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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.

Microbes produce a rich array of lipidic species that through their location in the cell wall and ability to mingle with host lipids represent a privileged class of immune-active molecules. Lipid-sensing immunity recognizes microbial lipids from pathogens and commensals causing immune responses. Yet microbial lipids are often heterogeneous, in limited supply and in some cases their structures are incompletely defined. Total synthesis can assist in structural determination, overcome supply issues, and provide access to high-purity, homogeneous samples and analogues. This account highlights synthetic approaches to lipidic species from pathogenic and commensal bacteria and fungi that have supported immunological studies involving lipid sensing through the pattern recognition receptor Mincle and cell-mediated immunity through the CD1-T cell axis.

Natural killer T (NKT) cells constitute a unique subset of T lymphocytes characterized by specifically interacting with antigenic glycolipids conjugated to the CD1d receptor on antigen-presenting cells. Functionally, NKT cells are capable of performing either effector or suppressor immune responses, depending on their production of proinflammatory or anti-inflammatory cytokines, respectively. Effector NKT cells are subdivided into three subsets, termed NKT1, NKT2, and NKT17, based on the cytokines they produce and their similarity to the cytokine profile produced by Th1, Th2, and Th17 lymphocytes, respectively. Recently, a new subgroup of NKT cells termed NKT10 has been described, which cooperates and interacts with other immune cells to promote immunoregulatory responses. Although the tissue-specific functions of NKT cells have not been fully elucidated, their activity has been associated with the pathogenesis of different inflammatory diseases with immunopathogenic similarities to periodontitis, including osteolytic pathologies such as rheumatoid arthritis and osteoporosis. In the present review, we revise and discuss the pathogenic characteristics of NKT cells in these diseases and their role in the pathogenesis of periodontitis; particularly, we analyze the potential regulatory role of the IL-10-producing NKT10 cells., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Samanta Melgar-Rodríguez et al.)

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8 + cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. SIGNIFICANCE: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells., (©2021 American Association for Cancer Research.)

Antibody-mediated modulation of major histocompatibility complex (MHC) molecules, or MHC class I-like molecules, could constitute an effective immunotherapeutic approach. We describe how single-domain antibodies (VHH), specific for the human MHC class I-like molecule CD1d, can modulate the function of CD1d-restricted T cells and how one VHH (1D12) specifically induced strong type I natural killer T (NKT) cell activation. The crystal structure of the VHH1D12-CD1d(α-GalCer)-NKT T-cell receptor (TCR) complex revealed that VHH1D12 simultaneously contacted CD1d and the type I NKT TCR, thereby stabilizing this interaction through intrinsic bispecificity. This led to greatly enhanced type I NKT cell-mediated antitumor activity in in vitro, including multiple myeloma and acute myeloid leukemia patient-derived bone marrow samples, and in vivo models. Our findings underscore the versatility of VHH molecules in targeting composite epitopes, in this case consisting of a complexed monomorphic antigen-presenting molecule and an invariant TCR, and represent a generalizable antitumor approach., (© 2020. The Author(s), under exclusive licence to Springer Nature America, Inc.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

α-Galactosylceramides are glycosphingolipids that show promise in cancer immunotherapy. After presentation by CD1d, they activate natural killer T cells (NKT), which results in the production of a variety of pro-inflammatory and immunomodulatory cytokines. Herein, we report the synthesis and biological evaluation of photochromic derivatives of KRN-7000, the activity of which can be modulated with light. Based on established structure-activity relationships, we designed photoswitchable analogues of this glycolipid that control the production of pro-inflammatory cytokines, such as IFN-γ. The azobenzene derivative α-GalACer-4 proved to be more potent than KRN-7000 itself when activated with 370 nm light. Photolipids of this type could improve our mechanistic understanding of cytokine production and could open new directions in photoimmunotherapy., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

α-Galactosylceramide (α-GalCer) is recognized by the CD1d proteins on antigen-presenting cells at the ceramide moiety and the galactose moiety is presented to iNKT cells, which stimulates the immune responses. However, the immune suppression by repeated injections of α-GalCer has discouraged its development as an anti-cancer agent. To overcome the shortcoming by spatiotemporal restriction of its exposure, we synthesized the photochromic azobenzene-incorporated analogues and tested the photo-immunoregulation effect in its binding to CD1d. FACS analyses indicated that some of these analogues enhanced the affinity to CD1d on photo-irradiation by about 20%. A docking simulation suggests that the photochromic molecule should be bulkier for a clearer discrimination between on and off states., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

High-throughput TCR sequencing allows interrogation of the human TCR repertoire, potentially connecting TCR sequences to antigenic targets. Unlike the highly polymorphic MHC proteins, monomorphic Ag-presenting molecules such as MR1, CD1d, and CD1b present Ags to T cells with species-wide TCR motifs. CD1b tetramer studies and a survey of the 27 published CD1b-restricted TCRs demonstrated a TCR motif in humans defined by the TCR β-chain variable gene 4-1 (TRBV4-1) region. Unexpectedly, TRBV4-1 was involved in recognition of CD1b regardless of the chemical class of the carried lipid. Crystal structures of two CD1b-specific TRBV4-1 + TCRs show that germline-encoded residues in CDR1 and CDR3 regions of TRBV4-1-encoded sequences interact with each other and consolidate the surface of the TCR. Mutational studies identified a key positively charged residue in TRBV4-1 and a key negatively charged residue in CD1b that is shared with CD1c, which is also recognized by TRBV4-1 TCRs. These data show that one TCR V region can mediate a mechanism of recognition of two related monomorphic Ag-presenting molecules that does not rely on a defined lipid Ag., (Copyright © 2019 by The American Association of Immunologists, Inc.)

