CD1d Complexes Research Articles

A single immunization with cellular vaccine confers dual protection against SARS-CoV-2 and cancer.

The efficacy of current coronavirus disease 2019 (COVID‐19) vaccines has been demonstrated; however, emerging evidence suggests insufficient protection in certain immunocompromised cancer patients. We previously developed a cell‐based anti‐cancer vaccine platform involving artificial adjuvant vector cells (aAVCs) capable of inducing a strong adaptive response by enhancing the innate immunity. aAVCs are target antigen‐transfected allogenic cells that simultaneously express the natural killer T‐cell ligand–CD1d complex on their surface. In the present study, we applied this system for targeting the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein (CoV‐2‐S) using CoV‐2‐S‐expressing aAVCs (aAVC‐CoV‐2) and evaluated the immune response in a murine model. A single dose of aAVC‐CoV‐2 induced a large amount of CoV‐2‐S‐specific, multifunctional CTLs in addition to CD4+ T‐cell‐dependent anti‐CoV‐2‐S‐specific Abs. CoV‐2‐S‐specific CTLs infiltrated the lung parenchyma and persisted as long‐term memory T cells. Furthermore, we immunized mice with CoV‐2‐S‐ and tumor‐associated antigen (TAA)‐co‐expressing aAVCs (aAVC‐TAA/CoV‐2) and evaluated whether the anti‐SARS‐CoV‐2 and antitumor CTLs were elicited. We found that the aAVC‐TAA/CoV‐2‐S therapy exerted apparent antitumor effects and induced CoV‐2‐S‐specific CTLs. These findings suggest aAVC‐TAA/CoV‐2‐S therapy as a promising vaccine candidate for preventing COVID‐19, as well as enhancing the effectiveness of cancer therapies.

Chemical Biology of αGalCer: A Chemist's Toolbox for the Stimulation of Invariant Natural Killer T (iNKT) Cells

AbstractOften referred to as the “Swiss Army knife” of the immune system, invariant natural killer T (iNKT) cells are a subpopulation of T lymphocytes stimulated by the synthetic glycolipid α‐galactosylceramide (αGalCer) when in complex with the CD1d receptor of antigen presenting cells. Through their ability to produce TH1 and TH2 cytokines and co‐stimulate several other lymphocytes, iNKT cells have emerged as central players in directing the immune response in a range of physiological processes, such as infections, autoimmune diseases, and cancer. Over the years, synthetic chemistry has advanced the field of iNKT cell stimulation with the development of more efficient approaches to prepare αGalCer, and, additionally, with the chemical synthesis of αGalCer analogs in the search of better TH1/TH2 cytokine skewing compounds for therapeutic applications. Here, we review the strategies for the synthesis of αGalCer and its analogs, including synthetic probes, together with the most important advances in the understanding of the mechanism of action of these compounds, as a guide to the available tools for interrogating the iNKT cell−αGalCer−CD1d complex and inspiration for future research.

A nano-engager for iNKT cells in cancer.

Invariant natural killer T cells (iNKT cells) are innate-like CD1d-restricted T cells that have NK cell–like properties and bear an invariant T cell receptor (iTCR). iNKT cells have shown potential for cancer immunotherapy. A study now shows that stabilization of the iTCR–CD1d complex via a single-chain bi-specific antibody stimulates iNKT cell–mediated anti-tumor immunity.

Structural Dynamics of the Lipid Antigen-Binding Site of CD1d Protein

CD1 molecules present lipid antigens to T-cells in early stages of immune responses. Whereas CD1‒lipid‒T-cell receptors interactions are reasonably understood, molecular details on initial trafficking and loading of lipids onto CD1 proteins are less complete. We present a molecular dynamics (MD) study of human CD1d, the isotype that activates iNKT cells. MD simulations and calculations of properties and Poisson-Boltzmann electrostatic potentials were used to explore the dynamics of the antigen-binding domain of the apo-form, CD1d complexes with three lipid–antigens that activate iNKT cells and CD1d complex with GM2AP, a protein that assists lipid loading onto CD1 molecules in endosomes/lysosomes. The study was done at pH 7 and 4.5, values representative of strongly acidic environments in endosomal compartments. Our findings revealed dynamic features of the entrance to the hydrophobic channels of CD1d modulated by two α helices with sensitivity to the type of lipid. We also found lipid- and pH-dependent dynamic changes in three exposed tryptophans unique to CD1d among the five human CD1 isotypes. On the basis of modelled structures, our data also revealed external effects produced by the helper protein GM2AP only when it interacts in its open form, thus suggesting that the own assistant protein also adapts conformation to association with CD1d.

