Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α-galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly proinflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here, we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α-galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6″ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings and suggested basic principles for capturing useful properties of sphinganine analogs of synthetic iNKT cell activators in the design of immunotherapeutic agents.

Considering the responses of selected C6″-substituted compounds in human cell lines and the strongly biased Th1 response elicited by the sphinganine-containing AH03-1, we have explored whether combining a C6″ substitution with a sphinganine variant of α-GalCer could provide useful synergistic effects. Herein we report studies on a α-GalCer analog, designated AH10-7, which incorporates a C6″ hydrocinnamoyl ester and lacks the C4′-OH of the sphingoid base. Using a combination of in vitro studies and in vivo analysis with wild-type and partially humanized mice, we found that the dual modifications in AH10-7 led to preservation of substantial potency in both mouse and human models of iNKT cell activation while maintaining the Th1-biasing property of other sphinganine derivatives. Structural studies by X-ray crystallography and in silico modeling provided a mechanistic basis for the effect of the C6″ substitution on enhancing presentation of AH10-7 by human CD1d. Our results, along with another recently published study of combining C6″ substitutions with other Th1-biasing modifications (), provide a rare example of two separate glycolipid modifications that synergize to create an analog of KRN7000 with potentially useful properties, suggesting a new approach to rational design of iNKT cell T cell receptor (TCR) ligands.

The first Th1-biasing compound reported was α-C-GalCer ( Figure 1 A), which showed marked superiority to KRN7000 in mouse cancer and infection models (). However, in studies using cell culture models of human iNKT cell responses, α-C-GalCer has been found to be only weakly stimulatory, suggesting that it may not be suitable for development as a human therapeutic (). Another strongly Th1-biasing iNKT cell agonist that was previously identified is the aminodiol (sphinganine) analog (AH03-1 in Figure 1 A) of KRN7000, which has activities similar to α-C-GalCer in mouse models (). AH03-1 and other related sphinganine analogs have generally been found to be highly active in vivo in mice or in vitro with mouse cells (), although two previous studies found that they were not efficient activators in cell culture models of human iNKT cell responses (). Among the more recently reported Th1-biasing analogs of α-GalCer are those in which the sugar has been modified by C6″-substituted amides, carbamates, and ureas. Some, such as NU-α-GalCer and PyrC-α-GalCer ( Figure 1 A), show promising anti-tumor activity in mice and elicit a Th1-biased response in studies using human iNKT cells in vitro ().

Despite the potent immune activating properties of KRN7000, there has been relatively limited success to date in advancing it into clinical use. Small phase 1 trials in human cancer patients have shown, at best, limited evidence of clinical benefit (), and there are features of the response to KRN7000 in vivo that may render it suboptimal for tumor immunotherapy and other potential applications (). A major issue in this regard is the tendency for KRN7000 to elicit high levels of both T helper 1 (Th1) and Th2 associated cytokines, which may have directly conflicting activities leading to ineffective and unpredictable immune responses. This problem has been addressed through the development of structural analogs of KRN7000 that stimulate iNKT cell responses that are more biased toward purely Th1 or Th2 cytokine predominance (). In general, analogs that bias toward interferon-γ (IFNγ) and other Th1-type cytokines following iNKT cell activation have been associated with favorable therapeutic effects in mouse models of cancer and infectious diseases (), whereas Th2-biasing analogs may be more suitable for treatment of autoimmune or inflammatory conditions (). Thus, it is well recognized that identifying ligands that selectively elicit Th1 or Th2 cytokines will likely be crucial for effective therapeutic applications of iNKT cell activation ().

Invariant natural killer T (iNKT) cells are a subset of unconventional T cells that participate in both adaptive and innate immunity (). A major feature that sets them apart from conventional T cells, which recognize peptides presented by major histocompatibility complex (MHC) molecules, is their recognition of lipid-based antigens presented by the MHC class I-like CD1d protein (). Since the discovery over two decades ago that natural forms of α-galactosylceramide (α-GalCer) known as agelasphins and their synthetic analogs are potent stimulators of iNKT cell responses with anti-cancer activities (), there has been increasing interest in incorporating iNKT cell activators into strategies for immunotherapy and vaccination (). The first α-GalCer developed from systematic structure-activity relationship studies was KRN7000 ( Figure 1 A) (). In mouse models, KRN7000 also showed potent anti-tumor properties, as well as considerable promise for the treatment of infectious and autoimmune diseases ().