iNKT cells are CD1d-restricted T cells recognizing lipid antigens. The prototypic iNKT cell-agonist α-galactosylceramide (α-GalCer) alongside compounds with similar structures induces robust proliferation and cytokine production of iNKT cells and protects against cancer in vivo . Monoclonal antibodies (mAbs) that detect CD1d-α-GalCer complexes have provided critical information for understanding of antigen presentation of iNKT cell agonists. Although most iNKT cell agonists with antitumor properties are α-linked glycosphingolipids that can be detected by anti-CD1d-α-GalCer mAbs, β-ManCer, a glycolipid with a β-linkage, induces strong antitumor immunity via mechanisms distinct from those of α-GalCer. In this study, we unexpectedly discovered that anti-CD1d-α-GalCer mAbs directly recognized β-ManCer-CD1d complexes and could inhibit β-ManCer stimulation of iNKT cells. The binding of anti-CD1d-α-GalCer mAb with β-ManCer-CD1d complexes was also confirmed by plasmon resonance and could not be explained by α-anomer contamination. The binding of anti-CD1d-α-GalCer mAb was also observed with CD1d loaded with another β-linked glycosylceramide, β-GalCer (C26:0). Detection with anti-CD1d-α-GalCer mAbs indicates that the interface of the β-ManCer-CD1d complex exposed to the iNKT cell TCR can assume a structure like that of CD1d-α-GalCer, despite its disparate carbohydrate structure. These results suggest that certain β-linked monoglycosylceramides can assume a structural display similar to that of CD1d-α-GalCer and that the data based on anti-CD1d-α-GalCer binding should be interpreted with caution., (Copyright © 2019 Clark, Yau, Bloom, Wang, Venzon, Suzuki, Pasquet, Compton, Cardell, Porcelli, Painter, Zajonc, Berzofsky and Terabe.)

Type I natural killer T (NKT) cells are a population of innate like T lymphocytes that rapidly respond to α-GalCer presented by CD1d via the production of both pro- and anti-inflammatory cytokines. While developing novel α-GalCer analogs that were meant to be utilized as potential adjuvants because of their production of pro-inflammatory cytokines (Th1 skewers), we generated α-galactosylsphingamides (αGSA). Surprisingly, αGSAs are not potent antigens in vivo despite their strong T-cell receptor (TCR)-binding affinities. Here, using surface plasmon resonance (SPR), antigen presentation assays, and X-ray crystallography (yielding crystal structures of 19 different binary (CD1d-glycolipid) or ternary (CD1d-glycolipid-TCR) complexes at resolutions between 1.67 and 2.85 Å), we characterized the biochemical and structural details of αGSA recognition by murine NKT cells. We identified a molecular switch within murine (m)CD1d that modulates NKT cell activation by αGSAs. We found that the molecular switch involves a hydrogen bond interaction between Tyr-73 of mCD1d and the amide group oxygen of αGSAs. We further established that the length of the acyl chain controls the positioning of the amide group with respect to the molecular switch and works synergistically with Tyr-73 to control NKT cell activity. In conclusion, our findings reveal important mechanistic insights into the presentation and recognition of glycolipids with polar moieties in an otherwise apolar milieu. These observations may inform the development αGSAs as specific NKT cell antagonists to modulate immune responses., (© 2019 Wang et al.)

iNKT cells recognize CD1d/α-galactosylceramide (α-GalCer) complexes via their invariant TCR receptor and stimulate the immune response. Many α-GalCer analogues have been investigated to interrogate this interaction. Following our previous work related to the modification of the hydrogen bond network between α-GalCer and CD1d, we have now focused our attention on the synthesis of 3-deoxy-3,3-difluoro- and 3,4-dideoxy-3,3,4,4-tetrafluoro-α-GalCer analogues, and studied their ability to stimulate human iNKT cells. In each case, deoxygenation at the indicated positions was accompanied by difluoro introduction in order to evaluate the resulting electronic effect on the stability of the ternary CD1d/Galcer/TCR complex which has been rationalized by modeling study. With deoxy-difluorination at the 3-position, the two epimeric 4-OH analogues were investigated to establish their capacity to compensate for the lack of the hydrogen bond donating group at the 3-position. The 3,4-dideoxytetrafluoro analogue was of interest to highlight the amide NH-bond hydrogen bond properties., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

