CD1d-dependent Manner Research Articles

Role of CD1d and iNKT cells in regulating intestinal inflammation.

Invariant natural killer T (iNKT) cells, a subset of unconventional T cells that recognize glycolipid antigens in a CD1d-dependent manner, are crucial in regulating diverse immune responses such as autoimmunity. By engaging with CD1d-expressing non-immune cells (such as intestinal epithelial cells and enterochromaffin cells) and immune cells (such as type 3 innate lymphoid cells, B cells, monocytes and macrophages), iNKT cells contribute to the maintenance of immune homeostasis in the intestine. In this review, we discuss the impact of iNKT cells and CD1d in the regulation of intestinal inflammation, examining both cellular and molecular factors with the potential to influence the functions of iNKT cells in inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.

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.

Protective colonic iNKT cells are expanded by Goblet cell-associated antigen passages in CD1d-dependent manner in adults

Abstract Invariant natural killer T (iNKT) cells are unique innate immune cell which secrete cytokines and immune mediators. These cells recognize self or microbial glycolipids presented in the context of CD1d. iNKT cells can contribute to host protection or pathogenesis during intestinal inflammation. Colonic iNKT cells are established under the influence of the microbiota in early life and the current understanding is that colonic iNKT cells are fixed and not manipulable in later life. Goblet cells (GCs) or/and goblet cell associated passages (GAPs) deliver luminal antigens and maintain peripheral T regulatory cells in the steady state. However, GAPs in the colon are absent in adult mice due to GCs microbial sensing. We found that the glycolipids can be delivered through GAPs and that inducing GAPs in the colon in adult mice resulted in significant iNKT cell expansion. Deletion of GCs or deletion of CD1d on GCs prevented iNKT cell expansion suggesting a role for colonic GCs in presenting glycolipids to iNKT cells. Single cell RNA sequencing of colonic-iNKT cells showed significantly increased iNKT2 and iNKT1 subsets after colonic GAP induction. Proteomic analysis of these iNKT cells further confirmed the characteristics of expanded iNKT subsets. Moreover, iNKT cells expanding after colonic GAP induction persisted and were protective in DSS-induced colitis. Adoptive transfer of the expanded colonic iNKT cells protected in the DSS-induced colitis model, indicating that expanded colonic iNKT cells were sufficient for protection in this model. These findings indicate that colonic iNKT cells are not fixed during early life but can be expanded in a GAP dependent and GC CD1d dependent manner and can be protective in some models of colitis. Crohn's and Colitis Foundation

Lipid sensing by gut CD1d calibrates peripheral serotonin release

Abstract The crosstalk between the immune and the neuroendocrine system is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones release in response to environmental cues, such as self 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, promotes peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. CD1d ligation on EC cells transduces a reverse signal and restrains potassium conductance, leading to calcium influx and 5-HT secretion. Together, we show that by coupling with iNKT cells, gut chemosensory cells perceive lipid antigens via CD1d to calibrate 5-HT release, modulating intestinal and systemic physiology.

Invariant natural killer T cells minimally influence gut microbiota composition in mice

ABSTRACT Invariant Natural Killer T (iNKT) cells are unconventional T cells that respond to glycolipid antigens found in microbes in a CD1d-dependent manner. iNKT cells exert innate-like functions and produce copious amounts of cytokines, chemokines and cytotoxic molecules within only minutes of activation. As such, iNKT cells can fuel or dampen inflammation in a context-dependent manner. In addition, iNKT cells provide potent immunity against bacteria, viruses, parasites and fungi. Although microbiota-iNKT cell interactions are not well-characterized, mounting evidence suggests that microbiota colonization early in life impacts iNKT cell homeostasis and functions in disease. In this study, we showed that CD1d−/− and Vα14 Tg mice, which lack and have increased numbers of iNKT cells, respectively, had no significant alterations in gut microbiota composition compared to their littermate controls. Furthermore, specific iNKT cell activation by glycolipid antigens only resulted in a transient and minimal shift in microbiota composition when compared to the natural drift found in our colony. Our findings demonstrate that iNKT cells have little to no influence in regulating commensal bacteria at steady state. Abbreviations: iNKT: invariant Natural Killer T cell; αGC: α-galactosylceramide

Human iNKT Cells Modulate Macrophage Survival and Phenotype.