The in silico docking procedure provided structures from which binding energies could be calculated using a hybrid quantum mechanics/molecular mechanics (QM/MM) protocol (). The computed binding energies ( Figure 7 E) correlated with experimental bioactivity trends ( Figures 2 and 3 ). Consistent with the observed trends in biological potencies, AH03-1 was found to have a less favorable binding energy relative to KRN7000 in the human versus mouse ternary complex ( Figure 7 E). Specifically, the sphingoid base in AH03-1 undergoes a displacement due to the absence of the 4′-OH that results in a net reduction of stabilizing van der Waals contacts with Tyr73 from CD1d. In contrast, the glycolipids with carbohydrate modifications (AH10-7 and AH15-1) were predicted to have more favorable binding in either the human or mouse system, suggesting that functionally relevant contacts realized through carbohydrate modification compensated for the lost interactions of the sphingoid base 4′-OH. In this manner, specific interactions established by the C6″-hydrocinnamoyl moiety improved binding energetics and correlated with the increased potency of AH10-7 for hCD1d presentation.

The crystal structure of the 2C12 TCR-mCD1d-AH10-7 ternary complex provided an opportunity to use molecular modeling to further explore how the sphingoid base and sugar modifications synergize to generate the observed effects of AH10-7 on iNKT cell activation. We used grid-based ligand docking simulations to examine the interactions of similar ligands with and without the 4′-OH (KRN7000 and AH03-1) and the C6″ modification (AH15-1 and AH10-7). As a validation of this approach, we showed that the simulated binding pose of AH10-7 reproduced well the 2C12 TCR-mCD1d-AH10-7 ternary complex structure obtained by X-ray crystallography ( Figure 7 A). Docking also predicted that this ligand and its phytosphingosine congener AH15-1 adopt a similar binding mode in complexes with hCD1d and the iNKT TCR ( Figure S3 ). As in the experimentally determined structure ( Figure 5 ), the most favored docking poses modeled for AH10-7 in the ternary mouse complex showed that Lys65, His68, and Met162 formed the sides, and Met69 the floor of a cleft on mouse CD1d above the A′ pocket to accommodate the hydrocinnamoyl substituent ( Figure 7 A). This cleft provides a hydrophobic shelter for the hydrocinnamoyl group that enables π-cation and π-π interactions with the side chain ammonium of mCD1d-Lys65 and imidazole of mCD1d-His68. By contrast, the favored poses of AH10-7 and AH15-1 modeled in the human ternary complex interdigitated the phenyl of the hydrocinnamoyl group between hCD1d-His68 and hCD1d-Trp153 to potentially realize π-π stacking interactions with both of these residues ( Figures 7 B and S3 ).

(A–D) QM/MM structures of AH10-7 (red), AH03-1 (green), and KRN7000 (blue). The crystallographic pose of AH10-7 is also shown in black in (A). H-bonding, ion-π stacking, and π-π interactions are indicated by yellow, green, and cyan dashed lines, respectively. (A) and (C) correspond with the mCD1d-TCR complexes, while (B) and (D) correspond with hCD1d-TCR complexes.

To directly assess the binding affinity of the iNKT cell TCR to the mCD1d protein loaded with AH10-7 versus KRN7000, we analyzed binding by surface plasmon resonance using the same soluble NKT TCR and mCD1d proteins used to generate the crystal structures of the ternary complexes ( Figure 6 A). Similar binding affinities of the TCR were observed for both AH10-7 and KRN7000 loaded mCD1d complexes (K∼100–110 nM), which was consistent with the close superposition of the galactose ring and other components of the TCR docking site in the crystal structures ( Figure 6 B). Comparison of AH10-7 with other C6″-substituted α-GalCer derivatives previously reported to show high levels of iNKT cell stimulatory activity indicated that all of these preserved a similar positioning of the galactose ring (and Figure 6 C), underscoring the importance of this feature in maintaining TCR binding affinity.

(C) Superposition of the 2C12 TCR-mCD1d-AH10-7, 2C12 TCR-mCD1d-BnNH-GSL, and 2C12 TCR-mCD1d-NU-α-GalCer. For clarity, only mCD1d (light gray) from the 2C12 TCR-mCD1d-AH10-7 ternary complex is shown. The lipid-antigens AH10-7, BnNH-GSL-1′, and NU-α-GalCer are colored in black, pale green, and light blue, respectively.