CD1d 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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glycosphingolipids and glycerophospholipids bind CD1d. Glycosphingolipid-reactive invariant NKT-cells (iNKT) exhibit myriad immune effects, however, little is known about the functions of phospholipid-reactive T cells (PLT). We report that the normal mouse immune repertoire contains αβ T cells, which recognize self-glycerophospholipids such as phosphatidic acid (PA) in a CD1d-restricted manner and don't cross-react with iNKT-cell ligands. PA bound to CD1d in the absence of lipid transfer proteins. Upon in vivo priming, PA induced an expansion and activation of T cells in Ag-specific manner. Crystal structure of the CD1d:PA complex revealed that the ligand is centrally located in the CD1d-binding groove opening for TCR recognition. Moreover, the increased flexibility of the two acyl chains in diacylglycerol ligands and a less stringent-binding orientation for glycerophospholipids as compared with the bindings of glycosphingolipids may allow glycerophospholipids to readily occupy CD1d. Indeed, PA competed with α-galactosylceramide to load onto CD1d, leading to reduced expression of CD1d:α-galactosylceramide complexes on the surface of dendritic cells. Consistently, glycerophospholipids reduced iNKT-cell proliferation, expansion, and cytokine production in vitro and in vivo. Such superior ability of self-glycerophospholipids to compete with iNKT-cell ligands to occupy CD1d may help maintain homeostasis between the diverse subsets of lipid-reactive T cells, with important pathogenetic and therapeutic implications., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Activated NKT cells can stimulate antigen-presenting cells leading to enhanced peptide antigen-specific immunity. However, administration of potent NKT cell agonists like α-galactosylceramide (α-GalCer) can be associated with release of high levels of cytokines, and in some situations, hepatotoxicity. Here we show that it is possible to provoke sufficient NKT cell activity to stimulate strong antigen-specific T cell responses without these unwanted effects. This was achieved by chemically conjugating antigenic peptides to α-galactosylphytosphingosine (α-GalPhs), an NKT cell agonist with very weak activity based on structural characterisation and biological assays. Conjugation improved delivery to antigen-presenting cells in vivo, while use of a cathepsin-sensitive linker to release the α-GalPhs and peptide within the same cell promoted strong T cell activation and therapeutic anti-tumour responses in mice. The conjugates activated human NKT cells and enhanced human T cell responses to a viral peptide in vitro. Accordingly, we have demonstrated a means to safely exploit the immunostimulatory properties of NKT cells to enhance T cell activation for virus- and tumour-specific immunity.

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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Invariant Natural Killer T-cells (iNKT-cells) are an attractive target for immune response modulation, as upon CD1d-mediated stimulation with KRN7000, a synthetic α-galactosylceramide, they produce a vast amount of cytokines. Here we present a synthesis that allows swift modification of the phytosphingosine side chain by amidation of an advanced methyl ester precursor. The resulting KRN7000 derivatives, termed α-galactosylsphingamides, were evaluated for their capacity to stimulate iNKT-cells. While introduction of the amide-motif in the phytosphingosine chain is tolerated for CD1d binding and TCR recognition, the studied α-galactosylsphingamides showed compromised antigenic properties.

In this review, we discuss small-molecule, carbohydrate-based immunostimulants that target Toll-like receptor 4 (TLR-4) and cluster of differentiation 1D (CD1d) receptors. The design and use of these molecules in immunotherapy as well as results from their use in clinical trials are described. How these molecules work and their utilization as vaccine adjuvants are also discussed. Future applications and extensions for the use of these analogues as therapeutic agents will be outlined., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The CD1d protein is a nonpolymorphic MHC class I-like protein that controls the activation of natural killer T (NKT) cells through the presentation of self- and foreign-lipid ligands, glycolipids, or phospholipids, leading to the secretion of various cytokines. The CD1d contains a large hydrophobic lipid binding pocket: the A' pocket of CD1d, which recognizes hydrophobic moieties of the ligands, such as long fatty acyl chains. Although lipid-protein interactions typically rely on hydrophobic interactions between lipid chains and the hydrophobic sites of proteins, we showed that the small polar regions located deep inside the hydrophobic A' pocket could be used for the modulation of the lipid binding. A series of the ligands, α-galactosyl ceramide (α-GalCer) derivatives containing polar groups in the acyl chain, was synthesized, and the structure-activity relationship studies demonstrated that simple modification from a methylene to an amide group in the long fatty acyl chain, when introduced at optimal positions, enhanced the CD1d recognition of the glycolipid ligands. Formation of hydrogen bonds between the amide group and the polar residues was supported by molecular dynamics (MD) simulations and WaterMap calculations. The computational studies suggest that localized hydrating water molecules may play an important role in the ligand recognition. Here, the results showed that confined polar residues in the large hydrophobic lipid binding pockets of the proteins could be potential targets to modulate the affinity for its ligands.