CD1d-restricted invariant Natural Killer T (iNKT) cells are unconventional innate-like T cells whose functions highly depend on the interactions they establish with other immune cells. Although extensive studies have been reported on the communication between iNKT cells and macrophages in mice, less data is available regarding the relevance of this crosstalk in humans. Here, we dove into the human macrophage-iNKT cell axis by exploring how iNKT cells impact the survival and polarization of pro-inflammatory M1-like and anti-inflammatory M2-like monocyte-derived macrophages. By performing in vitro iNKT cell-macrophage co-cultures followed by flow cytometry analysis, we demonstrated that antigen-stimulated iNKT cells induce a generalized activated state on all macrophage subsets, leading to upregulation of CD40 and CD86 expression. CD40L blocking with a specific monoclonal antibody prior to co-cultures abrogated CD40 and CD86 upregulation, thus indicating that iNKT cells required CD40-CD40L co-stimulation to trigger macrophage activation. In addition, activated iNKT cells were cytotoxic towards macrophages in a CD1d-dependent manner, killing M1-like macrophages more efficiently than their naïve M0 or anti-inflammatory M2-like counterparts. Hence, this work highlighted the role of human iNKT cells as modulators of macrophage survival and phenotype, untangling key features of the human macrophage-iNKT cell axis and opening perspectives for future therapeutic modulation.

Goblet cells regulate expansion of colonic iNKT cells in CD1d-dependent manner

Abstract Invariant natural killer T (iNKT) cells are innate-like T cells that secrete a wide array of cytokines and immune mediators. These cells recognize self or microbial ligands presented by cells expressing CD1d, and can contribute to host protection or/and pathogenesis during intestinal inflammation. The colonic iNKT cell population is established in early life under the influence of the microbiota and current understanding is that this iNKT cells are not manipulable in later life. Previous studies have identified that goblet cell associated passages (GAPs) play a role in luminal antigen delivery and the induction and maintenance of peripherally induced T regulatory cells in the steady state. Colonic GAPs are largely absent in adult mice due to goblet cell (GC) microbial sensing, which inhibits GAP formation. We hypothesized, that when present, colonic GAPs may deliver glycolipids to stimulate colonic iNKT cells. We found that the glycolipids can be delivered through GAPs and that inducing colonic GAPs in adult mice using pharmacological inhibitors or transgenic mouse models resulted in significant iNKT cell expansion. Further deletion of CD1d on GCs inhibited iNKT cell expansion suggesting a role for colonic GCs in presenting glycolipids to iNKT cells. Single cell RNA sequencing of sorted colonic iNKT cells showed significantly expanded iNKT2 and iNKT1 subsets after colonic GAP induction. Furthermore, the iNKT cells expanding after opening colonic GAPs were protective in DSS-induced colitis. Our findings suggest that the GAP function and CD1d expression by GCs plays a role in modulating colonic iNKT cell subsets in adulthood and can be protective in some colitis models.

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.

Lipid dysregulation and antigen presentation during intestinal inflammation

Abstract Inflammatory bowel diseases are associated with a loss of epithelial barrier integrity that can lead to an imbalance in gut homeostasis and severe inflammation. Studies in mice have shown that CD1d, a lipid antigen-presenting molecule, and natural killer T (NKT) cells play important roles in forming and maintaining homeostasis in the intestines. However, it is currently unknown what specific lipids are presented to NKT cells during homeostasis or inflammation in the gut. We hypothesize that during inflammation, CD1d ligands are replaced with pathogenic lipids that alter the activation of NKT cells and contribute to inflammation. Mice were administered dextran sodium sulfate (DSS) in their drinking water for 7 days to induce colitis symptoms, then switched to normal water for an additional 7 days to recover. Lipid species were measured in colons from day 7 inflamed mice and day 14 recovered mice. Compared to control mice, the lipid composition of inflamed colon was significantly altered. Day 7 enriched ceramide and hexosylceramide species remained elevated at the recovery time-point. These lipids stimulated inflammatory cytokine production in lamina propria cultures in a CD1d-dependent manner. To determine if these lipids were being generated endogenously, we measured the gene expression of several key enzymes in the hexosylceramide synthesis pathway. Expression of Degs1 and Ugcg were up-regulated in lamina propria cells during colitis, suggesting that immune cells could be responsible for the altered lipid composition we observed in DSS treated mice. Overall, we have demonstrated that colitis-induced lipid alterations in lamina propria cells of the gut can contribute to intestinal inflammation through CD1d antigen presentation.