(B) Superposition of the CD1d/glycolipid complexes from the 2C12 TCR-mCD1d-AH10-7 and 2C12 TCR-mCD1d-KRN7000 ternary structures. The superposition of both structures is based on the Cα alignment of the mCD1d. In the 2C12 TCR-mCD1d-AH10-7 complex, mCD1d (cartoon) and AH10-7 (sticks) are colored in black, while the 2C12 TCR-mCD1d-KRN7000, mCD1d, and KRN7000 are colored in wheat.

(A) Affinity measurements of the 2C12 αβ TCR against mCD1d-AH10-7 and mCD1d-KRN7000. The relative binding affinities for each TCR were measured by surface plasmon resonance. Error bars show SEM of two replicates (n = 2). RU, response units.

The interactions of the 2C12 iNKT TCR with KRN7000 and AH10-7 bound in mCD1d were exclusively mediated by the TCR α chain. In both ternary complexes, the galactose was located in a similar position and interacted exclusively with the CDR1α and CDR3α loops, with the C2″ and C3″ hydroxyl groups of the sugar forming hydrogen bonds with residues Arg95α and Asn30α, respectively ( Figure 5 C). It was noteworthy that the hydrocinnamoyl ester moiety of AH10-7 was positioned over the A′ pocket of mCD1d between Met69 and Met162 and interacted with Thr159 via van der Waals contacts. This pointed to a role for the C6″ substitution in strengthening the binding of AH10-7 to mCD1d and stabilizing the position of the galactosyl head group for TCR recognition.

The distinct electron density not accounted for by polypeptide residues in the mCD1d-binding groove enabled us to model unambiguously AH10-7 and KRN7000 in both ternary complexes ( Figures 5 C and S1 ). For both glycolipids, the hydrophobic N-acyl and sphingoid base chains were positioned deep within the A′ and F′ pockets of mCD1d, respectively, whereas the galactose polar moiety largely protruded out of the binding groove to interact with the TCR. Although the sphinganine of AH10-7 and phytosphingosine of KRN7000 adopted similar positions within the F′ pockets, we noted that the 4′-OH in the phytosphingosine chain enabled an additional hydrogen bond to Asp80 of mCD1d ( Figure 5 B). The static crystal structures did not detect rearrangement of the overall positioning of the glycolipid or significant impact on the mCD1d protein structure as a result of the presence or absence of this additional bond. However, as indicated by modeling studies (see below), the impact of removing the 4′-OH may be greater in hCD1d, potentially contributing to the apparent reduction in human iNKT cell responses promoted by AH03-1.

The overall docking mode adopted by the 2C12 iNKT TCR to recognize mCD1d presenting KRN7000 and AH10-7 was conserved and was similar to the one previously observed in other crystal structures of iNKT TCR-CD1d-α-GalCer ternary complexes () ( Figures 5 B and S2 ). In both complexes, the 2C12 iNKT TCR adopted a parallel docking mode (docking angle of ∼15°) over the F′ pocket of mCD1d. Upon 2C12 iNKT TCR ligation, the total buried surface area (BSA) of the TCR at the interface in both ternary complexes was ∼800 Å. The TCR α chain contributed the most to the BSA (60% of total), and this was mainly through the complementarity-determining region (CDR) 3α (44% of BSA). Here, the CDR3α bridged the α1- and α2-helices of mCD1d, with Asp94α and Arg95α hydrogen bonded to Arg79 and Asp80 ( Figure 5 B, left). The contribution of the TCR β chain to the binding interface was more limited and was evenly shared, mainly by the CDR2β, CDR3β, and two framework residues (Tyr48β and Glu56β) ( Figure 5 B, right), each accounting for a 10%–15% BSA ( Figure S2 ).

In order to gain structural insights into the molecular recognition of AH10-7 in comparison with KRN7000, we generated complexes in vitro of mCD1d loaded with these glycolipids and co-crystallized them with the 2C12 iNKT TCR () We used X-ray crystallography to determine the three-dimensional structures of these ternary complexes to 3.2 Å resolution for AH10-7 and 2.6 Å for KRN7000 ( Figure 5 A and Table S1 ). In both complexes, the electron density at the molecular interface between the mCD1d-glycolipid complexes and the 2C12 iNKT TCR was unambiguous. Most notably, distinct electron density corresponding with the in vitro loaded glycolipid antigens emerged from within the A′- and F′-portals of mCD1d and protruded out from the antigen-binding cleft ( Figures 5 A and S1 ).

(C) Molecular interactions of the 2C12 αβ TCR with the AH10-7 and KRN7000 glycolipid antigens. The AH10-7 and KRN7000 ligands are shown as sticks and colored in black and pale yellow, respectively. Hydrogen bond interactions are shown as magenta dashed lines (see also Figures S1 and S2 ).