Mouse CD1d is a nonclassical MHC molecule able to present lipids and glycolipids to a specialized subset of T cells known as natural killer T cells. The antigens presented by CD1d have been shown to cover a broad range of chemical structures and to follow precise rules determining the potency of the antigen in the context of T cell activation. Together with lipids, initial reports suggested that CD1d can also bind and present hydrophobic peptides with (F/W)XX(I/L/M)XXW. However, the exact location of peptide binding and the molecular basis for the required motif are currently unknown. Here we present the crystal structure of the first peptide identified to bind CD1d, p99, and show that it binds in the antigen-binding groove of CD1d in a manner compatible with its presentation to T cell receptors. Interestingly, the peptide adopts an α-helical conformation, which orients the motif residues toward its deep binding groove, therefore explaining the molecular requirements for peptide binding. Moreover, we demonstrate that a lipopeptide version of the same peptide is able to bind CD1d in a similar conformation, identifying another class of molecules binding this antigen-presenting molecule., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The cotton rat (Sigmodon hispidus) belongs to the rodent family of Cricetidae and provides a powerful model to study the pathogenesis of human respiratory viruses and measles virus. Recent studies in other rodent models have suggested a role for invariant natural killer T (iNKT) cells in antiviral immunity and vaccination against respiratory virus infections. Using new experimental tools, we provide the first evidence for a functional CD1d cell molecule (crCD1d) and iNKT T-cell receptor in cotton rats. The crCD1d cDNA sequence was identified and crCD1d transductants showed that monoclonal antibody WTH-2 stains crCD1d as efficiently as mouse or rat CD1d. The expression of crCD1d was clearly weaker for thymocytes and B cells, and higher for T cells, which is different to what is found in murine species. The antigen-presenting capacity of crCD1d was demonstrated with crCD1d-immunoglobulin dimers loaded with the glycolipid PBS57, which bound iNKT T-cell receptors. Evidence for functional cotton rat iNKT cells was provided by detection of interferon-γ and interleukin-4 in cultures of splenocytes stimulated with PBS57 and α-galactosylceramide and by specific staining of about 0·2% of splenocytes with PBS57-loaded crCD1d dimers. Canonical AV14/AJ18 rearrangements were identified and found to contain multiple members of the AV14 (AV11) family. One of them was expressed and found to bind CD1d dimers. In summary, these data provide the first evidence for functional CD1d molecules and iNKT T-cell receptors in cotton rats and provide the tools to analyse them both in the cotton rat model of infectious diseases., (© 2015 John Wiley & Sons Ltd.)

CD1d molecules are MHC class I-like molecules that present glycolipids to iNKT cells. The highly conserved interaction between CD1d:α-Galactosylceramide (αGC) complexes and the iNKT TCR not only defines this population of αβ T cells but can also be used for its direct identification. Therefore, CD1d oligomers are a widely used tool for iNKT cell related investigations. To this end, the lipid chains of the antigen have to be inserted into the hydrophobic pockets of the CD1d binding cleft, often with help of surfactants. In this study, we investigated the influence of different surfactants (Triton X-100, Tween 20, Tyloxapol) on in vitro loading of CD1d molecules derived from four different species (human, mouse, rat and cotton rat) with αGC and derivatives carrying modifications of the acyl-chain (DB01-1, PBS44) and a 6-acetamido-6-deoxy-addition at the galactosyl head group (PBS57). We also compared rat CD1d dimers with tetramers and staining of an iNKT TCR transductant was used as readout for loading efficacy. The results underlined the importance of CD1d loading efficacy for proper analysis of iNKT TCR binding and demonstrated the necessity to adjust loading conditions for each oligomer/glycolipid combination. The efficient usage of surfactants as a tool for CD1d loading was revealed to be species-specific and depending on the origin of the CD1d producing cells. Additional variation of surfactant-dependent loading efficacy between tested glycolipids was influenced by the acyl-chain length and the modification of the galactosyl head group with PBS57 showing the least dependence on surfactants and the lowest degree of species-dependent differences.

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., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The ability of different glycosphingolipids (GSLs) to activate type I natural killer T cells (NKT cells) has been known for 2 decades. The possible therapeutic use of these GSLs has been studied in many ways; however, studies are needed in which the efficacy of promising GSLs is compared under identical conditions. Here, we compare five unique GSLs structurally derived from α-galactosylceramide. We employed biophysical and biological assays, as well as x-ray crystallography to study the impact of the chemical modifications of the antigen on type I NKT cell activation. Although all glycolipids are bound by the T cell receptor of type I NKT cells in real time binding assays with high affinity, only a few activate type I NKT cells in in vivo or in vitro experiments. The differences in biological responses are likely a result of different pharmacokinetic properties of each lipid, which carry modifications at different parts of the molecule. Our results indicate a need to perform a variety of assays to ascertain the therapeutic potential of type I NKT cell GSL activators., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

α-GalCer is a potent immunomodulatory molecule that is presented to NKT cells via the CD1 antigen-presenting system. We hypothesized that when used as an adjuvant α-GalCer would induce protective immune responses against Rhodococcus equi, an important pathogen of young horses. Here we demonstrate that the equine CD1d molecule shares most features found in CD1d from other species and has a suitable lipid-binding groove for presenting glycolipids to NKT cells. However, equine CTL stimulated with α-GalCer failed to kill cells infected with R. equi, and α-GalCer did not increase killing by CTL co-stimulated with R. equi antigen. Likewise, α-GalCer did not induce the lymphoproliferation of equine PBMC or increase the proliferation of R. equi-stimulated cells. Intradermal injection of α-GalCer in horses did not increase the recruitment of lymphocytes or cytokine production. Furthermore, α-GalCer-loaded CD1d tetramers, which have been shown to be broadly cross-reactive, did not bind equine lymphocytes. Altogether, our results demonstrate that in contrast to previously described species, horses are unable to respond to α-GalCer. This raises questions about the capabilities and function of NKT cells and other lipid-specific T lymphocytes in horses., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

CD1d is a nonpolymorphic, MHC class I-like molecule that presents phospholipid and glycosphingolipid Ags to a subset of CD1d-restricted T cells called invariant NKT (iNKT) cells. This CD1d-iNKT cell axis regulates nearly all aspects of both the innate and adaptive immune responses. Expression of CD1d on B cells is suggestive of the ability of these cells to present Ag to, and form cognate interactions with, iNKT cells. In this article, we summarize key evidence regarding the role and regulation of CD1d in normal B cells and in humoral immunity. We then extend the discussion to B cell disorders, with emphasis on autoimmune disease, viral infection, and neoplastic transformation of B lineage cells, in which CD1d expression can be altered as a mechanism of immune evasion and can have both diagnostic and prognostic importance. Finally, we highlight current and future therapeutic strategies that aim to target the CD1d-iNKT cell axis in B cells., (Copyright © 2014 by The American Association of Immunologists, Inc.)

