herein, we report that prolonged psychological stress caused by physical confinement results in robust upregulation of T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), an immune checkpoint receptor that controls antitumor and antiviral immune responses. Elevated TIGIT expression was found not only on NK and conventional T cells, but also on iNKT and MAIT cells. Stress-provoked TIGIT upregulation was reversed through treatment with the glucocorticoid receptor (GR) antagonist RU486, but not with 6-hydroxydopamine that induces chemical sympathectomy. A Cre/Lox gene targeting model in which GR was ablated in cells expressing Lck under its proximal promoter revealed that TIGIT upregulation in stressed animals stems from direct GR signaling in T and iNKT cells. In fact, long-term oral administration of exogenous corticosterone (CS) to wild-type C57BL/6 (B6) mice was sufficient to increase TIGIT expression levels on T and iNKT cells. In vitro treatment with CS also potently and selectively upregulated TIGIT, but not CTLA-4 or LAG-3, on mouse iNKT and MAIT hybridomas. These results were recapitulated using primary hepatic iNKT and MAIT cells from wild-type B6 and B6.MAITCAST mice, respectively. Subjecting B6.MAITCAST mice to physical restraint also raised the frequency of TIGIT+ cells among hepatic MAIT cells in a GR-dependent manner. Finally, we found that TIGIT is similarly upregulated in a chronic variable stress model in which animals are exposed to unpredictable heterotypic stressors without developing habituation. Taken together, our findings link, for the first time to our knowledge, GR signaling to TIGIT expression. We propose that glucocorticoid hormones dampen immune responses, in part, by enhancing TIGIT expression across multiple critical subsets of effector lymphocytes, including innate-like T cells. Therefore, TIGIT may constitute an attractive target in immune-enhancing interventions for sustained physiological stress.

Differential Activation of Hepatic Invariant NKT Cell Subsets Plays a Key Role in Progression of Nonalcoholic Steatohepatitis.

Innate immune mechanisms play an important role in inflammatory chronic liver diseases. In this study, we investigated the role of type I or invariant NKT (iNKT) cell subsets in the progression of nonalcoholic steatohepatitis (NASH). We used α-galactosylceramide/CD1d tetramers and clonotypic mAb together with intracytoplasmic cytokine staining to analyze iNKT cells in choline-deficient l-amino acid-defined (CDAA)-induced murine NASH model and in human PBMCs, respectively. Cytokine secretion of hepatic iNKT cells in CDAA-fed C57BL/6 mice altered from predominantly IL-17+ to IFN-γ+ and IL-4+ during NASH progression along with the downmodulation of TCR and NK1.1 expression. Importantly, steatosis, steatohepatitis, and fibrosis were dependent upon the presence of iNKT cells. Hepatic stellate cell activation and infiltration of neutrophils, Kupffer cells, and CD8+ T cells as well as expression of key proinflammatory and fibrogenic genes were significantly blunted in Jα18-/- mice and in C57BL/6 mice treated with an iNKT-inhibitory RAR-γ agonist. Gut microbial diversity was significantly impacted in Jα18-/- and in CDAA diet-fed mice. An increased frequency of CXCR3+IFN-γ+T-bet+ and IL-17A+ iNKT cells was found in PBMC from NASH patients in comparison with nonalcoholic fatty liver patients or healthy controls. Consistent with their in vivo activation, iNKT cells from NASH patients remained hyporesponsive to ex-vivo stimulation with α-galactosylceramide. Accumulation of plasmacytoid dendritic cells in both mice and NASH patients suggest their role in activation of iNKT cells. In summary, our findings indicate that the differential activation of iNKT cells play a key role in mediating diet-induced hepatic steatosis and fibrosis in mice and its potential involvement in NASH progression in humans.

Peripheral Dopamine Controlled by Gut Microbes Inhibits Invariant Natural Killer T Cell-Mediated Hepatitis.

Neurotransmitters have been shown to regulate immune responses, and thereby are critically related to autoimmune diseases. Here we showed that depletion of dopaminergic neurons significantly promoted activation of hepatic iNKT cells and augmented concanavalin A (Con A)-induced liver injury. The suppressive effect of dopamine on iNKT cells was mediated by D1-like receptor-PKA pathway. Clearance of gut microbiota by antibiotic cocktail reduced synthesis of dopamine in intestines and exacerbated liver damage, and that could be restored by recovery of gut microbiota or replenishment of D1-like receptor agonist. Our results demonstrate that peripheral dopamine controlled by gut microbes inhibits IL4 and IFNγ production in iNKT cells and suppresses iNKT cell-mediated hepatitis. Together, we propose a gut microbe-nervous system-immune system regulatory axis in modulating autoimmune hepatitis.

Orchestrating liver repair: A newly discovered function of hepatic iNKT cells.

Orchestrating liver repair: A newly discovered function of hepatic iNKT cells.

