Dietary and Microbial Oxazoles Induce Intestinal Inflammation by Modulating Aryl Hydrocarbon Receptor Responses.

Genome-wide association studies have identified risk loci associated with the development of inflammatory bowel disease, while epidemiological studies have emphasized that pathogenesis likely involves host interactions with environmental elements whose source and structure need to be defined. Here, we identify a class of compounds derived from dietary, microbial, and industrial sources that are characterized by the presence of a five-membered oxazole ring and induce CD1d-dependent intestinal inflammation. We observe that minimal oxazole structures modulate natural killer T cell-dependent inflammation by regulating lipid antigen presentation by CD1d on intestinal epithelial cells (IECs). CD1d-restricted production of interleukin 10 by IECs is limited through activity of the aryl hydrocarbon receptor (AhR) pathway in response to oxazole induction of tryptophan metabolites. As such, the depletion of the AhR in the intestinal epithelium abrogates oxazole-induced inflammation. In summary, we identify environmentally derived oxazoles as triggers of CD1d-dependent intestinal inflammatory responses that occur via activation of the AhR in the intestinal epithelium.

Lipids regulate peripheral serotonin release via gut CD1d.

The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.

Lipidomic scanning of self-lipids identifies headless antigens for natural killer T cells.

To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d-TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR-ceramide-CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the "sphingomyelin cycle" that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.

The immune system in neurological diseases: What innate-like T cells have to say.

The immune system classically consists of 2 lines of defense, innate and adaptive, both of which interact with one another effectively to protect us against any pathogenic threats. Importantly, there is a diverse subset of cells known as innate-like T cells that act as a bridge between the innate and adaptive immune systems and are pivotal players in eliciting inflammatory immune responses. A growing body of evidence has demonstrated the regulatory impact of these innate-like T cells in central nervous system (CNS) diseases and that such immune cells can traffic into the brain in multiple pathological conditions, which can be typically attributed to the breakdown of the blood-brain barrier. However, until now, it has been poorly understood whether innate-like T cells have direct protective or causative properties, particularly in CNS diseases. Therefore, in this review, our attention is focused on discussing the critical roles of 3 unique subsets of unconventional T cells, namely, natural killer T cells, γδ T cells, and mucosal-associated invariant T cells, in the context of CNS diseases, disorders, and injuries and how the interplay of these immune cells modulates CNS pathology, in an attempt to gain a better understanding of their complex functions.

Ox-LDL-induced CD80+ macrophages expand pro-atherosclerotic NKT cells via CD1d in atherosclerotic mice and hyperlipidemic patients.

Atherosclerosis is an inflammatory disease of blood vessels involving the immune system. Natural killer T (NKT) cells, as crucial components of the innate and acquired immune systems, play critical roles in the development of atherosclerosis. However, the mechanism and clinical relevance of NKT cells in early atherosclerosis are largely unclear. The study investigated the mechanism influencing NKT cell function in apoE deficiency-induced early atherosclerosis. Our findings demonstrated that there were higher populations of NKT cells and interferon-gamma (IFN-γ)-producing NKT cells in the peripheral blood of patients with hyperlipidemia and in the aorta, blood, spleen, and bone marrow of early atherosclerotic mice compared with the control groups. Moreover, we discovered that the infiltration of CD80+ macrophages and CD1d expression on CD80+ macrophages in atherosclerotic mice climbed remarkably. CD1d expression increased in CD80+ macrophages stimulated by oxidized low-density lipoprotein (ox-LDL) ex vivo and in vitro. Ex vivo coculture of macrophages with NKT cells revealed that ox-LDL-induced CD80+ macrophages presented lipid antigen α-Galcer (alpha-galactosylceramide) to NKT cells via CD1d, enabling NKT cells to express more IFN-γ. Furthermore, a greater proportion of CD1d+ monocytes and CD1d+CD80+ monocytes were found in peripheral blood of hyperlipidemic patients compared with that of healthy donors. Positive correlations were found between CD1d+CD80+ monocytes and NKT cells or IFN-γ+ NKT cells in hyperlipidemic patients. Our findings illustrated that CD80+ macrophages stimulated NKT cells to secrete IFN-γ via CD1d-presenting α-Galcer, which may accelerate the progression of early atherosclerosis. Inhibiting lipid antigen presentation by CD80+ macrophages to NKT cells may be a promising immune target for the treatment of early atherosclerosis.NEW & NOTEWORTHY This work proposed the ox-LDL-CD80+ monocyte/macrophage-CD1d-NKT cell-IFN-γ axis in the progression of atherosclerosis. The proinflammatory IFN-γ+ NKT cells are closely related to CD1d+CD80+ monocytes in hyperlipidemic patients. Inhibiting CD80+ macrophages to present lipid antigens to NKT cells through CD1d blocking may be a new therapeutic target for atherosclerosis.