A number of potentially bioactive molecules can be found in nature. In particular, marine organisms are a valuable source of bioactive compounds. The activity of an α-galactosylceramide was first discovered in 1993 via screening of a Japanese marine sponge (Agelas mauritanius). Very rapidly, a synthetic glycololipid analogue of this natural molecule was discovered, called KRN7000. Associated with the CD1d protein, this α-galactosylceramide 1 (KRN7000) interacts with the T-cell antigen receptor to form a ternary complex that yields T helper (Th) 1 and Th2 responses with opposing effects. In our work, we carried out molecular dynamics simulations (11.5 µs in total) involving eight different ligands (conducted in triplicate) in an effort to find out correlation at the molecular level, if any, between chemical modulation of 1 and the orientation of the known biological response, Th1 or Th2. Comparative investigations of human versus mouse and Th1 versus Th2 data have been carried out. A large set of analysis tools was employed including free energy landscapes. One major result is the identification of a specific conformational state of the sugar polar head, which could be correlated, in the present study, to the biological Th2 biased response. These theoretical tools provide a structural basis for predicting the very different dynamical behaviors of α-glycosphingolipids in CD1d and might aid in the future design of new analogues of 1.

Background: T cell receptors (TCRs) can recognize diverse lipid and metabolite antigens presented by MHC-like molecules CD1 and MR1, and the molecular basis of many of these interactions has not been determined. Here we applied our protein docking algorithm TCRFlexDock, previously developed to perform docking of TCRs to peptide-MHC (pMHC) molecules, to predict the binding of αβ and γδ TCRs to CD1 and MR1, starting with the structures of the unbound molecules., Results: Evaluating against TCR-CD1d complexes with crystal structures, we achieved near-native structures in the top 20 models for two out of four cases, and an acceptable-rated prediction for a third case. We also predicted the structure of an interaction between a MAIT TCR and MR1-antigen that has not been structurally characterized, yielding a top-ranked model that agreed remarkably with a characterized TCR-MR1-antigen structure that has a nearly identical TCR α chain but a different β chain, highlighting the likely dominance of the conserved α chain in MR1-antigen recognition. Docking performance was improved by re-training our scoring function with a set of TCR-pMHC complexes, and for a case with an outlier binding mode, we found that alternative docking start positions improved predictive accuracy. We then performed unbound docking with two mycolyl-lipid specific TCRs that recognize lipid-bound CD1b, which represent a class of interactions that is not structurally characterized. Highly-ranked models of these complexes showed remarkable agreement between their binding topologies, as expected based on their shared germline sequences, while differences in residue-level interactions with their respective antigens point to possible mechanisms underlying their distinct specificities., Conclusions: Together these results indicate that flexible docking simulations can provide accurate models and atomic-level insights into TCR recognition of MHC-like molecules presenting lipid and other small molecule antigens.

Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications., (© 2014 by the Society for the Study of Reproduction, Inc.)

CD1 molecules belong to non-polymorphic MHC class I-like proteins and present lipid antigens to T cells. Five different CD1 genes (CD1a-e) have been identified and classified into two groups. Group 1 include CD1a-c and present pathogenic lipid antigens to αβ T cells reminiscence of peptide antigen presentation by MHC-I molecules. CD1d is the only member of Group 2 and presents foreign and self lipid antigens to a specialized subset of αβ T cells, NKT cells. NKT cells are involved in diverse immune responses through prompt and massive production of cytokines. CD1d-dependent NKT cells are categorized upon the usage of their T cell receptors. A major subtype of NKT cells (type I) is invariant NKT cells which utilize invariant Vα14-Jα18 TCR alpha chain in mouse. The remaining NKT cells (type II) utilize diverse TCR alpha chains. Engineered CD1d molecules with modified intracellular trafficking produce either type I or type II NKT cell-defects suggesting the lipid antigens for each subtypes of NKT cells are processed/generated in different intracellular compartments. Since the usage of TCR by a T cell is the result of antigen-driven selection, the intracellular metabolic pathways of lipid antigen are a key in forming the functional NKT cell repertoire.

The self-reactivity of their T-cell antigen receptor (TCR) is thought to contribute to the development of immune regulatory cells, such as invariant NK T cells (iNKT). In the mouse, iNKT cells express TCRs composed of a unique Vα14-Jα18 rearrangement and recognize lipid antigens presented by CD1d molecules. We created mice expressing a transgenic TCR-β chain that confers high affinity for self-lipid/CD1d complexes when randomly paired with the mouse iNKT Vα14-Jα18 rearrangement to study their development. We show that although iNKT cells undergo agonist selection, their development is also shaped by negative selection in vivo. In addition, iNKT cells that avoid negative selection in these mice express natural sequence variants of the canonical TCR-α and decreased affinity for self/CD1d. However, limiting the affinity of the iNKT TCRs for "self" leads to inefficient Egr2 induction, poor expression of the iNKT lineage-specific zinc-finger transcription factor PLZF, inadequate proliferation of iNKT cell precursors, defects in trafficking, and impaired effector functions. Thus, proper development of fully functional iNKT cells is constrained by a limited range of TCR affinity that plays a key role in triggering the iNKT cell-differentiation pathway. These results provide a direct link between the affinity of the TCR expressed by T-cell precursors for self-antigens and the proper development of a unique population of lymphocytes essential to immune responses.