Dynamic plasticity of the lipid antigen-binding site of CD1d is crucially favoured by acidic pH and helper proteins

CD1 molecules present lipid antigens for recognition by T-cell receptors (TCRs). Although a reasonably detailed picture of the CD1-lipid-TCR interaction exists, the initial steps regarding lipid loading onto and exchange between CD1 proteins remain elusive. The hydrophobic nature of lipids and the fact that CD1 molecules are unable to extract lipids from membranes raise the need for the assistance of helper proteins in lipid trafficking. However, the experimental study of this traffic in the endosomal compartments at which it occurs is so challenging that computational studies can help provide mechanistic insight into the associated processes. Here we present a multifaceted computational approach to obtain dynamic structural data on the human CD1d isotype. Conformational dynamics analysis shows an intrinsic flexibility associated with the protein architecture. Electrostatic properties together with molecular dynamics results for CD1d complexes with several lipids and helper proteins unravel the high dynamic plasticity of the antigen-binding site that is crucially favoured by acidic pH and the presence of helper proteins.

Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

The ins and outs of type I iNKT cell development

Natural killer T (NKT) cells are innate-like lymphocytes that bridge the gap between the innate and adaptive immune responses. Like innate immune cells, they have a mature, effector phenotype that allows them to rapidly respond to threats, compared to adaptive cells. NKT cells express T cell receptors (TCRs) like conventional T cells, but instead of responding to peptide antigen presented by MHC class I or II, NKT cell TCRs recognize glycolipid antigen in the context of CD1d. NKT cells are subdivided into classes based on their TCR and antigen reactivity. This review will focus on type I iNKT cells that express a semi invariant Vα14Jα18 TCR and respond to the canonical glycolipid antigen, α-galactosylceramide. The innate-like effector functions of these cells combined with their T cell identity make their developmental path quite unique. In addition to the extrinsic factors that affect iNKT cell development such as lipid:CD1d complexes, co-stimulation, and cytokines, this review will provide a comprehensive delineation of the cell intrinsic factors that impact iNKT cell development, differentiation, and effector functions – including TCR rearrangement, survival and metabolism signaling, transcription factor expression, and gene regulation.

Potent Th2 Cytokine Bias of Natural Killer T Cell by CD1d Glycolipid Ligands: Anchoring Effect of Polar Groups in the Lipid Component

AbstractTh2‐biasing CD1d ligands are attractive potential candidates for adjuvants and therapeutic drugs. However, the number of potent ligands is limited, and their biasing mechanism remain unclear. Herein, a series of novel Th2‐biasing CD1d glycolipid ligands, based on modification of their lipid part, have been identified. These have shown high binding affinities and efficient Th2 cytokine production. Importantly, the truncated acyl chain containing variants still retain their binding affinities and agonistic activities, which can be associated with an “anchoring effect,” that is, formation of a buried hydrogen bond between a polar group on the acyl chain and the CD1d lipid‐binding pocket. The analysis indicated that the appearance rates of ligand–CD1d complexes on the cell surface were involved in Th2‐biasing responses. The designed ligands, having the anchor in the shorter lipid part, are one of the most potent Th2‐biasing ligands among the known ligands.