Targeting colorectal cancer metastasis-initiating cells through hepatic iNKT cells

Abstract Aims To investigate the immune microenvironment modulation of iNKT cells for recognition and elimination of colorectal metastasis-initiating cell (MIC). Methods Different subtypes of human colorectal cancer cells and iNKT cells were analyzed by BD LSRFortessa™, TMA, and immunofluorescence(n=138). Microfluidic circulating tumor cell enrichment, Ex vivo 3D co-culture, Quansys Multiplex array, survival data analysis, and Elisa array were performed. Results There is a subtype of colorectal cancer stem cell referred as a metastasis-initiating cell (MIC), which do not express upregulated mesenchymal genes but express elevated levels of CD36 (the fatty acid receptor) and other lipid metabolism genes. Intriguingly, those cells overexpress NKG2DL, Various immune cells express NKG2D receptors, including natural killer (NK) cells, CD8+ T cells, and human iNKT cells. Concerning the NKT cells, there is an increase of NKG2D expression on liver resident iNKT cells (P < 0.05). We further observed that hepatic iNKT cells located at the invasive margin had been activated as shown by increased IFN-γ, TNF-α, granzyme B and Ki-67 expression. Furthermore, transmembrane CD1d density and intra/tmCXCL16 expression were also elevated in CD36+colorectal cancer stem cells, those cells showed significantly elevated α-GalCer: CD1d complex formation than other cancer stem cells in an ex vivo iNKT cells activation assay. CD36+cancer stem cells show susceptibility to iNKT cells induced cytotoxicity.

Tim-3 blockade promotes iNKT cell function to inhibit HBV replication.

Increased expression of T cell immunoglobulin and mucin domain‐3 (Tim‐3) on invariant natural killer T (iNKT) cells is reported in chronic hepatitis B virus (HBV) infection. However, whether Tim‐3 regulates iNKT cells in chronic HBV condition remains unclear. In this study, our results showed that the expression of Tim‐3 was up‐regulated on hepatic iNKT cells from HBV‐transgenic (Tg) mice or iNKT cells stimulated with α‐galactosylceramide (α‐Galcer). Compared with Tim‐3− iNKT cells, Tim‐3+ iNKT cells expressed more IFN‐γ, IL‐4 and CD107a, indicating a strong relationship between Tim‐3 and iNKT cell activation. Constantly, treatment of Tim‐3 blocking antibodies significantly enhanced the production of IFN‐γ, TNF‐α, IL‐4 and CD107a in iNKT cells both in vivo and in vitro. This Tim‐3− mediated suppression of iNKT cells was further confirmed in Tim‐3 knockout (KO) mice. Moreover, Tim‐3 blockade promoted α‐Galcer‐triggered inhibition of HBV replication, displaying as the decreased HBV DNA and HBsAg level in serum, and down‐regulated pgRNA expression in liver tissues. Collectively, our data, for the first time, demonstrated the potential role of Tim‐3 blockade in promoting iNKT cell‐mediated HBV inhibition. Therefore, combination of α‐Galcer with Tim‐3 blockade might be a promising approach in chronic hepatitis B therapy.

CD1d-Restricted pathways in hepatocytes control local natural killer T cell homeostasis and hepatic inflammation

Invariant natural killer T (iNKT) cells recognize lipid antigens presented by CD1d and play a central role in regulating immunity and inflammation in peripheral tissues. However, the mechanisms which govern iNKT cell homeostasis after thymic emigration are incompletely understood. Here we demonstrate that microsomal triglyceride transfer protein (MTP), a protein involved in the transfer of lipids onto CD1d, regulates liver iNKT cell homeostasis in a manner dependent on hepatocyte CD1d. Mice with hepatocyte-specific loss of MTP exhibit defects in the function of CD1d and show increased hepatic iNKT cell numbers as a consequence of altered iNKT cell apoptosis. Similar findings were made in mice with hepatocyte-specific loss of CD1d, confirming a critical role of CD1d in this process. Moreover, increased hepatic iNKT cell abundance in the absence of MTP is associated with susceptibility to severe iNKT cell-mediated hepatitis, thus demonstrating the importance of CD1d-dependent control of liver iNKT cells in maintaining immunological homeostasis in the liver. Together, these data demonstrate an unanticipated role of parenchymal cells, as shown here for hepatocytes, in tissue-specific regulation of CD1d-restricted immunity and further suggest that alterations in lipid metabolism may affect iNKT cell homeostasis through effects on CD1d-associated lipid antigens.

Antibody-dependent fragmentation is a newly identified mechanism of cell killing in vivo

The prevailing view is that therapeutic antibodies deplete cells through opsonization and subsequent phagocytosis, complement-dependent lysis or antibody-dependent cellular-cytotoxicity. We used high resolution in vivo imaging to identify a new antibody-dependent cell death pathway where Kupffer cells ripped large fragments off crawling antibody-coated iNKT cells. This antibody-dependent fragmentation process resulted in lethality and depletion of crawling iNKT cells in the liver sinusoids and lung capillaries. iNKT cell depletion was Fcy-receptor dependent and required iNKT cell crawling. Blood, spleen or joint iNKT cells that did not crawl were not depleted. The antibody required high glycosylation for sufficiently strong binding of the iNKT cells to the Fc Receptors on Kupffer cells. Using an acetaminophen overdose model, this approach functionally depleted hepatic iNKT cells and affected the severity of liver injury. This study reveals a new mechanism of antibody-dependent killing in vivo and raises implications for the design of new antibodies for cancer and auto-reactive immune cells.