Insights into the CD1 lipidome.

CD1 isoforms are MHC class I-like molecules that present lipid-antigens to T cells and have been associated with a variety of immune responses. The lipid repertoire bound and presented by the four CD1 isoforms may be influenced by factors such as the cellular lipidome, subcellular microenvironment, and the properties of the binding pocket. In this study, by shotgun mass spectrometry, we performed a comprehensive lipidomic analysis of soluble CD1 molecules. We identified 1040 lipids, of which 293 were present in all isoforms. Comparative analysis revealed that the isoforms bind almost any cellular lipid.CD1a and CD1c closely mirrored the cellular lipidome, while CD1b and CD1d showed a preference for sphingolipids. Each CD1 isoform was found to have unique lipid species, suggesting some distinct roles in lipid presentation and immune responses. These findings contribute to our understanding of the role of CD1 system in immunity and could have implications for the development of lipid-based therapeutics.

NKT cells promote Th1 immune bias to dengue virus that governs long-term protective antibody dynamics.

NKT cells are innate-like T cells, recruited to the skin during viral infection, yet their contributions to long-term immune memory to viruses are unclear. We identified granzyme K, a product made by cytotoxic cells including NKT cells, as linked to induction of Th1-associated antibodies during primary dengue virus (DENV) infection in humans. We examined the role of NKT cells in vivo using DENV-infected mice lacking CD1d-dependent (CD1ddep) NKT cells. In CD1d-KO mice, Th1-polarized immunity and infection resolution were impaired, which was dependent on intrinsic NKT cell production of IFN-γ, since it was restored by adoptive transfer of WT but not IFN-γ-KO NKT cells. Furthermore, NKT cell deficiency triggered immune bias, resulting in higher levels of Th2-associated IgG1 than Th1-associated IgG2a, which failed to protect against a homologous DENV rechallenge and promoted antibody-dependent enhanced disease during secondary heterologous infections. Similarly, Th2 immunity, typified by a higher IgG4/IgG3 ratio, was associated with worsened human disease severity during secondary infections. Thus, CD1ddep NKT cells establish Th1 polarity during the early innate response to DENV, which promotes infection resolution, memory formation, and long-term protection from secondary homologous and heterologous infections in mice, with consistent associations observed in humans. These observations illustrate how early innate immune responses during primary infections can influence secondary infection outcomes.

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.

Cutting Edge: Redundant Roles for MHC Class II-, CD1d-, and MR1-restricted T Cells in Clearing Bartonella Infection.

The importance of unconventional T cells for mucosal immunity is firmly established but for systemic bacterial infection remains less well defined. In this study, we explored the role of various T cell subsets in murine Bartonella infection, which establishes persistent bacteremia unless controlled by antibacterial Abs. We found that αß T cells are essential for Ab production against and clearance of B. taylorii, whereas MHC class I (MHC-I)- or MHC class II (MHC-II)-deficient mice eliminated B. taylorii infection with normal kinetics. Similarly, animals lacking either CD1d or MR1 suppressed bacteremia with normal kinetics. Interestingly, mice with a combined deficiency of either MHC-II and CD1d or MHC-II and MR1 failed to clear the infection, indicating that the combination of CD1d- and MR1-restricted T cells can compensate for the lack of MHC-II in this model. Our data document a previously underappreciated contribution of unconventional T cells to the control of systemic bacterial infection, supposedly as helper cells for antibacterial Ab production.

Rationally Designed Highly Potent NKT Cell Agonists with Different Cytokine Selectivity through Hydrogen-Bond Interaction.

Synthetic α-galactosylceramide (αGalCer) and its analogues as powerful agonists for natural killer T (NKT) cell manipulation have received significant attention in immunotherapy and adjuvant development. However, identifying new potent NKT cell agonists, especially those with Th1 selectivity that promote anticancer effects, remains a challenging task. In this work, we introduced a sulfonamide group into the acyl chain of αGalCer to form additional hydrogen bonds to intensify the glycolipid/CD1d interaction. Two compounds GCS-11 and GCS-12 demonstrated remarkable potency while exhibiting different cytokine induction patterns. Compared to αGalCer, the Th1-biased GCS-11 exhibited a 6-fold increase in IFN-γ but not IL-4, while the Th1/2-balanced GCS-12 elicited 7- and 5-fold increase in IFN-γ and IL-4, respectively, in vivo. These findings place them among the most potent NKT cell agonists, with superior antitumor effects. Therefore, hydrogen-bond-involved derivatization could be a powerful strategy to develop potent and polarized NKT cell agonists for various immunotherapies.