Targeting colorectal cancer metastasis-initiating cells through hepatic iNKT cells

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

A whole genome mouse siRNA screen to identify novel genes involved in lipid antigen presentation

Abstract Although most T lymphocytes recognize peptides presented by major histocompatibility complex (MHC)-encoded class I and class II molecules, there also are significant populations of T cells that recognize nonpeptide antigens. Prominent among these T lymphocytes are the type I or invariant natural killer T cells (iNKT cells). These T lymphocytes recognize lipids presented by CD1d, a nonpolymorphic, class I-like, antigen-presenting molecule. We have carried out a whole genome siRNA screen in a macrophage cell line for genes that affect the presentation of a potent glycosphingolipid antigen, GalGal Cer, to iNKT cells. In order to stimulate iNKT cells, this antigen requires internalization and lysosomal carbohydrate antigen processing to remove the terminal galactose. After several rounds of validation, functional classification and gene expression analysis, we have identified genes that lead to altered antigen presentation in macrophages. A majority of the identified genes do not perturb surface CD1d expression, but we can demonstrate they effect the formation of surface CD1d complexes with the stimulating glycolipid. Members of the HOPS and ESCRT complexes have been identified to play an important role in Cd1d dependent antigen presentation. Interestingly, our data show that ABCC1 and several other ABC family transporters are involved in lipid antigen presentation by CD1d. CD1d and MHC-II antigen presentation pathways both depends on antigen loading in the lysosome, therefore we also analyzed the role of these genes in MHC-II antigen presentation. Although the majority of genes identified doesn’t affect MHC-II antigen presentation, there are a few molecules that affect both antigen presentation pathways.

Immune microenvironment modulation via the interaction of human invariant natural killer T cell (iNKT cells) and Kupffer cells (KC), dendritic cells (DC) in colorectal liver metastasis

Abstract Aims To investigate the immune microenvironment modulation via the interaction between human iNKT cells and KC, DC in colorectal liver metastasis. Methods Different subtypes of iNKT cells, KC and DC from the core of tumor (CT), tumor invasive margin (IM), and adjacent normal liver tissue (NL) were analyzed by BD LSRFortessa™, TMA, and immunofluorescence(n=138). Ex vivo 3D co-culture, Quansys Multiplex array, and Elisa array were performed. Results In metastasis, CXCR6+iNKT positively correlated with better prognosis. In addition, iNKT cells were enriched at IM, especially iNKT2. Intriguingly, although there were few iNKT cells in CT, the iNKT/Lymphocytes rate increased to ~5 folds and expressed elevated IL9, IFNγ, and FasL compared to NL and IM. This implied the critical role of iNKT cells in tumor control. Both DC and KC could drive iNKT activation, the proportion of BDCA-3+DCs significantly increased from NL to CT; though they expressed higher CXCL16 compared to other subtypes, the capability of activating iNKT cells via CD1d was less strong than plasmacytoid DC(pDC). pDCs showed significantly elevated α-GalCer: CD1d complex formation in an ex vivo iNKT cells activation assay, which implied its critical role of activating capability of pDC. However, pDCs, as well as BDCA-1+DCs, decreased from NL to CT. In this study, KC was found also activating iNKT cells. Interestingly, iNKT expressed Th2 cytokines when activated by KC, however, Th1 biased response could be found when activated with DC. In addition, KC subsets also differed with location. Furthermore, transmembrane CD1d density and intra/tmCXCL16 expression, Th1/2/17 biased immune responses of KC and DC were associated with the lipid metabolism preferences of these cells.

“Endocytic pH regulates cell surface localization of glycolipid antigen loaded CD1d complexes”

Invariant natural killer T (iNKT) cells recognize glycolipid antigens presented by CD1d, an antigen presenting protein structurally similar to MHC class I. Stimulation of iNKT cells by glycolipid antigens can induce strong immune responses in vivo, with rapid production of a wide variety of cytokines including those classically associated with either T helper type 1 (Th1) or type 2 (Th2) responses. Alterations in the lipid tails or other portions of CD1d-presented glycolipid ligands can bias the iNKT response towards production of predominantly Th1 or Th2 associated cytokines. However, the mechanism accounting for this structure-activity relationship remains controversial. The Th1-biasing glycolipids have been found to consistently form complexes with CD1d that preferentially localize to plasma membrane cholesterol rich microdomains (lipid rafts), whereas CD1d complexes formed with Th2-biasing ligands are excluded from these microdomains. Here we show that neutralization of endosomal pH enhanced localization of CD1d complexes containing Th2-biasing glycolipids to plasma membrane lipid rafts of antigen presenting cells (APC). Transfer of APCs presenting these “stabilized” CD1d/αGC complexes into mice resulted in immune responses with a more prominent Th1-like bias, characterized by increased NK cell transactivation and interferon-γ production. These findings support a model in which low endosomal pH controls stability and lipid raft localization of CD1d-glycolipid complexes to regulate the outcome of iNKT cell mediated responses.