Invariant NKT Cells From Donor Lymphocyte Infusions (DLI-iNKTs) Promote ex vivo Lysis of Leukemic Blasts in a CD1d-Dependent Manner.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative treatment option for hematologic malignancies but relapse remains the most common cause of death. Infusion of donor lymphocytes (DLIs) can induce remission and prolong survival by exerting graft-vs.-leukemia (GVL) effects. However, sufficient tumor control cannot be established in all patients and occurrence of graft-vs.-host disease (GVHD) prevents further dose escalation. Previous data indicate that invariant natural killer T (iNKT) cells promote anti-tumor immunity without exacerbating GVHD. In the present study we investigated lysis of leukemic blasts through iNKT cells from donor-derived lymphocytes for leukemia control and found that iNKT cells constitute about 0.12% of cryopreserved donor T cells. Therefore, we established a 2-week cell culture protocol allowing for a robust expansion of iNKT cells from cryopreserved DLIs (DLI-iNKTs) that can be used for further preclinical and clinical applications. Such DLI-iNKTs efficiently lysed leukemia cell lines and primary patient AML blasts ex vivo in a dose- and CD1d-dependent manner. Furthermore, expression of CD1d on target cells was required to release proinflammatory cytokines and proapoptotic effector molecules. Our results suggest that iNKT cells from donor-derived lymphocytes are involved in anti-tumor immunity after allo-HCT and therefore may reduce the risk of relapse and improve progression-free and overall survival.

TLR9-mediated dendritic cell activation uncovers mammalian ganglioside species with specific ceramide backbones that activate invariant natural killer T cells.

CD1d-restricted invariant natural killer T (iNKT) cells represent a heterogeneous population of lipid-reactive T cells that are involved in many immune responses, mediated through T-cell receptor (TCR)–dependent and/or independent activation. Although numerous microbial lipid antigens (Ags) have been identified, several lines of evidence have suggested the existence of relevant Ags of endogenous origin. However, the identification of their precise nature as well as the molecular mechanisms involved in their generation are still highly controversial and ill defined. Here, we identified two mammalian gangliosides—namely monosialoganglioside GM3 and disialoganglioside GD3—as endogenous activators for mouse iNKT cells. These glycosphingolipids are found in Toll-like receptor-stimulated dendritic cells (DC) as several species varying in their N-acyl fatty chain composition. Interestingly, their ability to activate iNKT cells is highly dependent on the ceramide backbone structure. Thus, both synthetic GM3 and GD3 comprising a d18:1-C24:1 ceramide backbone were able to activate iNKT cells in a CD1d-dependent manner. GM3 and GD3 are not directly recognized by the iNKT TCR and required the Ag presenting cell intracellular machinery to reveal their antigenicity. We propose a new concept in which iNKT cells can rapidly respond to pre-existing self-molecules after stress-induced structural changes in CD1d-expressing cells. Moreover, these gangliosides conferred partial protection in the context of bacterial infection. Thus, this report identified new biologically relevant lipid self-Ags for iNKT cells.