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.

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.

A Humanized Mouse Model Coupled with Computational Analysis Identifies Potent Glycolipid Agonist of Invariant NKT Cells.

Invariant natural killer T (iNKT) cells play an important role in many innate and adaptive immune responses, with potential applications in cancer immunotherapy. The glycolipid KRN7000, an α-galactosylceramide, potently activates iNKT cells but has shown limited anticancer effects in human clinical trials conducted so far. In spite of almost three decades of structure-activity relationship studies, no alternative glycolipid has yet emerged as a superior clinical candidate. One reason for the slow progress in this area is that standard mouse models do not accurately reflect the specific ligand recognition by human iNKT cells and their requirements for activation. Here we evaluated a series of KRN7000 analogues using a recently developed humanized mouse model that expresses a human αTCR chain sequence and human CD1d. In this process, a more stimulatory, previously reported but largely overlooked glycolipid was identified, and its activity was probed and rationalized via molecular simulations.

Motility and tumor infiltration are key aspects of invariant natural killer T cell anti-tumor function.

Dysfunction of invariant natural killer T (iNKT) cells contributes to immune resistance of tumors. Most mechanistic studies focus on their static functional status before or after activation, not considering motility as an important characteristic for antigen scanning and thus anti-tumor capability. Here we show via intravital imaging, that impaired motility of iNKT cells and their exclusion from tumors both contribute to the diminished anti-tumor iNKT cell response. Mechanistically, CD1d, expressed on macrophages, interferes with tumor infiltration of iNKT cells and iNKT-DC interactions but does not influence their intratumoral motility. VCAM1, expressed by cancer cells, restricts iNKT cell motility and inhibits their antigen scanning and activation by DCs via reducing CDC42 expression. Blocking VCAM1-CD49d signaling improves motility and activation of intratumoral iNKT cells, and consequently augments their anti-tumor function. Interference with macrophage-iNKT cell interactions further enhances the anti-tumor capability of iNKT cells. Thus, our findings provide a direction to enhance the efficacy of iNKT cell-based immunotherapy via motility regulation.

NKT cells in the antitumor response: the ß version?

NKT cells recognize glycolipids presented by CD1d-expressing antigen-presenting cells (APCs) and include type I NKT cells with antitumor function and type II NKT cells, which have been reported to suppress the antitumor response. Some type II NKT cells recognize sulfatide, a glycosphingolipid with a sulfate modification of the sugar. Type I NKT cells recognize different glycosphingolipids. In this issue of the JCI, Nishio and colleagues showed that APCs could process sulfatide antigens, analogous to protein processing for peptide-reactive T cells. Antigen processing in lysosomes removed sulfate to generate a glycosphingolipid that stimulated type I NKT cells and thereby turned an antigen with no antitumor activity into one that not only stimulated type I NKT cells but also stimulated antitumor responses. These findings may extend to the development of glycolipid antigens that could stimulate anticancer responses via antigen processing by APCs.

Intrinsic factors and CD1d1 but not CD1d2 expression levels control invariant natural killer T cell subset differentiation.

Invariant natural killer T (NKT) cell subsets are defined based on their cytokine-production profiles and transcription factors. Their distribution is different in C57BL/6 (B6) and BALB/c mice, with a bias for NKT1 and NKT2/NKT17 subsets, respectively. Here, we show that the non-classical class I-like major histocompatibility complex CD1 molecules CD1d2, expressed in BALB/c and not in B6 mice, could not account for this difference. We find however that NKT cell subset distribution is intrinsic to bone marrow derived NKT cells, regardless of syngeneic CD1d-ligand recognition, and that multiple intrinsic factors are likely involved. Finally, we find that CD1d expression levels in combination with T cell antigen receptor signal strength could also influence NKT cell distribution and function. Overall, this study indicates that CD1d-mediated TCR signals and other intrinsic signals integrate to influence strain-specific NKT cell differentiation programs and subset distributions.

Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer.

In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding ß-galactosylceramide (ßGalCer) without sulfate. C24:2 induced IFN-γ-dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid's function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.

Type 1 invariant natural killer T cells in chronic inflammation and tissue fibrosis.

Chronic tissue inflammation often results in fibrosis characterized by the accumulation of extracellular matrix components remodeling normal tissue architecture and function. Recent studies have suggested common immune mechanisms despite the complexity of the interactions between tissue-specific fibroblasts, macrophages, and distinct immune cell populations that mediate fibrosis in various tissues. Natural killer T (NKT) cells recognizing lipid antigens bound to CD1d molecules have been shown to play an important role in chronic inflammation and fibrosis. Here we review recent data in both experimental models and in humans that suggest a key role of type 1 invariant NKT (iNKT) cell activation in the progression of inflammatory cascades leading to recruitment of neutrophils and activation of the inflammasome, macrophages, fibroblasts, and, ultimately, fibrosis. Emerging evidence suggests that iNKT-associated mechanisms contribute to type 1, type 2 and type 3 immune pathways mediating tissue fibrosis, including idiopathic pulmonary fibrosis (IPF). Thus, targeting a pathway upstream of these immune mechanisms, such as the inhibition of iNKT activation, may be important in modulating various fibrotic conditions.

Do antigen-presenting CD1a, CD1b, CD1c, and CD1d molecules bind different self-lipids?

Humans express four different lipid antigen-presenting molecules, CD1a, CD1b, CD1c, and CD1d, that are differentially expressed on antigen-presenting cells and which recycle through different endosomal compartments. Huang et al. now answer the question on whether the four CD1 isoforms selectively bind certain lipids.

Antigen Priming Induces Functional Reprogramming in iNKT Cells via Metabolic and Epigenetic Regulation: An Insight into iNKT Cell-Based Antitumor Immunotherapy.

Dysfunction of intratumoral invariant natural killer T (iNKT) cells hinders their antitumor efficacy, but the underlying mechanisms and the relationship with endogenous antigen priming remain to be explored. Here, we report that antigen priming leads to metabolic reprogramming and epigenetic remodeling, which causes functional reprogramming in iNKT cells, characterized by limited cytokine responses upon restimulation but constitutive high cytotoxicity. Mechanistically, impaired oxidative phosphorylation (OXPHOS) in antigen-primed iNKT cells inhibited T-cell receptor signaling, as well as elevation of glycolysis, upon restimulation via reducing mTORC1 activation, and thus led to impaired cytokine production. However, the metabolic reprogramming in antigen-primed iNKT cells was uncoupled with their enhanced cytotoxicity; instead, epigenetic remodeling explained their high expression of granzymes. Notably, intratumoral iNKT cells shared similar metabolic reprogramming and functional reprogramming with antigen-primed iNKT cells due to endogenous antigen priming in tumors, and thus recovery of OXPHOS in intratumoral iNKT cells by ZLN005 successfully enhanced their antitumor responses. Our study deciphers the influences of antigen priming-induced metabolic reprogramming and epigenetic remodeling on functionality of intratumoral iNKT cells, and proposes a way to enhance efficacy of iNKT cell-based antitumor immunotherapy by targeting cellular metabolism.

Anti-CD1d treatment suppresses immunogenic maturation of lung dendritic cells dependent on lung invariant natural killer T cells in asthmatic mice.