(B) Molecular interactions of the 2C12 TCR α and β chains with mCD1d. The CDR loops are colored as in (A), and the β-chain framework residues are colored in pale green (structures shown are from the 2C12 αβ TCR-mCD1d-AH10-7 complex and are essentially identical in the KRN7000-containing complex).

The preferential lipid raft localization of CD1d-α-GalCer complexes formed with AH10-7 suggested that this compound was likely to stimulate a Th1 cytokine bias in vivo, such as that previously shown for α-C-GalCer and AH03-1 in mice (). Measurement of serum levels of IFNγ and IL-4 after glycolipid injection into mice showed a significant enhancement of IFNγ without increases in IL-4 for AH10-7 compared with KRN7000 ( Figure 4 B). In contrast, AH15-1, which retains the 4′-OH group in the sphingoid base, produced levels of both cytokines that were similar to those elicited by KRN7000. The altered ratio of IFNγ to IL-4 stimulated by AH10-7 was consistent with a Th1-biasing effect for this glycolipid that was significantly augmented compared with KRN7000 or AH15-1, although less pronounced than with AH03-1. Given the association of Th1-biasing properties with improved anti-cancer effects in mice (), we tested the impact of AH10-7 compared with KRN7000 in the mouse B16-F10 melanoma model of metastatic cancer (). This revealed strong anti-tumor activity of AH10-7 in both wild-type and hCD1d-KI mice ( Figure 4 C), which was at least as great as for KRN7000 in this model.

Previous studies have attributed a strong Th1-type cytokine bias to the sphinganine derivative AH03-1 in mice and correlated this with its presentation by CD1d proteins that localized preferentially to lipid raft microdomains in the plasma membrane of antigen-presenting cells (). We tested whether AH10-7 preserved the lipid raft localization by measuring the detergent sensitivity to elution of α-GalCer-mCD1d complexes from the surface of dendritic cells using a previously described flow cytometry-based assay () ( Figure 4 A). As previously shown, complexes of CD1d with the Th1-biasing sphinganine derivative AH03-1 showed greater resistance to detergent elution from the plasma membrane compared with KRN7000, consistent with strong localization to lipid raft microdomains. For comparison, the strongly Th2-biasing glycolipid α-GalCer C20:2 ( Figure 1 A) showed rapid and nearly complete elution from the plasma membrane. AH10-7 behaved similarly to AH03-1 in this assay, showing strong lipid raft localization of its presentation. This was attributable to its sphinganine modification, since a phytosphingosine variant of AH10-7 (AH15-1, Figure 1 B) behaved no differently than KRN7000 in this assay ( Figure 4 A).

Asterisks indicate significant differences compared with KRN7000 for (A) and (B), or for the indicated comparisons in (C). *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001 (ANOVA with Dunnet post-test for multiple comparisons).

(C) In vivo antitumor activity of glycolipid AH10-7. Wild-type C57BL/6 or hCD1d-KI mice were injected i.v. with 3 × 10 5 B16-F10 F10 melanoma cells. Two days later, groups of 11–15 mice received i.v. injections of KRN7000, AH10-7 (4 nmol), or vehicle. Animals were sacrificed 15 days later, lungs were removed, and the numbers of tumor nodules were counted visually. Images on the left are representative lungs from wild-type C57BL/6 or hCD1d-KI from each of the treatment groups. Graphs on the right show mean values ±1 SE for mice treated with vehicle or glycolipids in the wild-type mice (filled bars) or hCD1d-KI mice (hatched bars). This experiment was carried out twice in wild-type mice, and once in hCD1d-KI mice.

(B) Serum cytokine levels following systemic injection of glycolipids. Wild-type C57BL/6 mice were injected intravenously (i.v.) with 4 nmol of each glycolipid or control vehicle (N = 3 mice per group). Serum was collected at 2 and 24 hr post injection. Mean values ±1 SE for IFNγ levels at 24 hr and IL-4 levels at 2 hr are shown (solid bars), and also the calculated ratios of these cytokines for each glycolipid (hatched bars). Results shown are representative of three separate experiments.

