CD1d Ligand Research Articles

Abstract CT275: Phase 1 clinical update of allogeneic invariant natural killer T cells (iNKTs), agenT-797, alone or in combination with pembrolizumab or nivolumab in patients with advanced solid tumors

Abstract Introduction: AgenT-797 is an allogeneic iNKT cell therapy and represents a novel, scalable, off-the-shelf approach against solid tumors. iNKTs are a unique subset of T cells, that mediate antitumor responses by direct killing, targeting CD1d and other ligands in the tumor microenvironment, and by activating host immune cells. We conducted a clinical trial to investigate agenT-797 activity as single agent and in combination (combo) with PD-1 blockade after prior progression on PD-1 therapy. Methods: Patients (pts) with relapsed or refractory solid tumors were treated with single IV infusion of agenT-797 (no lymphodepletion) at 4.3 × 10^6 or 1.4 × 10^7 cells/kg, as monotherapy or in combo with pembrolizumab (pembro) or nivolumab (nivo). Dose escalation followed 3+3 scheme. Endpoints included safety, persistence of agenT-797, objective responses, duration of response, progression-free survival, and time to response. Adverse events (AEs) were reported per CTCAE v5.0. Dose limiting toxicities (DLTs) were evaluated. AgenT-797 persistence was assessed by assays utilizing SNPs and cfDNA analysis. Serum biomarkers were measured with MSD V-PLEX cytokine assays. On-treatment tumor biopsy was obtained for multiplex immunofluorescence staining and next generation sequencing. Results: As of February 5th, 32 pts (median age 62y, range 30-83) were treated with agenT-797 monotherapy (n=26) or combo (n=6) with pembro or nivo. Pts had median 4 lines of prior therapy (range 1-13). Tumor types included pancreatic (6), NSCLC (4), rectal (4), cholangiocarcinoma/biliary duct (4), and other (14)*. Tolerability was favorable, with no cytokine release syndrome, neurotoxicity, and no DLTs. Treatment-related AEs included 16.7% (5) grade 1, 3.3% (1) grade 2, and 3.3% (1) grade 3 (anemia). At data cutoff, agenT-797 monotherapy and combo revealed early clinical activity. Among 29 evaluable pts, a confirmed partial response in MSI-H gastric cancer refractory to PD-1 treated with agenT-797 + nivo (remains ongoing >6 months) and 8 pts had stable disease (SD). Prolonged SD (>3 months) for 3 monotherapy pts. Overall response rate 20% (1/5) for pts treated with agenT-797 + pembro or nivo. AgenT-797 was detected in peripheral blood up to day 8 post infusion. Preliminary data identified transient increase in serum IFNgamma levels day 2 post infusion. Conclusion: AgenT-797 was well tolerated as monotherapy and in combo with PD-1 (pembro or nivo). Anti-tumor activity in combo with nivo was observed in gastric cancer. Results support the potential of a novel therapeutic strategy employed by agenT-797 to enhance antitumor immunity in PD-1 refractory tumors. Enrollment ongoing and correlative studies will be presented. *PD-1 refractory NSCLC, pancreatic, rectal, cholangiocarcinoma, cervical, gastric, ocular melanoma, renal, and upper tract urothelial Citation Format: Benedito Carneiro, Benjamin Garmezy, John T. Hamm, Rachel E. Sanborn, Trisha Wise-Draper, Anthony El- Khoueiry, Breelyn Wilky, Dave S.B Hoon, Alexa Buffa, Xavier Michelet, Marco Purbhoo, Mark A. Exley, David Einstein. Phase 1 clinical update of allogeneic invariant natural killer T cells (iNKTs), agenT-797, alone or in combination with pembrolizumab or nivolumab in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT275.

Chemical Synthesis and Molecular Interaction Analysis of α-Galactosyl Ceramide Derivatives as CD1d Ligands.

CD1d is a non-classical major histocompatibility complex (MHC) protein, responsible for lipid antigen presentation, which presents lipids to natural killer T (NKT) cells. Various CD1d lipid ligands have been reported, including microbial and endogenous glycolipids/phospholipids. Among them, an α-galactosylceramide (α-GalCer), a representative CD1d ligand, is one of the most potent ligands and its derivatives have been developed. In this chapter, the chemistry of α-GalCer and its derivatives are described with an emphasis on their chemical syntheses and molecular interaction analysis with CD1d are described.