Our previous findings show that invariant natural killer T (iNKT)cells can promote immunogenic maturation of lung dendritic cells (LDCs) to enhance Th2 cell responses in asthma. It has been accepted that recognition of glycolipid antigens presented by CD1d molecules by the T cell receptors of iNKT cells leads to iNKT cell activation. Therefore, we examine the immunoregulatory influences of anti-CD1d treatment on Th2 cell response and immunogenic maturation of LDCs and subsequently explored whether these influences were dependent on lung iNKT cells in asthmatic mice. We discoveredthat in wild-type mice sensitized and challenged with house dust mite or ovalbumin (OVA), anti-CD1d treatment inhibited Th2 cell response and immunogenic maturation of LDCs. LDCs from asthmatic mice with anti-CD1d treatment had a markedly decreased influence on Th2 cell responses in vivo and in vitro. Furthermore, anti-CD1d treatment reduced the abundance and activation of lung iNKT cells in asthmatic mice. Moreover, in asthmatic iNKT cell-deficient Jα18-/- mice, anti-CD1d treatment did not influence Th2 cell responses and immunogenic maturation of LDCs. Meanwhile, the quantity of CD40L+ iNKT cells in asthmatic mice was significant decreased by anti-CD1d treatment. Finally, the inhibition of anti-CD1d treatment on LDC immunogenic maturation and Th2 cell responses in asthmatic mice was reversed by anti-CD40 treatment. Our data suggest that anti-CD1d treatment can suppress Th2 cell responses through inhibiting immunogenic maturation of LDCs dependent on lung iNKT cells, which couldbe partially related to the downregulation of CD40L expression on lung iNKT cells in asthmatic mice.

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.

CD1 and iNKT cells mediate immune responses against the GBS hemolytic lipid toxin induced by a non-toxic analog.

Although hemolytic lipids have been discovered from many human pathogens including Group B Streptococcus (GBS), strategies that neutralize their function are lacking. GBS is a leading cause of pregnancy-associated neonatal infections, and adult GBS infections are on the rise. The GBS hemolytic lipid toxin or granadaene, is cytotoxic to many immune cells including T and B cells. We previously showed that mice immunized with a synthetic nontoxic analog of granadaene known as R-P4 had reduced bacterial dissemination during systemic infection. However, mechanisms important for R-P4 mediated immune protection was not understood. Here, we show that immune serum from R-P4-immunized mice facilitate GBS opsonophagocytic killing and protect naïve mice from GBS infection. Further, CD4+ T cells isolated from R-P4-immunized mice proliferated in response to R-P4 stimulation in a CD1d- and iNKT cell-dependent manner. Consistent with these observations, R-P4 immunized mice lacking CD1d or CD1d-restricted iNKT cells exhibit elevated bacterial burden. Additionally, adoptive transfer of iNKT cells from R-P4 vaccinated mice significantly reduced GBS dissemination compared to adjuvant controls. Finally, maternal R-P4 vaccination provided protection against ascending GBS infection during pregnancy. These findings are relevant in the development of therapeutic strategies targeting lipid cytotoxins.

CD1d-dependent natural killer T-cells inactivation aggravates sepsis-induced myocardial injury via T lymphocytes infiltration and IL-6 production in mice.

Myocardial edema mediated by endothelial dysfunction plays an important role in sepsis-induced cardiomyopathy (SIC); however, its mechanism is unclear. The current study aimed to provide evidence on the cardioprotection of CD1d-dependent natural killer T (NKT) cells and clarify the possible mechanism in a mouse model of sepsis. Wild-type (WT) and CD1d-dependent NKT-cells inactivation (CD1dko) mice were subjected to sepsis induced by intraperitoneal injection of lipopolysaccharide (LPS). The NKT-cells number and CD1d expression were both increased in the hearts and blood of WT mice after LPS treatment. Compared with WT mice, CD1dko mice exhibited remarkably accelerated LPS-induced mortality, cardiac dysfunction, myocardial injury, endothelial apoptosis, microvascular damage, microvascular permeability and cardiac edema. Mechanistically, CD1d deficiency further increased LPS-induced accumulation of T lymphocytes in the myocardium and upregulation of IL-6 protein levels. Administration of an IL-6 neutralizing antibody to CD1dko mice improved cardiac dysfunction, myocardial injury and edema induced by LPS. Our study identified that CD1d-dependent NKT-cells inactivation exacerbated SIC via T lymphocytes infiltration and IL-6 production. Hence, activation of CD1d-dependent NKT cells may be a potential candidate strategy for SIC treatment.