(A) Enhanced plasma membrane lipid raft association of CD1d proteins presenting 4′-deoxyphytosphingosine variant AH10-7. Mouse JAWS II dendritic cells were preincubated with glycolipids to allow loading of CD1d molecules and then labeled by cell surface binding of fluorochrome conjugated mCD1d/α-GalCer complex-specific mAb L363. Cells were then treated with 0.05% Triton X-100, and elution of cell surface CD1d/glycolipid complexes was monitored over time by flow cytometry. Starting values for mean fluorescence intensity (MFI) were normalized to 100%, and the change in MFI values over time are plotted for cells loaded with KRN7000 and the other indicated glycolipids. Values at 32.5 s post detergent addition (vertical dashed lines) for a minimum of four replicate samples were averaged, and the mean ± 1 SE for percentage residual MFI is plotted in the bar graph on the right. Results shown are representative of three separate experiments.

We further investigated the differential presentation of AH10-7 and AH03-1 by hCD1d using fully human cell culture systems. Stimulation of human iNKT cell clones with the glycolipids in the presence of human monocyte-derived DCs or hCD1d-transfected HeLa cells showed a significant diminution of activity compared with KRN7000 with the sphinganine derivative AH03-1, which was partially reversed by the C6″ substitution in AH10-7 ( Figure 2 C). In addition, we analyzed proliferation and expansion of primary iNKT cells in human peripheral blood mononuclear cell (PBMC) cultures from four normal blood donors, which showed a similarly enhanced activity of AH10-7 compared with the minimal expansion and proliferation induced by AH03-1 ( Figure 3 ). Taken together, these in vitro studies indicated that the 4′-OH group of the sphingoid base had an important influence on presentation of α-GalCer by hCD1d but not mCD1d, and that the defect in presentation of a sphinganine derivative by hCD1d could be significantly overcome by incorporation of the C6″ hydrocinnamoyl group in AH10-7.

(B) Graphs depict the fold expansion of iNKT cell percentages at the end of culture relative to the unstimulated sample (left), and the percentage of iNKT cells that have proliferated based on CFSE dilution (right). Each scatter point represents an independent donor sample, and bars show mean values for three or four donors.

(A) Carboxyfluorescein succinimidyl ester (CFSE)-labeled human PBMCs were pulsed with respective lipids and cultured for 7 days in the presence of 50 U/mL human IL-2. Representative fluorescence-activated cell sorting (FACS) plots from one donor show α-GalCer loaded CD1d tetramer-positive iNKT cells (red ovals) and tetramer-negative CD3 + T cells (blue rectangular regions), followed by the histograms of cells that have proliferated based on CFSE dilution. Blue histograms are tetramer-negative CD3 + T cells, and red histograms are tetramer-positive iNKT cells.

These findings with a canonical iNKT cell line expressing a single clonal antigen receptor (TCR) indicated that the addition of the C6″ hydrocinnamoyl group partially restored hCD1d presentation of a sphinganine derivative of KRN7000. To extend this to iNKT cells with more heterogeneous TCRs, we sorted iNKT cells from the spleens of both wild-type and hCD1d-KI mice and fused these to thymoma line BW5147αto generate polyclonal iNKT cell lines (). Similar to the results with the clonal mouse iNKT cell line, we found that a polyclonal iNKT cell line from wild-type mice showed nearly equivalent responses to the three glycolipids when presented by mCD1d, while a marked reduction in responses was seen for AH03-1 but not AH10-7 when presented by hCD1d ( Figure 2 B, left). A similar trend, although slightly less pronounced, was observed for a polyclonal iNKT cell line derived from hCD1d-KI mice ( Figure 2 B, right).

Initial assessment of bioactivity was done using cell culture with a mouse iNKT cell hybridoma (DN3A4-1.2) and bone marrow-derived dendritic cells (DCs) (). To assess presentation of the glycolipids by mouse (m) versus human (h) CD1d, we used bone marrow-derived DCs from either wild-type or human CD1d knock-in (hCD1d-KI) mice. The latter, in which the coding sequence of the mouse CD1D gene has been replaced by the orthologous human CD1D sequence, has been previously described as a partially humanized mouse model for the study of iNKT cell responses (). Measurement of interleukin (IL)-2 release as an indicator of iNKT cell activation showed similar potency over a wide range of concentrations for KRN7000 and both sphinganine derivatives (AH03-1 and AH10-7, Figure 2 A) when presented by mCD1d. In contrast, with hCD1d-expressing DCs, AH03-1 showed minimal ability to stimulate the mouse iNKT cell hybridoma, whereas AH10-7 retained partial activity relative to KRN7000.

Significant differences compared with response to KRN7000 in (B) and (C) are indicated by asterisks: *, p < 0.05; **, p < 0.01; ***, p < 0.001 (ANOVA with Dunnet post-test for multiple comparisons).