In this issue of Immunity, a study by Luoma et al. (2013) provides structural evidence for direct interactions of human Vδ1(+) T cell receptors with CD1d, capping a long trail of evidence that CD1 might be a major influence on γδ T cell biology., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The nature of the antigens recognized by γδ T cells and their potential recognition of major histocompatibility complex (MHC)-like molecules has remained unclear. Members of the CD1 family of lipid-presenting molecules are suggested ligands for Vδ1 TCR-expressing γδ T cells, the major γδ lymphocyte population in epithelial tissues. We crystallized a Vδ1 TCR in complex with CD1d and the self-lipid sulfatide, revealing the unusual recognition of CD1d by germline Vδ1 residues spanning all complementarity-determining region (CDR) loops, as well as sulfatide recognition separately encoded by nongermline CDR3δ residues. Binding and functional analysis showed that CD1d presenting self-lipids, including sulfatide, was widely recognized by gut Vδ1+ γδ T cells. These findings provide structural demonstration of MHC-like recognition of a self-lipid by γδ T cells and reveal the prevalence of lipid recognition by innate-like T cell populations., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The T cell repertoire comprises αβ and γδ T cell lineages. Although it is established how αβ T cell antigen receptors (TCRs) interact with antigen presented by antigen-presenting molecules, this is unknown for γδ TCRs. We describe a population of human Vδ1(+) γδ T cells that exhibit autoreactivity to CD1d and provide a molecular basis for how a γδ TCR binds CD1d-α-galactosylceramide (α-GalCer). The γδ TCR docked orthogonally, over the A' pocket of CD1d, in which the Vδ1-chain, and in particular the germ line-encoded CDR1δ loop, dominated interactions with CD1d. The TCR γ-chain sat peripherally to the interface, with the CDR3γ loop representing the principal determinant for α-GalCer specificity. Accordingly, we provide insight into how a γδ TCR binds specifically to a lipid-loaded antigen-presenting molecule.

CD1d-restricted T (natural killer T; NKT) cells are important for controlling herpesvirus infections. Interestingly, herpes simplex virus (HSV) can down-regulate CD1d-mediated activation of NKT cells. We have previously shown that the Thr322 residue in the cytoplasmic tail of human CD1d is important for its intracellular trafficking and functional expression. We proposed that the phosphorylation of T322 is a signal for CD1d lysosomal targeting and subsequent degradation. In the current study, we generated dual mutants by substituting the T322 and S323 residues of wild-type (WT) CD1d with Ala (non-phosphorylatable) or Asp (mimicking phosphorylation) and ectopically expressed them in human embryonic kidney 293 cells. We found that the surface expression levels of the CD1d mutants was in this order: T322AS323A > WT > T322A > S323A > S323D > T322D > T322DS323D. Our results therefore suggest that mimicking the phosphorylation of both T322 and S323 has a cumulative negative effect on the functional expression of CD1d. As previously reported, we also found that upon an HSV infection, antigen presentation by WT CD1d is reduced and the CD1d molecule is degraded. Interestingly, the T322A/S323A double mutation inhibited CD1d degradation and rescued CD1d-mediated antigen presentation following an HSV-1 infection. This suggests that the T322/S323 dyad may be phosphorylated, which then targets CD1d for lysosomal degradation post-infection as a means of immune evasion, explaining (at least in part) the reduced antigen presentation observed. Hence, our findings strongly suggest that T322 and S323 form a dual residue motif that can regulate the functional expression of CD1d during a viral infection., (© 2013 John Wiley & Sons Ltd.)

Invariant natural killer T cells (iNKT cells) are restricted by CD1d molecules and activated upon CD1d-mediated presentation of glycolipids to T cell receptors (TCRs) located on the surface of the cell. Because the cytokine response profile is governed by the structure of the glycolipid, we sought a method for labeling various glycolipids to study their in vivo behavior. The prototypical CD1d agonist, α-galactosyl ceramide (α-GalCer) 1, instigates a powerful immune response and the generation of a wide range of cytokines when it is presented to iNKT cell TCRs by CD1d molecules. Analysis of crystal structures of the TCR-α-GalCer-CD1d ternary complex identified the α-methylene unit in the fatty acid side chain, and more specifically the pro-S hydrogen at this position, as a site for incorporating a label. We postulated that modifying the glycolipid in this way would exert a minimal impact on the TCR-glycolipid-CD1d ternary complex, allowing the labeled molecule to function as a good mimic for the CD1d agonist under investigation. To test this hypothesis, the synthesis of a biotinylated version of the CD1d agonist threitol ceramide (ThrCer) was targeted. Both diastereoisomers, epimeric at the label tethering site, were prepared, and functional experiments confirmed the importance of substituting the pro-S, and not the pro-R, hydrogen with the label for optimal activity. Significantly, functional experiments revealed that biotinylated ThrCer (S)-10 displayed behavior comparable to that of ThrCer 5 itself and also confirmed that the biotin residue is available for streptavidin and antibiotin antibody recognition. A second CD1d agonist, namely α-GalCer C20:2 4, was modified in a similar way, this time with a fluorescent label. The labeled α-GalCer C20:2 analogue (11) again displayed functional behavior comparable to that of its unlabeled substrate, supporting the notion that the α-methylene unit in the fatty acid amide chain should be a suitable site for attaching a label to a range of CD1d agonists. The flexibility of the synthetic strategy, and late-stage incorporation of the label, opens up the possibility of using this labeling approach to study the in vivo behavior of a wide range of CD1d agonists.