The GM2 ganglioside inhibits iNKT cell responses in a CD1d-dependent manner

Invariant natural killer T (iNKT) cells are a subset of T lymphocytes that recognize lipid antigens presented on CD1d molecules at the surface of antigen-presenting cells. GM2 is a glycosphingolipid abundant in cellular membranes and known to bind CD1d molecules, but the functional consequences of this binding are not completely clarified.Herein, we analyzed the effect of GM2 in iNKT cell activation. We found that culturing antigen-presenting cells or total peripheral blood mononuclear cells with GM2 did not induce activation of human iNKT cells, implying that this lipid is not antigenic for human iNKT cells. To investigate if this lipid could inhibit iNKT cell activation, we simultaneously incubated antigen-presenting cells with GM2 and the iNKT cell antigen α-Galactosylceramide (α-GalCer) and used them to stimulate iNKT cells. We found that GM2 reduced human iNKT cell activation in a dose-dependent manner. An explanation for this effect could be a direct competition of GM2 with antigenic lipids for CD1d binding. This was demonstrated by the use of an antibody (L363) that stains mouse CD1d:α-GalCer complexes, as in the presence of GM2 the amount of CD1d:α-GalCer complexes are reduced. We further explored the consequences of chronic GM2 overload on human iNKT cells by analyzing iNKT cells in patients diagnosed with GM2 gangliosidoses. We found that pediatric patients present a higher frequency of circulating CD4+ iNKT cells and concomitant lower frequency of CD4−CD8− iNKTs. A lower percentage of iNKT cells expressing the NK marker CD161 was also observed in these patients. In contrast, in two adult patients studied, no differences on iNKT cell phenotype were observed.Altogether, this study uncovers a new role for GM2 in the modulation of iNKT cell activation, thus strengthening the central role of lipid metabolism in iNKT cell biology.

Key role for CD103+ DCs in activating lung iNKT cells during Streptococcus pneumoniae infection

Abstract Invariant natural killer T cells (iNKT) are profoundly important for protection against Streptococcus pneumoniae. Prior work has shown iNKT cells are rapidly activated in a CD1d-dependent manner, however the critical antigen presenting cells remain unknown. We have begun to uncover a dominant role for a more recently described iNKT cell subset: NKT17 cells. Within 14 hours of infection, approximately 60% of NKT17 cells have received a TCR signal, and 45% produce IL-17A. Notably, only a minor percentage of NKT1 cells are TCR-activated or produce IFN-γ. Here, by generating conditional CD1d knockout mice, our goal was to determine which cell types directly activate NKT17 cells during infection. In mice with a CD11c+ cell-specific deletion, there was a 50% reduction of IL-17A-producing NKT17 cells at 14 hours, and a significant increase in bacteria loads at 48 hours. While these mice lack CD1d on several cell types, we focused on CD103+ DCs. If CD103+ DCs are absent, mice are highly susceptible to S. pneumoniae infection, iNKT cells do not expand, and IL-17A is diminished. Using confocal and two-photon microscopy of live tissue sections and flow cytometry we investigated their role as APCs. CD103+ DCs engulf a GFP-expressing S. pneumoniae strain within 2 hours and are primarily located within the interstitial lung tissue, notably where most NKT17 cells localize. We next generated a mouse with a CD103+ DC-specific deletion of CD1d. These mice also showed a 50% reduction of IL-17A-producing NKT17 cells at 14 hours and an increase in bacteria loads at 48 hours. Continuing studies include microscopy of live lung sections to further investigate the direct interaction between CD103+ DCs and iNKT cells during S. pneumoniae infection.

NKT cell subsets involved in host defense from pulmonary Streptococcus pneumoniae infection

Abstract Invariant natural killer T cells (iNKT) are a population of innate-like T lymphocytes that have crucial roles in protective responses to infectious agents, particularly Streptococcus pneumoniae. Despite their abundance in the lungs, there is limited understanding of how iNKT cells mediate protection there. Prior work from our group has shown that after pulmonary S. pneumoniae infection, iNKT cells are rapidly activated in a CD1d-dependent manner. Further, others have defined iNKT1, 2, and 17 cell subsets, analogous to CD4+ Th1, Th2, and Th17 subsets. In this study we investigated the roles of lung iNKT cell subsets in protection from S. pneumoniae. In naïve C57BL/6 mouse lungs, we found NKT1 and NKT17 cells were the predominant iNKT cell subsets, with most NKT17 cells in the tissue and NKT1 cells in both the tissue and vasculature. We then utilized fluorescent bacteria, flow cytometry, and intravital microscopy to analyze the lungs during pulmonary infection. At 24 hours, the bacteria localized within the tissue, and were taken up primarily by alveolar macrophages and neutrophils. At 14 and 24 hours, a majority of the lung NKT17 cells were producing IL-17, while only a minor percentage of NKT1 cells were producing IFNγ. This correlated with data from Nur77GFP reporter mice that indicate TCR-mediated activation. At 24 hours, approximately 75% of NKT17 but only 30% of NKT1 cells had been TCR-activated; suggesting NKT17 cells may play a dominant role early in infection. Yet, by 24 hours, NKT1 cells expanded to become the major subset in the lungs, proposing they are also relevant for protection. Experiments are underway to determine when and where subsets are first activated, roles of other cytokines, and consequences of removing individual subsets.