Endocytic pH regulates cell surface localization of glycolipid antigen loaded CD1d complexes

Invariant natural killer T (iNKT) cells recognize glycolipid antigens presented by CD1d, an antigen presenting protein structurally similar to MHC class I. Stimulation of iNKT cells by glycolipid antigens can induce strong immune responses in vivo, with rapid production of a wide variety of cytokines including those classically associated with either T helper type 1 (Th1) or type 2 (Th2) responses. Alterations in the lipid tails or other portions of CD1d-presented glycolipid ligands can bias the iNKT response towards production of predominantly Th1 or Th2 associated cytokines. However, the mechanism accounting for this structure–activity relationship remains controversial. The Th1-biasing glycolipids have been found to consistently form complexes with CD1d that preferentially localize to plasma membrane cholesterol rich microdomains (lipid rafts), whereas CD1d complexes formed with Th2-biasing ligands are excluded from these microdomains. Here we show that neutralization of endosomal pH enhanced localization of CD1d complexes containing Th2-biasing glycolipids to plasma membrane lipid rafts of antigen presenting cells (APC). Transfer of APCs presenting these “stabilized” CD1d/αGC complexes into mice resulted in immune responses with a more prominent Th1-like bias, characterized by increased NK cell transactivation and interferon-γ production. These findings support a model in which low endosomal pH controls stability and lipid raft localization of CD1d-glycolipid complexes to regulate the outcome of iNKT cell mediated responses.

Pathogen-expanded CD11b+ invariant NKT cells feedback inhibit T cell proliferation via membrane-bound TGF-β1

Natural killer T cells (NKT cells) are effector cells, but also regulator of immune response, which either promote or suppress immune response through production of different cytokines. However, the subsets of NKT cells with definite phenotype and regulatory function need to be further identified. Furthermore, the mechanisms for NKT cells to regulate immune response remain to be fully elucidated. Here we identified CD11b+ invariant NKT (CD11b+ iNKT) cells as a new subset of regulatory NKT cells in mouse models with infection. αGalCer:CD1d complex+TCRβ+NK1.1+ NKT cells could be categorized to CD11b+ and CD11b− subsets. NKT cells are enriched in liver. During Listeria monocytogenes infection, hepatic CD11b+ iNKT cells were significantly induced and expanded, with peak expansion on day 8. CD11b+ iNKT cells were also expanded significantly in spleen and mesenteric lymph nodes. As compared to CD11b− iNKT cells, CD11b+ iNKT cells expressed higher levels of CD27, FasL, B7H1, CD69, and particularly higher level of membrane-bound TGF-β1 (mTGF-β1), but produced less IFN-γ, IL-4, IL-10 and TGF-β1. Hepatic CD11b+ iNKT cells suppressed antigen-nonspecific and OVA-specific CD4 and CD8 T cell proliferation through mTGF-β1 both in vitro and in vivo, meanwhile, they did not interfere with activation of CD4 T cells and cytotoxicity of the activated CD8 T cells. Thus, we have identified a new subset of pathogen-expanded CD11b+ invariant NKT cells which can feedback inhibit T cell response through cell-to-cell contact via cell surface (membrane-bound) TGF-β1, especially at the late stage of immune response against infection. CD11b+ regulatory iNKT cells may contribute to protect host from pathological injure by preventing immune overactivation.