Human CD4+ iNKT cell adoptive immunotherapy induces anti-tumour responses against CD1d-negative EBV-driven B lymphoma.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that are uniquely suitable as off-the-shelf cellular immunotherapies due to their lack of alloreactivity. Two major subpopulations of human iNKT cells have been delineated, a CD4- subset that has a TH1/cytolytic profile, and a CD4+ subset that appears polyfunctional and can produce both regulatory and immunostimulatory cytokines. Whether these two subsets differ in anti-tumour effects is not known. Using live cell imaging, we found that CD4- iNKT cells limited growth of CD1d+ Epstein-Barr virus (EBV)-infected B-lymphoblastoid spheroids in vitro, whereas CD4+ iNKT cells showed little or no direct anti-tumour activity. However, the effects of the two subsets were reversed when we tested them as adoptive immunotherapies in vivo using a xenograft model of EBV-driven human B cell lymphoma. We found that EBV-infected B cells down-regulated CD1d in vivo, and administering CD4- iNKT cells had no discernable impact on tumour mass. In contrast, xenotransplanted mice bearing lymphomas showed rapid reduction in tumour mass after administering CD4+ iNKT cells. Immunotherapeutic CD4+ iNKT cells trafficked to both spleen and tumour and were associated with subsequently enhanced responses of xenotransplanted human T cells against EBV. CD4+ iNKT cells also had adjuvant-like effects on monocyte-derived DCs and promoted antigen-dependent responses of human T cells in vitro. These results show that allogeneic CD4+ iNKT cellular immunotherapy leads to marked anti-tumour activity through indirect pathways that do not require tumour cell CD1d expression and that are associated with enhanced activity of antigen-specific T cells.

CD1d protects against hepatocyte apoptosis in non-alcoholic steatohepatitis.

BACKGROUND & AIMS: Hepatocyte apoptosis, a well-defined form of cell death in non-alcoholic steatohepatitis (NASH), is considered the primary cause of liver inflammation and fibrosis. However, the mechanisms underlying the regulation of hepatocyte apoptosis in NASH remain largely unclear. We explored the anti-apoptotic effect of hepatocyte CD1d in NASH. METHODS: Hepatocyte CD1d expression was analyzed in patients with NASH and mouse models. Hepatocyte-specific gene overexpression or knockdown and anti-CD1d crosslinking were used to investigate the anti-apoptotic effect of hepatocyte CD1d on lipotoxicity-, Fas-, and concanavalin (ConA)-mediated liver injuries. A high-fat diet, a methionine-choline-deficient diet, a Fas agonist, and ConA were used to induce lipotoxic and/or apoptotic liver injuries. Palmitic acid was used to mimic lipotoxicity-induced apoptosis in vitro. RESULTS: We identified a dramatic decrease in CD1d expression in hepatocytes of patients with NASH and mouse models. Hepatocyte-specific CD1d overexpression and knockdown experiments collectively demonstrated that hepatocyte CD1d protected against hepatocyte apoptosis and alleviated hepatic inflammation and injuries in NASH mice. Furthermore, decreased JAK2-STAT3 signaling was observed in NASH patient livers. Mechanistically, anti-CD1d crosslinking on hepatocytes induced tyrosine phosphorylation of the CD1d cytoplasmic tail, leading to the recruitment and phosphorylation of JAK2. Phosphorylated JAK2 activated STAT3 and subsequently reduced apoptosis in hepatocytes, which was associated with an increase in anti-apoptotic effectors (Bcl-xL and Mcl-1) and a decrease in pro-apoptotic effectors (cleaved-caspase 3/7). Moreover, anti-CD1d crosslinking effectively protected against Fas- or ConA-mediated hepatocyte apoptosis and liver injury in mice. CONCLUSIONS: Our study uncovered a previously unrecognized anti-apoptotic CD1d-JAK2-STAT3 axis in hepatocytes that conferred hepatoprotection and highlighted the potential of hepatocyte CD1d-directed therapy for liver injury and fibrosis in NASH, as well as in other liver diseases associated with hepatocyte apoptosis. IMPACT AND IMPLICATIONS: Excessive and/or sustained hepatocyte apoptosis is critical in driving liver inflammation and injury. The mechanisms underlying the regulation of hepatocyte apoptosis in non-alcoholic steatohepatitis (NASH) remain largely unclear. Here, we found that CD1d expression in hepatocytes substantially decreases and negatively correlates with the severity of liver injury in patients with NASH. We further revealed a previously unrecognized anti-apoptotic CD1d-JAK2-STAT3 signaling axis in hepatocytes, which confers significant protection against liver injury in NASH and acute liver diseases. Thus, hepatocyte CD1d-targeted therapy could be a promising strategy to manipulate liver injury in both NASH and other hepatocyte apoptosis-related liver diseases.

Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d.

Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αß T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug-like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αßTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1-TRAJ6) that was conserved across donors. By trapping a CD1d-type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.

Varicella zoster virus downregulates expression of the non-classical antigen presentation molecule CD1d.

BACKGROUND: The non-classical antigen presentation molecule CD1d presents lipid antigens to invariant natural killer T (iNKT) cells. Activation of these cells triggers a rapid cytokine response providing an interface between innate and adaptive immune responses. The importance of CD1d and iNKT cells in varicella zoster virus (VZV) infection has been emphasised by clinical reports of individuals with CD1d or iNKT cell deficiencies experiencing severe, disseminated varicella post-vaccination. METHODS: Three strains of VZV, VZV-S, rOka, and VZV rOka-66S were used to infect Jurkat cells. Flow cytometry of VZV- and mock-infected cells assessed the modulatory impact of VZV on CD1d. Infected cell-supernatant and transwell coculture experiments explored the role of soluble factors in VZV-mediated immunomodulation. CD1d transcripts were assessed by RT-qPCR. RESULTS: Surface and intracellular flow cytometry demonstrated CD1d was strikingly downregulated by VZV-S and rOka in both infected and VZV antigen-negative cells compared to mock. CD1d downregulation is cell-contact-dependant and CD1d transcripts are targeted by VZV. Mechanistic investigations using rOka-66S (unable to express the viral kinase ORF66), implicate this protein in CD1d modulation in infected cells. CONCLUSIONS: VZV implements multiple mechanisms targeting both CD1d transcript and protein. This provides evidence of VZV interaction with and manipulation of the CD1d-iNKT cell axis.

Diversification of a Novel α-Galactosyl Ceramide Hotspot Boosts the Adjuvant Properties in Parenteral and Mucosal Vaccines.

The development of potent adjuvants is an important step for improving the performance of subunit vaccines. CD1d agonists, such as the prototypical α-galactosyl ceramide (α-GalCer), are of special interest due to their ability to activate iNKT cells and trigger rapid dendritic cell maturation and B-cell activation. Herein, we introduce a novel derivatization hotspot at the α-GalCer skeleton, namely the N-substituent at the amide bond. The multicomponent diversification of this previously unexplored glycolipid chemotype space permitted the introduction of a variety of extra functionalities that can either potentiate the adjuvant properties or serve as handles for further conjugation to antigens toward the development of self-adjuvanting vaccines. This strategy led to the discovery of compounds eliciting enhanced antigen-specific T cell stimulation and a higher antibody response when delivered by either the parenteral or the mucosal route, as compared to a known potent CD1d agonist. Notably, various functionalized α-GalCer analogues showed a more potent adjuvant effect after intranasal immunization than a PEGylated α-GalCer analogue previously optimized for this purpose. Ultimately, this work could open multiple avenues of opportunity for the use of mucosal vaccines against microbial infections.

Dynamics of IL-15/IL-15R-α expression in response to HSV-1 infection reveal a novel mode of viral immune evasion counteracted by iNKT cells.