α-Galatosylceramide and retinoic acid regulate splenic follicular B cell differentiation and antibody production in a CD1d-dependent manner

Abstract α-Galactosylceramide (αGalCer) and all-trans-retinoic acid (RA) are each potent immune regulators able to increase immune responses in various conditions. αGalCer is particularly known for its direct activation of invariant NKT cells and B cells. In the present study, we used wildtype (WT) and CD1d knockout (KO) mice to characterize the specificity of αGalCer and its interaction with RA on B cell activation. WT and CD1dKO spleen cells were cultured ± αGalCer (100 ng/ml), or LPS (100 ng/ml) as a direct B-cell stimulus, and RA (20 nM), and B cell proliferation and differentiation were determined by flow cytometry. αGalCer and LPS both markedly increased the CD19+ B cell division. Whereas αGalCer mainly enriched spleen follicular CD23+ B cells, LPS increased the marginal zone CD21+ B cell population. The effects of αGalCer were completely absent in CD1dKO mice. However, although spleen B cells of CD1dKO mice failed to respond to αGalCer, these cells had a higher rate of proliferation compared to cells of WT mice, suggesting that CD1d molecules may have immune tonic effects in addition to reacting to αGalCer. Moreover, RA worked together with αGalCer to increase the proportions of IgG+ and CD138+ cells among CD23+ B cells, suggesting enhanced B-cell differentiation in splenic follicles. An in vivo immunization study using Keyhole limpet hemocyanin (KLH, 50 μg, ip) further confirmed that administration of RA (37 μg, po) and αGalCer (2 μg, sc), given at the time of primary immunization, increased antibody production in both the primary and secondary responses, which was not observed in CD1dKO mice. Thus, immune enhancement by αGalCer requires CD1d, and, when combined with RA promotes follicular B cell differentiation and increased antibody production.

The biliary epithelium presents antigens to and activates natural killer T cells.

Cholangiocytes express antigen-presenting molecules, but it has been unclear whether they can present antigens. Natural killer T (NKT) cells respond to lipid antigens presented by the major histocompatibility complex class I-like molecule CD1d and are abundant in the liver. We investigated whether cholangiocytes express CD1d and present lipid antigens to NKT cells and how CD1d expression varies in healthy and diseased bile ducts. Murine and human cholangiocyte cell lines as well as human primary cholangiocytes expressed CD1d as determined by flow cytometry and western blotting. Murine cholangiocyte cell lines were able to present both exogenous and endogenous lipid antigens to invariant and noninvariant NKT cell hybridomas and primary NKT cells in a CD1d-dependent manner. A human cholangiocyte cell line, cholangiocarcinoma cell lines, and human primary cholangiocytes also presented exogenous CD1d-restricted antigens to invariant NKT cell clones. CD1d expression was down-regulated in the biliary epithelium of patients with late primary sclerosing cholangitis, primary biliary cirrhosis, and alcoholic cirrhosis compared to healthy controls. Cholangiocytes express CD1d and present antigens to NKT cells and CD1d expression is down-regulated in diseased biliary epithelium, findings which show that the biliary epithelium can activate an important lymphocyte subset of the liver. This is a potentially important immune pathway in the biliary system, which may be capable of regulating inflammation in the context of biliary disease.

The regulatory role of invariant NKT cells in tumor immunity.