The Actin Cytoskeleton Controls the Activation of Invariant Natural Killer T Cells by Fine-Tuning CD1d Nanoscale Aggregation on Antigen Presenting Cells

Invariant Natural killer T (iNKT) cells are a subset of lipid-specific T cells, restricted by the MHC-I class like molecule CD1d. A recent study showed a correlation between the co-localization of lipid-loaded CD1d molecules with lipid rafts on the membrane of antigen presenting cells, and their capacity of eliciting secretion of Th1-cytokines by stimulated iNKT cells [1]. Thus, this study suggested that not only the structure of CD1d bound to an exogenous lipid could influence Cd1d-mediated immunity, but also its partitioning on the membrane. Here, we address the spatiotemporal behaviour of α-Galactosylceramide loaded CD1d complexes on the cell membrane of human myeloid cells using multiple colour high-speed single-particle tracking (100 Hz) combined with an iNKT T Cell Receptor-Qdot conjugate as imaging probe. Furthermore, we complement these studies using STED nanoscopy to obtain nanoscale images of CD1d spatial organization. Our results indicate a direct role of the actin cytoskeleton in actively segregating CD1d nanoclusters on the cell membrane resulting in an inhibition of the activation of iNKT cells [2]. As a whole, our work proposes a new paradigm of biophysical interaction between CD1d presenting cells and NKT cells which deviates significantly from classical MHCI/II complexes and CD8/CD4-T Cells interactions.[1] J. S. Im et al., Immunity, 30, 888, 2009.[2] J. A. Torreno-Pina et al., in preparation.

Nanomapping of CD1d–glycolipid complexes on THP1 cells by using simultaneous topography and recognition imaging

CD1d molecule, a monomorphic major histocompatibility complex class I-like molecule, presents different types of glycolipids to invariant natural killer T (iNKT) cells that play an important role in immunity to infection and tumors, as well as in regulating autoimmunity. Here, we present simultaneous topography and recognition imaging (TREC) analysis to detect density, distribution and localization of single CD1d molecules on THP1 cells that were loaded with different glycolipids. TREC was conducted using magnetically coated atomic force microscopy tips functionalized with a biotinylated iNKT cell receptor (TCR). The recognition map revealed binding sites visible as dark spots, resulting from oscillation amplitude reduction during specific binding between iNKT TCR and the CD1d-glycolipid complex. THP1 cells were pulsed with three different glycolipids (α-GalCer, C20 and OCH12) for 4 and 16 hr. Whereas CD1d-α-GalCer and CD1d-C20:2 complexes on cellular membrane formed smaller microdomains up to ~10 000 nm(2) (dimension area), OCH12 loaded CD1d complexes presented larger clusters with a dimension up to ~30 000 nm(2). Moreover, the smallest size of recognition spots was about 25 nm, corresponding to a single CD1d binding site. TREC successfully revealed the distribution and localization of CD1d-glycolipid complexes on THP1 cell with single molecule resolution under physiological conditions.

α-Galactosylceramide analogues in cancer immunotherapeutics (P2102)

Abstract Natural Killer T cells (NKT) initiate potent anti-tumor responses upon activation by glycolipid antigens presented by CD1d, a non-classical MHC class I-like antigen presenting molecule. Anti-tumor responses are induced following NKT cell activation through their direct production of cytokines such as IFN-γ, and through a variety of indirect effects including dendritic cell maturation, the activation of NK cells and the cross-priming of tumor-specific CD8+ T cells. The glycolipid α-galactosylceramide (α-GC) is a CD1d ligand and potent NKT cell activator that enhances anti-tumor immune responses and decreases metastases in vivo. However, the response is short-lived, and causes liver toxicity and long term anergy of NKT cells. Here we report a novel class of chemically modified α-GCs, designated GCBs, that demonstrate unique binding properties to both mouse and human CD1d and enhanced NKT cell stimulation in vitro, as compared to previously described NKT cell activating ligands. A range of analyses have been carried out to characterize the structure and stability of GCB:CD1d complexes, revealing unique features that distinguish them from previously analyzed α-GC:CD1d complexes. Soluble GCB:CD1d complexes are currently being tested in vivo in mouse models for their ability to drive the sustained activation of NKT cells, which may provide a superior approach for development of improved cancer immunotherapies.