Herpes simplex virus type 1 (HSV-1) infects and persists in most of the human population. Interleukin-15 (IL-15) has an important role in the activation of cell-mediated immune responses and acts in complex with IL-15 receptor alpha (IL-15R-α) through cell surface transpresentation. Here, we have examined the IL-15/IL-15R-α complex response dynamics during HSV-1 infection in human keratinocytes. Surface expression of the IL-15/IL-15R-α complex rapidly increased in response to HSV-1, reaching a peak around 12 h after infection. This response was dependent on detection of viral replication by TLR3, and enhancement of IL15 and IL15RA gene expression. Beyond the peak of expression, levels of IL-15 and IL-15R-α gradually declined, reaching a profound loss of surface expression beyond 24 h of infection. This involved the loss of IL15 and IL15RA transcription. Interestingly, invariant natural killer T (iNKT) cells inhibited the viral interference with IL-15/IL-15R-α complex expression in an IFNγ-dependent manner. These results indicate that rapid upregulation of the IL-15/IL-15R-α complex occurs in HSV-1 infected keratinocytes, and that this response is targeted by viral interference. Shutdown of the IL-15 axis represents a novel mode of HSV-1 immune evasion, which can be inhibited by the host iNKT cell response.

Pathogenic NKT cells attenuate urogenital chlamydial clearance and enhance infertility.

Urogenital chlamydial infections continue to increase with over 127 million people affected annually, causing significant economic and public health pressures. While the role of traditional MHCI and II peptide presentation is well defined in chlamydial infections, the role of lipid antigens in immunity remains unclear. Natural killer (NK) T cells are important effector cells that recognize and respond to lipid antigens during infections. Chlamydial infection of antigen-presenting cells facilitates presentation of lipid on the MHCI-like protein, CD1d, which stimulates NKT cells to respond. During urogenital chlamydial infection, wild-type (WT) female mice had significantly greater chlamydial burden than CD1d-/- (NKT-deficient) mice, and had significantly greater incidence and severity of immunopathology in both primary and secondary infections. WT mice had similar vaginal lymphocytic infiltrate, but 59% more oviduct occlusion compared to CD1d-/- mice. Transcriptional array analysis of oviducts day 6 post-infection revealed WT mice had elevated levels of Ifnγ (6-fold), Tnfα (38-fold), Il6 (2.5-fold), Il1ß (3-fold) and Il17a (6-fold) mRNA compared to CD1d-/- mice. In infected females, oviduct tissues had an elevated infiltration of CD4+ -invariant NKT (iNKT) cells, however, iNKT-deficient Jα18-/- mice had no significant differences in hydrosalpinx severity or incidence compared to WT controls. Lipid mass spectrometry of surface-cleaved CD1d in infected macrophages revealed an enhancement of presented lipids and cellular sequestration of sphingomyelin. Taken together, these data suggest an immunopathogenic role for non-invariant NKT cells in urogenital chlamydial infections, facilitated by lipid presentation via CD1d via infected antigen-presenting cells.

CD1d-mediated lipid presentation by CD11c+ cells regulates intestinal homeostasis.

Intestinal homeostasis relies on a continuous dialogue between the commensal bacteria and the immune system. Natural killer T (NKT) cells, which recognize CD1d-restricted microbial lipids and self-lipids, contribute to the regulation of mucosal immunity, yet the mechanisms underlying their functions remain poorly understood. Here, we demonstrate that NKT cells respond to intestinal lipids and CD11c+ cells (including dendritic cells (DCs) and macrophages) are essential to mediate lipid presentation within the gut ultimately controlling intestinal NKT cell homeostasis and activation. Conversely, CD1d and NKT cells participate in the control of the intestinal bacteria composition and compartmentalization, in the regulation of the IgA repertoire and in the induction of regulatory T cells within the gut. These changes in intestinal homeostasis require CD1d expression on DC/macrophage populations as mice with conditional deletion of CD1d on CD11c+ cells exhibit dysbiosis and altered immune homeostasis. These results unveil the importance of CD11c+ cells in controlling lipid-dependent immunity in the intestinal compartment and reveal an NKT cell-DC crosstalk as a key mechanism for the regulation of gut homeostasis.

Invariant NKT cell-augmented GM-CSF-secreting tumor vaccine is effective in advanced prostate cancer model.