Invariant natural killer T (iNKT) cells are a unique population of T lymphocytes, which lie at the interface between the innate and adaptive immune systems, and are important mediators of immune responses and tumor surveillance. iNKT cells recognize lipid antigens in a CD1d-dependent manner; their subsequent activation results in a rapid and specific downstream response, which enhances both innate and adaptive immunity. The capacity of iNKT cells to modify the immune microenvironment influences the ability of the host to control tumor growth, making them an important population to be harnessed in the clinic for the development of anticancer therapeutics. Indeed, the identification of strong iNKT-cell agonists, such as α-galactosylceramide (α-GalCer) and its analogues, has led to the development of synthetic lipids that have shown potential in vaccination and treatment against cancers. In this Masters of Immunology article, we discuss these latest findings and summarize the major discoveries in iNKT-cell biology, which have enabled the design of potent strategies for immune-mediated tumor destruction.

Type II NKT-TFH Cells Against Gaucher Lipids Regulate B Cell Immunity and Inflammation

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency of acid β-glucosidase (GBA) that leads to the accumulation of two key sphingolipids, glucosylceramide (βGL1) and glucosylsphingosine (LGL1) in lysosomes of mononuclear phagocytes. Pathophysiology of classic GD is complex and includes chronic inflammation with an increased risk of B cell malignancies, pointing to immune dysregulation beyond the accumulation of lipid-loaded macrophages. Although an involvement of accumulated lipids has been implicated, the mechanisms linking sphingolipid accumulation with the observed clinical phenotypes are poorly understood.Natural Killer T (NKT) cells are a specialized lineage of T cells that recognize lipid/glycolipid antigens presented by MHC-like molecule CD1d. NKT cells are currently classified into two major subsets- type I or invariant NKT (iNKT) cells that express a conserved TCR and recognize α-galactosylceramide (α-GalCer) and type II or diverse NKT (dNKT) cells that utilize diverse TCR and do not recognize α-GalCer. Type II NKT cells comprise a major subset in humans and are increasingly implicated in immune regulation of diverse disease states. However in contrast to type I NKT cells, information on the functional properties of human type II NKT cells and their dysregulation in the context of disease is extremely limited.We utilized human and murine CD1d-tetramers loaded with GD-associated lipids (βGL1 and LGL1) to characterize the genomic and functional properties of T cells against these lipids. Analysis of human PBMCs revealed that βGL1-22/LGL1-tetramer+ cells are a distinct subset of CD1d-restricted T cells with a naïve phenotype and diverse Vβ receptor usage. Gene expression profiling as well as multiplex cytokine analysis demonstrated a distinct genomic and cytokine profile of these cells compared to classical type I NKT cells. Using both CD1d blocking experiments and CD1d-expressing C1R cells we could show that βGL1-22 and LGL1 could activate respective lipid-specific T cells in a CD1d dependent manner. Akin to human PBMCs, murine βGL1-22/LGL1 was shown to be a cognate antigen of type II NKT cells by performing double staining with α-GalCer and βGL1-22/LGL1-loaded-CD1d tetramers and using CD1d-/- and Jα18-/- mice. Transcriptional profile of βGL1-22 and LGL1-specific T cells revealed increased expression of genes associated with T-follicular-helper (TFH) phenotype. Therefore, we analyzed whether βGL1-22 or LGL1-specific type II NKT cells acquire TFH phenotype (CXCR5hi PD1hi ICOShi BCL6+IL-21+) upon immunization with βGL1-22 or LGL1. Separate groups of WT (C57BL/6), CD1d-/- and Jα18-/- mice were immunized with vehicle, α-GalCer, βGL1-22 or LGL1, after 7 days splenocytes and serum from each mice strain was assessed for upregulation of TFH markers and germinal center (GC) B cells by flow cytometry and antibodies by ELISA respectively. In contrast to type-I NKT cells, βGL1-22 and LGL1-specific NKT cells constitutively expressed TFH phenotype. The magnitude of TFH response was dictated by the amount of antigen and directly correlated with induction of GC B cells, hypergammaglobulinemia and production of anti-lipid antibodies. Complementarily, in vitro co culture of human βGL1-22 and LGL1-specific type II NKT cells with purified autologous B cells induced TFH markers in lipid specific T cells and led to expansion of plasmablasts and Ig secretion. In order to study changes in βGL1-22/LGL1-specific T cells in the setting of a disease wherein these ligands are altered, we analyzed patients and mouse models with GD. Compared to wild type control mice, GD mice exhibited >20-fold increase of LGL1-specific T cells. Concomitant to increase in LGL1-specific T cells in GD mice, notable up regulation of TFH markers and induction of anti-lipid antibodies was also observed. Similar to GD mice, sphingolipids accumulation in GD patients results in an increase of LGL1-specific type II NKT cells which correlated with biomarkers of disease severity and show reduction upon clinical improvement with enzyme replacement therapy.These data reveal and characterize a new subset of human and murine type II NKT-TFH cells against lipids dysregulated in Gaucher disease that regulate B cell immunity and inflammation and provide a mechanistic link between lipid storage and inflammation. DisclosuresPastores:Genzyme: Honoraria, Research Funding; Shire: Honoraria, Research Funding. Mistry:Genzyme, a Sanofi Company: Consultancy, Honoraria, Travel reimbursement, Research grants Other. Dhodapkar:Celgene: Research Funding.