(C) Left: responses of human iNKT cell clone HDD5 co-cultured with human monocyte-derived dendritic cells (hMDDC) and 100 nM of the indicated glycolipids. Supernatants were harvested after 24 hr of culture for measurement of IFNγ levels. Right: responses of human iNKT cell clone HDD3 to 100 nM of the indicated glycolipids presented by HeLa cells transfected with hCD1d. Results shown are means ± 1 SE for triplicate samples, and are representative of three separate experiments.

(B) Polyclonal iNKT cell hybridoma lines derived from wild-type C57BL/6 mice (left, B6 iNKT) or from hCD1d KI mice (right, KI iNKT) were co-cultured with either wild-type BMDCs (top, filled bars) or hCD1d-KI BMDCs (bottom, hatched bars) plus 100 nM of the indicated glycolipids. Activation of iNKT hybridoma cells was determined by measurement of IL-2 secretion at 18 hr. Results shown are means ±1 SE for duplicate samples, and are representative of two separate experiments.

(A) Activation of mouse iNKT cell hybridoma DNA3.4–1.2 by KRN7000 or 4′-deoxy variants AH03-1 and AH10-7. The indicated concentrations of glycolipids were added to co-cultures of DNA3.4–1.2 cells with bone marrow-derived dendritic cells (BMDCs) from either wild-type C57BL/6 mice (top, mCD1d) or from hCD1d-KI mice (bottom, hCD1d). Activation of iNKT hybridoma cells was determined by measurement of IL-2 levels in culture supernatants by ELISA after 18 hr of culture. Symbols show means and error bars represent ±1 SE for triplicate values, and results shown are representative of five separate experiments.

To examine the impact of linking an aromatic group to the sugar C6″ in combination with removal of the 4′-OH of the phytosphingosine chain, we synthesized the sphinganine-containing compound AH10-7 and, for comparison, its phytosphingosine congener AH15-1, as outlined in Figure 1 B. Both glycolipids were prepared by the glycosylation of known protected forms) and) of their respective ceramides ( Figure 1 B). Thiophenyl-activated carbohydrate, used for both glycosylations, was prepared by esterification of a previously described, partially protected thiophenyl galactose (). The coupling reactions betweenandorgave high α-selectivity, and the resultant α-GalCers were deprotected under standard conditions to give AH10-7 and AH15-1.

Discussion

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Yuan W. Human CD1d knock-in mouse model demonstrates potent antitumor potential of human CD1d-restricted invariant natural killer T cells. Our analysis also took advantage of a hCD1d KI mouse model that more faithfully replicates many of the features of human iNKT cell responses in comparison with standard wild-type mice (). This mouse strain was generated by replacing the mouse genomic segment containing the exons encoding mCD1d with the homologous human genomic sequence (). Expression of hCD1d in tissues of these mice is similar to its physiologically normal pattern, and they develop a population of endogenous iNKT cells that closely mirrors the numbers, phenotype, and functions of these cells in humans. The use of iNKT cell hybridomas and primary bone marrow-derived DCs from these mice allowed us to acquire further support for the importance of the 4′-OH for effective responses to α-GalCer in the context of hCD1d presentation. Most notably, AH10-7 was at least as active as KRN7000 for inducing anti-tumor responses against experimental metastases of the B16-F10 melanoma line in both wild-type and hCD1d-KI mice, suggesting that the pairing of structural modifications used in the design of this compound may be a promising path for development of human therapeutics.

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Elewaut D. Enhanced TCR footprint by a novel glycolipid increases NKT-dependent tumor protection. A variety of structural and modeling analyses were employed to better understand the molecular basis for the effects of the two modifications to the α-GalCer structure. These studies revealed that, similar to the previously described structure of other C6″-substituted forms of α-GalCer bound to CD1d (), the aromatic group and its linker to the galactose formed contacts that held it close to the surface of CD1d. This most likely stabilized the position of the galactose of the bound AH10-7, which was oriented almost identically to the analogous structure with KRN7000 ( Figures 6 B and S1 ). The complexes of mCD1d with either of these glycolipids bound had almost identical affinities for at least one mouse iNKT cell TCR, as shown using a soluble form of the TCR in surface plasmon resonance studies ( Figure 6 A). This is consistent with the similar potency of KRN7000 and AH10-7 for activation of iNKT T cells with presentation by mCD1d, as observed experimentally ( Figure 2 ).