Role for lysosomal phospholipase A2 in iNKT cell-mediated CD1d recognition

Invariant natural killer T (iNKT) cells recognize self lipid antigens presented by CD1d molecules. The nature of the self-antigens involved in the development and maturation of iNKT cells is poorly defined. Lysophospholipids are self-antigens presented by CD1d that are generated through the action of phospholipases A1 and A2. Lysosomal phospholipase A2 (LPLA2, group XV phospholipase A2) resides in the endocytic system, the main site where CD1d antigen acquisition occurs, suggesting that it could be particularly important in CD1d function. We find that Lpla2(-/-) mice show a decrease in iNKT cell numbers that is neither the result of a general effect on the development of lymphocyte populations nor of effects on CD1d expression. However, endogenous lipid antigen presentation by CD1d is reduced in the absence of LPLA2. Our data suggest that LPLA2 plays a role in the generation of CD1d complexes with thymic lipids required for the normal selection and maturation of iNKT cells.

Invariant NKT cell anergy is induced by a strong TCR-mediated signal plus co-stimulation

Vα14 TCR expressing invariant NK T (iNKT) cells recognize α-galactosylceramide (αGC)/CD1d complex and produce large amounts of various cytokines before the onset of the adaptive immunity. After stimulation with a high dose (2-5 μg) of αGC in vivo, iNKT cells in the spleen and liver become anergic in terms of the proliferation and cytokine production to subsequent stimulation. In this study, we monitor how iNKT anergy is induced. Anergized iNKT cells dramatically reduced the expression of IL-2Rα, and exogenous IL-2 restored the ability to proliferate and produce IL-4 but not to produce IFN-γ. Anergized iNKT cells expressed high levels of programmed death-1 (PD-1). However, iNKT cells in PD-1-deficient mice became anergic as a result of αGC injection, as do normal mice. Furthermore, anti-PD-1 blocking mAb was unable to restore their responsiveness. When iNKT cells were stimulated with immobilized anti-CD3 in the presence or absence of anti-CD28, they produced cytokines in a dose-dependent manner. Unlike in naive CD4 T cells, the strong TCR-mediated signaling with co-stimulation renders them anergic to any subsequent stimulation with αGC and spleen dendritic cells (DCs). Moreover, iNKT cells also became anergic after stimulation with phorbol-12-myristate-13-acetate + ionophore. Finally, the injection of αGC-pulsed DCs was more potent in inducing anergy than B cells. These results indicate that strong TCR-mediated activation with co-stimulation provides signals that induce the anergic state in iNKT cells.

Production and characterization of monoclonal antibodies against complexes of the NKT cell ligand α-galactosylceramide bound to mouse CD1d

The α-galactosylceramide (α-GalCer) known as KRN7000 remains the best studied ligand of the lipid-binding MHC class I-like protein CD1d. The KRN7000:CD1d complex is highly recognized by invariant natural killer T (iNKT) cells, an evolutionarily conserved subset of T lymphocytes that express an unusual semi-invariant T cell antigen receptor, and mediate a variety of proinflammatory and immunoregulatory functions. To facilitate the study of glycolipid antigen presentation to iNKT cells by CD1d, we undertook the production of mouse monoclonal antibodies (mAbs) specific for complexes of KRN7000 bound to mouse CD1d (mCD1d) proteins. Three such monoclonal antibodies were isolated that bound only to mCD1d proteins that were loaded with KRN7000 or closely-related forms of α-GalCer. These mAbs showed no reactivity with mCD1d proteins that were not loaded with α-GalCer, nor did they bind to complexes formed by loading mCD1d with the self-glycolipid and putative iNKT cell ligand isoglobotrihexosylceramide. These complex-specific monoclonal antibodies allow the direct detection and monitoring of complexes formed by the binding of KRN7000 and other α-GalCer analogues to mCD1d. The availability of these mAbs should facilitate a wide range of studies on the biology and potential clinical applications of CD1d-restricted iNKT cells.