Antigen-presenting Tcells provide critical B7 co-stimulation for thymic iNKT cell development via CD28-dependent trogocytosis.

Invariant natural killer T (iNKT) cell development in the thymus depends on Tcell receptor recognition of CD1dligand on CD4/CD8 double-positive thymocytes. We previously reported that B7-CD28 co-stimulation is required for thymic iNKT cell development, but the cellular and molecular mechanisms underlying this co-stimulatory requirement are not understood. Here we report that CD28 expression on CD1d-expressing antigen-presenting Tcells is required for thymic iNKT cell development. Mechanistically, antigen-presenting T cells provide co-stimulation through an unconventional mechanism, acquiring B7molecules via CD28-dependent trogocytosis from B7-expressing thymic epithelial cells, dendritic cells,and B cells and providing critical B7 co-stimulation to developing iNKT cells. Thus, the present study demonstrates a mechanism of B7 co-stimulation in thymic Tcell development by antigen-presenting Tcells.

Synthesis and Immunological Activity of Novel Oligo(ethylene glycol) Analogues of α-Galactosylceramide.

CD1d-arbitrated activation of i-NKT cells by α-galactosylceramide results in the effective secretion of Th1 and Th2 cytokines, with adjuvanticity skewed toward Th2 immunity. However, the polarization of immune response could be achieved by suitable modification of the glycolipid structure. In the current study, novel glycolipids with an amphiphilic oligo ethylene glycol lipid moiety bearing the benzyloxy group at the terminus on the acyl arm of sphingosine, exhibited CD1d ligand binding as quantified by IL-2 cytokine production. When immunized with quadrivalent split influenza virus in BALB/c mice, the novel ceramide analogues with a longer oligo (ethylene glycol) chain length induced significant levels of antibody (IgG) with Th1-polarized immune response.

Use of a Clostridioides difficile Murine Immunization and Challenge Model to Evaluate Single and Combination Vaccine Adjuvants Consisting of Alum and NKT Cell-Activating Ligands.

Adjuvant combinations may enhance or broaden the expression of immune responses to vaccine antigens. Information on whether established Alum type adjuvants can be combined with experimental CD1d ligand adjuvants is currently lacking. In this study, we used a murine Clostridioides difficile immunization and challenge model to evaluate Alum (Alhydrogel™), α-galactosylceramide (α-GC), and one of its analogs 7DW8-5 singly and in combination as vaccine adjuvants. We observed that the Alum/α-GC combination caused modest enhancement of vaccine antigen-specific IgG1 and IgG2b responses, and a broadening to include IgG2c that did not significantly impact overall protection. Similar observations were made using the Alum/7DW8-5 combination. Examination of the impact of adjuvants on NKT cells revealed expansion of invariant NKT (iNKT) cells with modest expansion of their iNKTfh subset and little effect on diverse NKT (dNKT) cells. Side effects of the adjuvants was determined and revealed transient hepatotoxicity when Alum/α-GC was used in combination but not singly. In summary these results showed that the Alum/α-GC or the Alum/7DW8-5 combination could exert distinct effects on the NKT cell compartment and on isotype switch to produce Th1-driven IgG subclasses in addition to Alum/Th2-driven subclasses. While Alum alone was efficacious in stimulating IgG-mediated protection, and α-GC offered no apparent additional benefit in the C. difficile challenge model, the work herein reveals immune response features that could be optimized and harnessed in other vaccine contexts.

Polar functional group-containing glycolipid CD1d ligands modulate cytokine-biasing responses and prevent experimental colitis

The MHC class I-like molecule CD1d is a nonpolymorphic antigen-presenting glycoprotein, and its ligands include glycolipids, such as α-GalCer. The complexes between CD1d and ligands activate natural killer T cells by T cell receptor recognition, leading to the secretion of various cytokines (IFN-γ, IL-4, IL-17A, etc.). Herein, we report structure–activity relationship studies of α-GalCer derivatives containing various functional groups in their lipid acyl chains. Several derivatives have been identified as potent CD1d ligands displaying higher cytokine induction levels and/or unique cytokine polarization. The studies also indicated that flexibility of the lipid moiety can affect the binding affinity, the total cytokine production level and/or cytokine biasing. Based on our immunological evaluation and investigation of physicochemical properties, we chose bisamide- and Bz amide-containing derivatives 2 and 3, and evaluated their in vivo efficacy in a DSS-induced model of ulcerative colitis. The derivative 3 that exhibits Th2- and Th17-biasing responses, demonstrated significant protective effects against intestinal inflammation in the DSS-induced model, after a single intraperitoneal injection.