The mechanism of bone marrow failure (BMF) in paroxysmal nocturnal hemoglobinuria (PNH) is not yet known. Because in PNH the biosynthesis of the glycolipid molecule glycosylphosphatidylinositol (GPI) is disrupted in hematopoietic stem and progenitor cells by a somatic mutation in the PIG-A gene, BMF might result from an autoimmune attack, whereby T cells target GPI in normal cells, whereas PIG-A mutant GPI-negative cells are spared. In a deliberate test of this hypothesis, we have demonstrated in PNH patients the presence of CD8(+) T cells reactive against antigen-presenting cells (APCs) loaded with GPI. These T cells were significantly more abundant in PNH patients than in healthy controls; their reactivity depended on CD1d expression and they increased upon coculture with CD1d-expressing, GPI-positive APCs. In GPI-specific T cells captured by CD1d dimer technology, we identified, through global T-cell receptor α (TCRα) analysis, an invariant TCRVα21 sequence, which was then found at frequencies higher than background in the TCR repertoire of 6 of 11 PNH patients. Thus, a novel, autoreactive, CD1d-restricted, GPI-specific T-cell population, enriched in an invariant TCRα chain, is expanded in PNH patients and may be responsible for BMF in PNH.

Isoglobotrihexosylceramide (iGb3) has been identified as a potent CD1d-presented self-antigen for mouse invariant natural killer T (iNKT) cells. The role of iGb3 in humans remains unresolved, however, as there have been conflicting reports about iGb3-dependent human iNKT-cell activation, and humans lack iGb3 synthase, a key enzyme for iGb3 synthesis. Given the importance of human immune responses, we conducted a human-mouse cross-species analysis of iNKT-cell activation by iGb3-CD1d. Here we show that human and mouse iNKT cells were both able to recognise iGb3 presented by mouse CD1d (mCD1d), but not human CD1d (hCD1d), as iGb3-hCD1d was unable to support cognate interactions with the iNKT-cell TCRs tested in this study. The structural basis for this discrepancy was identified as a single amino acid variation between hCD1d and mCD1d, a glycine-to-tryptophan modification within the α2-helix that prevents flattening of the iGb3 headgroup upon TCR ligation. Mutation of the human residue, Trp153, to the mouse ortholog, Gly155, therefore allowed iGb3-hCD1d to stimulate human iNKT cells. In conclusion, our data indicate that iGb3 is unlikely to be a major antigen in human iNKT-cell biology., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Together with peptides, T lymphocytes respond to hydrophobic molecules, mostly lipids, presented by the non-classical CD1 family (CD1a-e). These molecules have evolved complex and diverse binding grooves in order to survey different cellular compartments for self and exogenous antigens, which are then presented for recognition to T-cell receptors (TCRs) on the surface of T cells. In particular, most CD1d-presented antigens are recognized by a population of lymphocytes denominated natural killer T (NKT) cells, characterized by a strong immunomodulatory potential. Among NKT cells, two major subsets (type I and type II NKT cells) have been described, based on their TCR repertoire and antigen specificity. Here we review recent structural and biochemical studies that have shed light on the molecular details of CD1d-mediated antigen recognition by type I and II NKT cells, which are in many aspects distinct from what has been observed for peptide major histocompatibility complex-reactive TCRs., (© 2012 John Wiley & Sons A/S.)

Natural killer T cells (NKT cells) are divided into type I and type II subsets on the basis of differences in their T cell antigen receptor (TCR) repertoire and CD1d-antigen specificity. Although the mode by which type I NKT cell TCRs recognize CD1d-antigen has been established, how type II NKT cell TCRs engage CD1d-antigen is unknown. Here we provide a basis for how a type II NKT cell TCR, XV19, recognized CD1d-sulfatide. The XV19 TCR bound orthogonally above the A' pocket of CD1d, in contrast to the parallel docking of type I NKT cell TCRs over the F' pocket of CD1d. At the XV19 TCR-CD1d-sulfatide interface, the TCRα and TCRβ chains sat centrally on CD1d, where the malleable CDR3 loops dominated interactions with CD1d-sulfatide. Accordingly, we highlight the diverse mechanisms by which NKT cell TCRs can bind CD1d and account for the distinct antigen specificity of type II NKT cells.