Invariant natural killer T cells (iNKT cells) express a semi-invariant T cell receptor that recognizes certain glycolipids (including α-galactosylceramide, αGC) bound to CD1d, and can induce potent antitumor responses. Here, we assessed whether αGC could enhance the efficacy of a GM-CSF-producing tumor cell vaccine in the transgenic SV40 T antigen-driven TRAMP prostate cancer model. In healthy mice, we initially found that optimal T cell responses were obtained with αGC-pulsed TRAMP-C2 cells secreting GM-CSF and milk fat globule epidermal growth factor protein-8 (MFG-E8) with an RGD to RGE mutation (GM-CSF/RGE TRAMP-C2), combined with systemic low dose IL-12. In a therapeutic model, transgenic TRAMP mice were then castrated at ~ 20 weeks, followed by treatment with the combination vaccine. Untreated mice succumbed to tumor by ~ 40 weeks, but survival was markedly prolonged by vaccine treatment, with most mice surviving past 80 weeks. Prostates in the treated mice were heavily infiltrated with T cells and iNKT cells, which both secreted IFNγ in response to tumor cells. The vaccine was not effective if the αGC, IL-12, or GM-CSF secretion was eliminated. Finally, immunized mice were fully resistant to challenge with TRAMP-C2 cells. Together these findings support further development of therapeutic vaccines that exploit iNKT cell activation.

Protein abundance of the cytokine receptor γc controls the thymic generation of innate-like T cells.

Innate-like T (iT) cells comprise a population of immunoregulatory T cells whose effector function is imposed during their development in the thymus to provide protective immunity prior to antigen encounter. The molecular mechanism that drives the generation of iT cells remains unclear. Here, we report that the cytokine receptor γc plays a previously unappreciated role for thymic iT cells by controlling their cellular abundance, lineage commitment, and subset differentiation. As such, γc overexpression on thymocytes dramatically altered iT cell generation in the thymus, as it skewed the subset composition of invariant NKT (iNKT) cells and promoted the generation of IFNγ-producing innate CD8 T cells. Mechanistically, we found that the γc-STAT6 axis drives the differentiation of IL-4-producing iNKT cells, which in turn induced the generation of innate CD8 T cells. Collectively, these results reveal a cytokine-driven circuity of thymic iT cell differentiation that is controlled by the abundance of γc proteins.

Sterile activation of invariant natural killer T cells by ER-stressed antigen-presenting cells.

Invariant NKT (iNKT) cells have the unique ability to shape immunity during antitumor immune responses and other forms of sterile and nonsterile inflammation. Recent studies have highlighted a variety of classes of endogenous and pathogen-derived lipid antigens that can trigger iNKT cell activation under sterile and nonsterile conditions. However, the context and mechanisms that drive the presentation of self-lipid antigens in sterile inflammation remain unclear. Here we report that endoplasmic reticulum (ER)-stressed myeloid cells, via signaling events modulated by the protein kinase RNA-like ER kinase (PERK) pathway, increase CD1d-mediated presentation of immunogenic endogenous lipid species, which results in enhanced iNKT cell activation both in vitro and in vivo. In addition, we demonstrate that actin cytoskeletal reorganization during ER stress results in an altered distribution of CD1d on the cell surface, which contributes to enhanced iNKT cell activation. These results define a previously unidentified mechanism that controls iNKT cell activation during sterile inflammation.

The iNKT Cell-Macrophage Axis in Homeostasis and Disease.

Invariant natural killer T (iNKT) cells are CD1d-restricted, lipid-reactive T cells that exhibit preponderant immunomodulatory properties. The ultimate protective or deleterious functions displayed by iNKT cells in tissues are known to be partially shaped by the interactions they establish with other immune cells. In particular, the iNKT cell-macrophage crosstalk has gained growing interest over the past two decades. Accumulating evidence has highlighted that this immune axis plays central roles not only in maintaining homeostasis but also during the development of several pathologies. Hence, this review summarizes the reported features of the iNKT cell-macrophage axis in health and disease. We discuss the pathophysiological significance of this interplay and provide an overview of how both cells communicate with each other to regulate disease onset and progression in the context of infection, obesity, sterile inflammation, cancer and autoimmunity.

Tumor-Localized Administration of α-GalCer to Recruit Invariant Natural Killer T Cells and Enhance Their Antitumor Activity against Solid Tumors.