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.

Design and Discovery of Covalent α-GalCer Derivatives as Potent CD1d Ligands.

CD1d is a nonpolymorphic antigen-presenting protein responsible for the regulation of natural killer T (NKT) cell activation. α-Galactosyl ceramide (α-GalCer, KRN7000) is the representative CD1d ligand that can bind to the CD1d protein. The resulting complex is recognized by the T cell receptors of the NKT cell, inducing various immune responses. Previous structure-activity relationship studies of α-GalCer have revealed that the ability of NKT cells to induce cytokines depends on the ligand structure, and in particular, ligands that form more stable complexes with CD1d display potent activity. We focused on the Cys residue of the large hydrophobic pockets of CD1d (A' pocket) and developed α-GalCer derivatives containing groups that can form covalent bonds. The assay results revealed that these ligands displayed higher levels of cytokine production and Th2 cell-type cytokine polarization response. Furthermore, the LC-MS/MS analysis indicated that the chloroacetylamide-containing ligand was covalently bound to Cys12 of CD1d, which suggests that the enhanced activities result from the formation of a stable CD1d-ligand complex. To our knowledge, this is the first ligand that allows covalent bond formation to CD1d under physiological conditions.

Synthesis and biological activities of amino acids functionalized α-GalCer analogues

Invariant natural killer T-cells (iNKT-cells) are promising targets for manipulating the immune system, which can rapidly release a large amount of Th1 and Th2 cytokines upon the engagement of their T cell receptor with glycolipid antigens presented by CD1d. In this paper, we wish to report a novel series of α-GalCer analogues which were synthesized by incorporation of l-amino acid methyl esters in the C-6′ position of glycolipid. The evaluation of these synthetic analogues for their capacities to stimulate iNKT-cells into producing Th1 and Th2 cytokines both in vitro and in vivo indicated that they were potent CD1d ligands and could stimulate murine spleen cells into a higher release of the Th1 cytokine IFN-γ in vitro. In vivo, Gly-α-GalCer (1) and Lys-α-GalCer (3) showed more Th1-biased responses than α-GalCer, especially analogue 3 showed the highest selectivity for IFN-γ production (IFN-γ/IL-4 = 5.32) compared with α-GalCer (IFN-γ/IL-4 = 2.5) in vivo. These novel α-GalCer analogues might be used as efficient X-ray crystallographic probes to reveal the relationship between glycolipids and CD1d proteins in α-GalCer/CD1d complexes and pave the way for developing new potent immunostimulating agents.

Invariant natural killer T cells are functionally impaired in patients with systemic sclerosis

BackgroundSystemic sclerosis (SSc) is a potentially fatal autoimmune disease that leads to extensive fibrosis of the skin and internal organs. Invariant natural killer T (iNKT) cells are potent immunoregulatory T lymphocytes being able to orchestrate dysregulated immune responses. The purpose of this study was to evaluate numbers and function of iNKT cells in patients with SSc and to analyze their correlation with disease parameters.MethodsHuman iNKT cells from 88 patients with SSc and 33 healthy controls were analyzed by flow cytometry. Their proliferative capacity and cytokine production were investigated following activation with CD1d ligand α-galactosylceramide (α-GalCer).ResultsWe observed an absolute and relative decrease of iNKT cells in patients with SSc compared with healthy controls. Interestingly, the subtype of SSc, disease severity, or treatment with immunosuppressive drugs did not affect iNKT cell numbers. However, T helper (Th) cell immune polarization was biased towards a Th17 immunophenotype in SSc patients. Moreover, iNKT cells from patients with SSc showed a significantly decreased expansion capacity upon stimulation with α-GalCer.ConclusioniNKT cells are deficient and functionally impaired in patients with SSc. Therefore, adoptive transfer strategies using culture-expanded iNKT cells could be a novel approach to treat SSc patients.

Porcine Invariant Natural Killer T Cells: Functional Profiling and Dynamics in Steady State and Viral Infections.