Glycolipids presented by the major histocompatibility complex (MHC) class I homolog CD1d are recognized by natural killer T cells (NKT cells) characterized by either a semi-invariant T cell antigen receptor (TCR) repertoire (type I NKT cells or iNKT cells) or a relatively variable TCR repertoire (type II NKT cells). Here we describe the structure of a type II NKT cell TCR in complex with CD1d-lysosulfatide. Both TCR α-chains and TCR β-chains made contact with the CD1d molecule with a diagonal footprint, typical of MHC-TCR interactions, whereas the antigen was recognized exclusively with a single TCR chain, similar to the iNKT cell TCR. Type II NKT cell TCRs, therefore, recognize CD1d-sulfatide complexes by a distinct recognition mechanism characterized by the TCR-binding features of both iNKT cells and conventional peptide-reactive T cells.

A library of dimeric CD1d ligands, containing two α-galactosyl ceramide (α-GalCer) units linked by spacers of varying lengths has been synthesised. The key dimerisation reactions were carried out via copper-catalysed click reactions between a 6"-azido-6"-deoxy-α-galactosyl ceramide derivative and various diynes. Each α-GalCer dimer was tested for its ability to stimulate iNKT cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Huisgen [3+2] dipolar cycloaddition of 6″-azido-6″-deoxy-α-galactosyl ceramide 11 with a range of alkynes (or a benzyne precursor) yielded a series of triazole-containing α-galactosyl ceramide (α-GalCer) analogues in high yield. These α-GalCer analogues and the precursor azide 11 were tested for their ability to activate iNKT cells and stimulate IL-2 cytokine secretion in vitro, and IFN-γ and IL-4 cytokine secretion in vivo. Some of these analogues, specifically 11, 12b, 12f and 13, were more potent IL-2 stimulators than the prototypical CD1d agonist, α-GalCer 1. In terms of any cytokine bias, most of the triazole-containing analogues exhibited a small Th2 cytokine-biasing response relative to that shown by α-GalCer 1. In contrast, the cycloaddition precursor, namely azide 11, provided a small Th1 cytokine-biasing response., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Aim: To create a method of detecting typeII natural killer T (NKT) cells of mice., Methods: Biotinylated mouse CD1d monomers were mixed with sulfatide at a molar ratio of 1:3 (protein:lipid) and incubated at room temperature overnight, and then 80 μg of streptavidin-PE was added into 200 μg of the CD1d-sulfatide mixture and incubated at room temperature for 4 h to get sulfatide/CD1d tetramer. Flow cytometry was used to detect the percentage of typeII NKT cells in mononuclear cells (MNCs) of lung and spleen of normal mice, as well as the percentage of typeII NKT cells in spleen MNCs of mice after stimulated with sulfatide., Results: In normal mice, the percentage of typeII NKT cells accounted for (0.875±0.096)% and (1.175±0.263)% in MNCs of spleen and lung; the percentage in spleen MNCs after activated with sulfatide was (2.75±0.603)%, which significantly increased as compared with that in normal mice (P<0.01)., Conclusion: Sulfatide-loaded CD1d tetramer is an effective method of detecting typeII NKT cells in mice.

In the present study, surface CD1d, which is involved in immune cell interactions, was assessed for effects on hematopoiesis. Mouse BM hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) express CD1d. The numbers and cycling status of HPCs in the BM and spleen of different strains of cd1d(-/-) mice were enhanced significantly, suggesting that CD1d is a negative regulator of HPCs. In support of this, CD1d was required for the SCF and Flt3 ligand synergistic enhancement of CSF induction of HPC colony formation and for HPC response to myelosuppressive chemokines. Colony formation by immature subsets of HPCs was greatly enhanced when normal, but not cd1d(-/-), BM cells were pretreated with CD1d Abs in vitro. These effects required the full CD1d cytoplasmic tail. In contrast, long-term, but not short-term, repopulating HSC engraftment was impaired significantly, an effect that was minimally influenced by the presence of a truncated CD1d cytoplasmic tail. Pretreatment of normal BM cells with CD1d Abs greatly enhanced their engraftment of HSCs. The results of the present study implicate CD1d in a previously unrecognized regulatory role of normal and stressed hematopoiesis.

Invariant natural killer T (iNKT) cells are restricted by the non-polymorphic MHC class I-like protein, CD1d, and activated following presentation of lipid antigens bound to CD1d molecules. The prototypical iNKT cell agonist is α-galactosyl ceramide (α-GalCer). CD1d-mediated activation of iNKT cells by this molecule results in the rapid secretion of a range of pro-inflammatory (Th1) and regulatory (Th2) cytokines. Polarization of the cytokine response can be achieved by modifying the structure of the glycolipid, which opens up the possibility of using CD1d agonists as therapeutic agents for a range of diseases. Analysis of crystal structures of the T-cell receptor-α-GalCer-CD1d complex led us to postulate that amide isosteres of known CD1d agonists should modulate the cytokine response profile upon iNKT-cell activation. To this end, we describe the synthesis and biological activity of amide analogues of α-GalCer and its non-glycosidic analogue threitol ceramide (ThrCer). All of the analogues were found to stimulate murine and human iNKT cells by CD1d-mediated presentation to varying degrees; however, the thioamide and carbamate analogues of ThrCer were of particular interest in that they elicited a strongly polarized cytokine response (more interferon-gamma (IFN-γ), no interleukin-4 (IL-4)) in mice. While the ThrCer-carbamate analogue was shown to transactivate natural killer (NK) cells, a mechanism that has been used to account for the preferential production of IFN-γ by other CD1d agonists, this pathway does not account for the polarized cytokine response observed for the thioamide analogue.