Recognition of lysophosphatidylcholine by type II NKT cells and protection from an inflammatory liver disease.

Lipids presented by the MHC class I-like molecule, CD1d, are recognized by NK T (NKT) cells, which can be broadly categorized into two subsets. The well-characterized type I NKT cells express a semi-invariant TCR and can recognize both α- and β-linked glycolipids, whereas type II NKT cells are less well studied, express a relatively diverse TCR repertoire, and recognize β-linked lipids. Recent structural studies have shown a distinct mode of recognition of a self-glycolipid sulfatide bound to CD1d by a type II NKT TCR. To further characterize Ag recognition by these cells, we have used the structural data and screened other small molecules able to bind to CD1d and activate type II NKT cells. Using plate-bound CD1d and APC-based Ag presentation assay, we found that phospholipids such as lysophosphatidylcholine (LPC) can stimulate the sulfatide-reactive type II NKT hybridoma Hy19.3 in a CD1d-dependent manner. Using plasmon resonance studies, we found that this type II NKT TCR binds with CD1d-bound LPC with micromolar affinities similar to that for sulfatide. Furthermore, LPC-mediated activation of type II NKT cells leads to anergy induction in type I NKT cells and affords protection from Con A-induced hepatitis. These data indicate that, in addition to self-glycolipids, self-lysophospholipids are also recognized by type II NKT cells. Because lysophospholipids are involved during inflammation, our findings have implications for not only understanding activation of type II NKT cells in physiological settings, but also for the development of immune intervention in inflammatory diseases.

INKT Cell Production of GM-CSF Controls Mycobacterium tuberculosis

Invariant natural killer T (iNKT) cells are activated during infection, but how they limit microbial growth is unknown in most cases. We investigated how iNKT cells suppress intracellular Mycobacterium tuberculosis (Mtb) replication. When co-cultured with infected macrophages, iNKT cell activation, as measured by CD25 upregulation and IFNγ production, was primarily driven by IL-12 and IL-18. In contrast, iNKT cell control of Mtb growth was CD1d-dependent, and did not require IL-12, IL-18, or IFNγ. This demonstrated that conventional activation markers did not correlate with iNKT cell effector function during Mtb infection. iNKT cell control of Mtb replication was also independent of TNF and cell-mediated cytotoxicity. By dissociating cytokine-driven activation and CD1d-restricted effector function, we uncovered a novel mediator of iNKT cell antimicrobial activity: GM-CSF. iNKT cells produced GM-CSF in vitro and in vivo in a CD1d-dependent manner during Mtb infection, and GM-CSF was both necessary and sufficient to control Mtb growth. Here, we have identified GM-CSF production as a novel iNKT cell antimicrobial effector function and uncovered a potential role for GM-CSF in T cell immunity against Mtb.