Pigs are important livestock and comprehensive understanding of their immune responses in infections is critical to improve vaccines and therapies. Moreover, similarities between human and swine physiology suggest that pigs are a superior animal model for immunological studies. However, paucity of experimental tools for a systematic analysis of the immune responses in pigs represent a major disadvantage. To evaluate the pig as a biomedical model and additionally expand the knowledge of rare immune cell populations in swine, we established a multicolor flow cytometry analysis platform of surface marker expression and cellular responses for porcine invariant Natural Killer T cells (iNKT). In humans, iNKT cells are among the first line defenders in various tissues, respond to CD1d-restricted antigens and become rapidly activated. Naïve porcine iNKT cells were CD3+/CD4−/CD8+ or CD3+/CD4−/CD8− and displayed an effector- or memory-like phenotype (CD25+/ICOS+/CD5hi/CD45RA−/CCR7 ± /CD27+). Based on their expression of the transcription factors T bet and the iNKT cell-specific promyelocytic leukemia zinc finger protein (PLZF), porcine iNKT cells were differentiated into functional subsets. Analogous to human iNKT cells, in vitro stimulation of porcine leukocytes with the CD1d ligand α-galactosylceramide resulted in rapid iNKT cell proliferation, evidenced by an increase in frequency and Ki-67 expression. Moreover, this approach revealed CD25, CD5, ICOS, and the major histocompatibility complex class II (MHC II) as activation markers on porcine iNKT cells. Activated iNKT cells also expressed interferon-γ, upregulated perforin expression, and displayed degranulation. In steady state, iNKT cell frequency was highest in newborn piglets and decreased with age. Upon infection with two viruses of high relevance to swine and humans, iNKT cells expanded. Animals infected with African swine fever virus displayed an increase of iNKT cell frequency in peripheral blood, regional lymph nodes, and lungs. During Influenza A virus infection, iNKT cell percentage increased in blood, lung lymph nodes, and broncho-alveolar lavage. Our in-depth characterization of porcine iNKT cells contributes to a better understanding of porcine immune responses, thereby facilitating the design of innovative interventions against infectious diseases. Moreover, we provide new evidence that endorses the suitability of the pig as a biomedical model for iNKT cell research.

Structure-activity relationship studies of Bz amide-containing α-GalCer derivatives as natural killer T cell modulators

AbstractCD1d is a non-polymorphic antigen-presenting glycoprotein that recognizes glycolipids as ligands. Ligands bind to the hydrophobic grooves of CD1d, and the resulting ligand-CD1d complexes activate natural killer T (NKT) cells by means of T cell receptor recognition, leading to the secretion of various cytokines. However, details of the ligand recognition mechanism of a large hydrophobic ligand binding pocket and the relationship between cytokine induction and ligand structure are unclear. We report the synthesis of α-GalCer derivatives containing a Bz amide group having various substituting groups in the ceramide moiety, and the analysis of the structure-activity relationships. The assays reveal that the Bz amide-containing CD1d ligands function as NKT cell modulators displaying Th2 cytokine biasing responses. Furthermore, molecular dynamics simulation studies suggest that the phenyl groups can interact with the aromatic amino acid residues in the lipid binding pocket of CD1d.

Synthesis of 5-Thio-α-GalCer Analogues with Fluorinated Acyl Chain on Lipid Residue and Their Biological Evaluation.

Invariant natural killer T (iNKT) cells are a subclass of T cells that initiates the secretion of T helper 1 and 2 cytokines after recognizing CD1d protein presented glycolipid antigens. In this Letter, we designed and synthesized a novel series of CD1d ligand α-galactosylceramides (α-GalCers) in which the acyl chain backbone of the lipid was incorporated with fluorine atoms. The in vivo evaluation of immunostimulatory activities revealed that the synthesized α-5-thio-galactopyranosyl-N-perfluorooctanoyl phytosphingosine exhibited a remarkable potency toward selectively enhancing TH1 cytokine production with the IFN γ/IL-4 ratio of 9/1, while its perfluorotetradecanoyl counterpart showed TH2 profile with an IFN γ/IL-4 ratio of 0.59/1. The analogues synthesized here would be used as probes to study lipid-protein interactions in α-GalCer/CD1d complexes.