Invariant natural killer T (iNKT) cells have the capacity to mount potent anti-tumor reactivity and have therefore become a focus in the development of cell-based immunotherapy. iNKT cells attack tumor cells using multiple mechanisms with a high efficacy; however, their clinical application has been limited because of their low numbers in cancer patients and difficulties in infiltrating solid tumors. In this study, we aimed to overcome these critical limitations by using α-GalCer, a synthetic glycolipid ligand specifically activating iNKT cells, to recruit iNKT to solid tumors. By adoptively transferring human iNKT cells into tumor-bearing humanized NSG mice and administering a single dose of tumor-localized α-GalCer, we demonstrated the rapid recruitment of human iNKT cells into solid tumors in as little as one day and a significantly enhanced tumor killing ability. Using firefly luciferase-labeled iNKT cells, we monitored the tissue biodistribution and pharmacokinetics/pharmacodynamics (PK/PD) of human iNKT cells in tumor-bearing NSG mice. Collectively, these preclinical studies demonstrate the promise of an αGC-driven iNKT cell-based immunotherapy to target solid tumors with higher efficacy and precision.

Hepatocyte CD1d protects against liver immunopathology in mice with schistosomiasis japonica.

Schistosomiasis is a neglected tropical disease with over 250 million people infected worldwide. The main clinically important species Schistosoma mansoni (S. mansoni) and Schistosoma japonicum (S. japonicum) cause inflammatory responses against tissue-trapped eggs, resulting in formation of granulomas mainly in host liver. Persistent granulomatous response results in severe fibrosis in the liver, leading to irreversible impairment of the liver and even death of the host. CD1d, a highly conserved MHC class I-like molecule, is expressed by both haematopoietic and non-haematopoietic cells. CD1d on antigen-presenting cells (APCs) of haematopoietic origin presents pathogen-derived lipid antigens to natural killer T (NKT) cells, which enables them to rapidly produce large amounts of various cytokines and facilitate CD4+ T helper (Th) cell differentiation upon invading pathogens. Noteworthy, hepatocytes of non-haematopoietic origin have recently been shown to be involved in maintaining liver NKT cell homeostasis through a CD1d-dependent manner. However, whether hepatocyte CD1d-dependent regulation of NKT cell homeostasis also modulates CD4+ Th cell responses and liver immunopathology in murine schistosomiasis remains to be addressed. Here, we show in mice that CD1d expression on hepatocytes was decreased dramatically upon S. japonicum infection, accompanied by increased NKT cells, as well as upregulated Th1 and Th2 responses. Overexpression of CD1d in hepatocytes significantly decreased local NKT numbers and cytokines (IFN-γ, IL-4, IL-13), concomitantly with downregulation of both Th1 and Th2 responses and alleviation in pathological damage in livers of S. japonicum-infected mice. These findings highlight the potential of hepatocyte CD1d-targeted therapies for liver immunopathology control in schistosomiasis.

Contribution of the SYK Tyrosine kinase expression to human iNKT self-reactivity.

Invariant Natural Killer T (iNKT) cells are particular T lymphocytes at the frontier between innate and adaptative immunities. They participate in the elimination of pathogens or tumor cells, but also in the development of allergic reactions and autoimmune diseases. From their first descriptions, the phenomenon of self-reactivity has been described. Indeed, they are able to recognize exogenous and endogenous lipids. However, the mechanisms underlying the self-reactivity are still largely unknown, particularly in humans. Using a CD1d tetramer-based sensitive immunomagnetic approach, we generated self-reactive iNKT cell lines from blood circulating iNKT cells of healthy donors. Analysis of their functional characteristics in vitro showed that these cells recognized endogenous lipids presented by CD1d molecules through their TCR that do not correspond to α-glycosylceramides. TCR sequencing and transcriptomic analysis of T cell clones revealed that a particular TCR signature and an expression of the SYK protein kinase were two mechanisms supporting human iNKT self-reactivity. The SYK expression, strong in the most self-reactive iNKT clones and variable in ex vivo isolated iNKT cells, seems to decrease the activation threshold of iNKT cells and increase their overall antigenic sensitivity. This study indicates that a modulation of the TCR intracellular signal contributes to iNKT self-reactivity.