Development and functional evaluation of Th2-biased CD1d ligands containing modified lipid moiety

Development and functional evaluation of Th2-biased CD1d ligands containing modified lipid moiety

Differentiation of Langerhans Cells from Monocytes and Their Specific Function in Inducing IL-22-Specific Th Cells.

Human mucosal tissues and skin contain two distinct types of dendritic cell (DC) subsets, epidermal Langerhans cells (LCs) and dermal DCs, which can be distinguished by the expression of C-type lectin receptors, Langerin and DC-SIGN, respectively. Although peripheral blood monocytes differentiate into these distinct subsets, monocyte-derived LCs (moLCs) induced by coculture with GM-CSF, IL-4, and TGF-β1 coexpress both Langerin and DC-SIGN, suggesting that the environmental cues remain unclear. In this study, we show that LC differentiation is TGF-β1 dependent and that cofactors such as IL-4 and TNF-α promote TGF-β1-dependent LC differentiation into Langerin+DC-SIGN- moLCs but continuous exposure to IL-4 blocks differentiation. Steroids such as dexamethasone greatly enhanced TNF-α-induced moLC differentiation and blocked DC-SIGN expression. Consistent with primary LCs, dexamethasone-treated moLCs express CD1a, whereas monocyte-derived DCs (moDCs) express CD1b, CD1c, and CD1d. moDCs but not moLCs produced inflammatory cytokines after stimulation with CD1b and CD1d ligands mycolic acid and α-galactosylceramide, respectively. Strikingly, CD1a triggering with squalene on moLCs but not moDCs induced strong IL-22-producing CD4+ helper T cell responses. As IL-22 is an important cytokine in the maintenance of skin homeostasis, these data suggest that CD1a on LCs is involved in maintaining the immune barrier in the skin.

The Hypervariable Loops of Free TCRs Sample Multiple Distinct Metastable Conformations in Solution.

CD4+ and CD8+ αβ T cell antigen recognition is determined by the interaction between the TCR Complementarity Determining Region (CDR) loops and the peptide-presenting MHC complex. These T cells are known for their ability to recognize multiple pMHC complexes, and for a necessary promiscuity that is required for their selection and function in the periphery. Crystallographic studies have previously elucidated the role of structural interactions in TCR engagement, but our understanding of the dynamic process that occurs during TCR binding is limited. To better understand the dynamic states that exist for TCR CDR loops in solution, and how this relates to their states when in complex with pMHC, we simulated the 2C T cell receptor in solution using all-atom molecular dynamics in explicit water and constructed a Markov State Model for each of the CDR3α and CDR3β loops. These models reveal multiple metastable states for the CDR3 loops in solution. Simulation data and metastable states reproduce known CDR3β crystal conformations, and reveal several novel conformations suggesting that CDR3β bound states are the result of search processes from nearby pre-existing equilibrium conformational states. Similar simulations of the invariant, Type I Natural Killer T cell receptor NKT15, which engages the monomorphic, MHC-like CD1d ligand, demonstrate that iNKT TCRs also have distinct states, but comparatively restricted degrees of motion.

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.

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

Th2-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.

NKT cells in cardiovascular diseases

Despite life-style advice and the prescription of cholesterol-lowering and anti-thrombotic drugs, cardiovascular diseases are still the leading cause of death worldwide. Therefore, there is an urgent need for new therapeutic strategies focussing on atherosclerosis, the major underlying pathology of cardiovascular diseases characterized by an accumulation of lipids in an inflamed arterial/vessel wall. CD1d-restricted lipid-sensing natural killer T (NKT) cells, bridging the innate and adaptive immunity, and CD1d-expressing antigen-presenting cells are detected in atherosclerotic lesions of mice and humans. In this review we will summarize studies that point to a critical role for NKT cells in the pathogenesis of atherosclerosis and other cardiovascular diseases by the secretion of pro-atherogenic cytokines and cytotoxins. These pro-atherogenic NKT cells are potential targets for new therapeutic strategies in the prevention and treatment of cardiovascular diseases. Additionally, proteins transferring lipids during atherosclerosis, which are also important in the loading of lipids onto CD1d and possible endogenous ligands responsible for the activation of NKT cells during atherosclerosis will be discussed.

Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain

A novel series of CD1d ligand α-galactosylceramides (α-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of α-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid–protein interactions in KRN7000/CD1d complexes.