Abstract
α-Galactosylceramide (α-GalCer) is a prototypical antigen recognized by natural killer T (NKT) cells, a subset of T cells crucial for immune regulation. Despite its significance, the complex structure-activity relationship of α-GalCer and its analogs remains poorly understood, particularly in defining the structural determinants of NKT cell responses. In this study, we designed and synthesized potential immunomodulatory ligands targeting NKT cells, inspired by glycolipids derived from the gut symbiont Bacteroides fragilis. A series of α-GalCer analogs with terminal iso-branched sphinganine backbones was developed through rational modification of the acyl chain. Our results identified the C3′ hydroxyl group as a structural element that impairs glycolipid presentation by CD1d, as evidenced by reduced IL-2 secretion and weak competition with a potent CD1d ligand. Notably, among C3′-deoxy α-GalCer analogs, those containing an α-chloroacetamide group exhibited robust NKT cell activation with Th2 selectivity. Computational docking and mass spectrometry analyses further confirmed the substantial interaction of α-chloroacetamide analogs to CD1d. These findings underscore the potential of leveraging microbiota-derived glycolipid structures to selectively modulate NKT cell functions for therapeutic purposes.
Introduction
α-Galactosylceramide (α-GalCer) is a representative glycolipid antigen recognized by natural killer T (NKT) cells, a specialized T cell subset essential for immune homeostasis through rapid cytokine production1,2,3. The mechanism involves α-GalCer binding to CD1d, an MHC class I-like glycoprotein expressed on antigen-presenting cells such as dendritic cells4. This CD1d–glycolipid complex activates the T cell receptor (TCR) signaling pathway in NKT cells4,5,6. Structural variations in α-GalCer derivatives significantly influence the cytokine profile secreted by activated NKT cells, spanning Th1 cytokines (e.g., IFN-γ, TNF-α) to Th2 cytokines (e.g., IL-4, IL-10)7,8,9. Th1-promoting ligands have potential as antitumor agents10,11,12,13,14 or vaccine adjuvants15, while Th2-biased ligands are promising for autoimmune disease therapies16,17.
Since the discovery of KRN7000 (1), a potent immunostimulatory NKT cell ligand derived from the marine sponge Agelas mauritianus, structure-activity relationship (SAR) studies have focused on optimizing α-GalCer analogs to develop potent and selective glycolipids (Fig. 1a)7,8,9,18,19,20,21,22,23,24. Structural variants with two lipid chains–sphingoid chain and acyl chain–have been thoroughly explored as key determinants of the hydrophobic interactions between α-GalCers and CD1d binding grooves. For example, OCH (2), a truncated analog of KRN7000 (1) with a half-length sphingosine chain, induces Th2-skewed immune responses forming a weaker antigen–CD1d complex16,22. Notably, functional group-incorporated lipid chain analogs have demonstrated potent and selective NKT cell responses through non-covalent or covalent interactions at the binding interface. Examples include α-GalCer analogs with heteroaromatic moieties in the sphingosine backbone (3)25,26 or α-chloroacetamide anchoring moieties in the acyl chain (4)27, both of which elicit potent Th2-selective responses.
*Fig. 1: Structures of α-GalCer analogs.*
figure 1
a Chemical structures of α-GalCer analogs inducing different NKT cell responses–KRN7000 (1), OCH (2), α-GalCer with pyrazole sphingosine backbone (3), α-GalCer with chloroacetamide acyl chain (4), and two representative Bacteroides fragilis-derived α-GalCers (BfaGCs, 5–6). b Crystal structure of representative immunomodulatory BfaGC with mCD1d (PDB: 7M72). Two hydrophobic pockets (A′ pocket and F′ pocket) of CD1d are shaded in brown. The ligand is shown in green sticks, and Cys12 and Thr159 are shown in pink. Hydrogen bonding is indicated as a red dotted line. c Chemical structures of BfaGC (5) and three α-GalCer analogs (7–9) with iso-branched sphinganine backbone and rationally designed acyl chains. C3′ hydroxyl groups were highlighted in pink, and chloroacetamide moieties were shown in blue.
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We recently reported the total synthesis of 24 α-GalCer analogs inspired by Bacteroides fragilis glycolipids (BfaGCs), identified their natural structures, and demonstrated their immunomodulatory activities as distinct ligands of NKT cells (Fig. 1a)28. These BfaGC analogs reduced pro-inflammatory cytokine production, revealing their therapeutic potential for autoimmune diseases28,29,30. Our study identified the terminal iso-branching of the sphinganine backbone in BfaGC analogs, derived from dietary branched-chain amino acids, as a critical feature for immunomodulatory responses28. However, the impact of acyl chains on the NKT cell modulatory activity of iso-branched sphinganine analogs remains underexplored, necessitating further investigation into structural variants beyond naturally occurring microbial glycolipids.
To address this, we hypothesized that modifying the acyl chains could enhance the NKT cell immunomodulatory responses of natural BfaGC analogs with iso-branched sphinganines. Guided by the X-ray co-crystal structure of CD1d with a representative BfaGC, SB2217 (PDB: 7M72, Fig. 1b), we focused on hydroxyl groups in BfaGC lipid chains. Specifically, the unique C3′ hydroxyl group in the fatty acyl chain of BfaGC forms a hydrogen bond with the CD1d residue Thr159. Our previous research provided a clue that the C3′ hydroxyl group can be removed for NKT cell activation28. Notably, the removal of this hydroxyl group resulted in a modest enhancement of IL-2 secretion, giving prominence to further investigation28. Additionally, we examined interactions between the acyl chains and polar residues within the CD1d A′ pocket. Inspired by literature on acyl chain functionalization27,31,32,33,34,35,36,37,38, we explored incorporating covalent anchoring moieties into glycolipid structures to stabilize the CD1d–ligand complex and enhance potency.
Covalent chemical probes, which offer high binding affinity and prolonged action, have gained attention for their potential to improve therapeutic outcomes39,40,41. CD1d is a promising candidate for such probes due to the presence of Cys12 in its A′ pocket, which can serve as a covalent binding site27,34. Fujimoto et al. demonstrated that glycolipids bearing an α-chloroacetamide warhead form strong covalent bonds with the CD1d residue Cys1227. Based on these insights, we designed α-GalCer analogs incorporating α-chloroacetamide moieties in the acyl chain to achieve enhanced anchoring and ligand potency.
We initially synthesized three α-GalCer analogs (7–9) based on the natural ligand 5, featuring C16:0-equivalent acyl chains (Fig. 1c). Surprisingly, the C3′ hydroxyl group impaired α-GalCer presentation by CD1d and attenuated NKT cell activation. Conversely, introducing an α-chloroacetamide moiety significantly enhanced NKT cell activation by strengthening CD1d binding. Building on these findings, we synthesized six C3′-deoxy analogs (10–15) to optimize covalent binding moieties for robust immunomodulatory responses. Among these, analog 9, containing an α-chloroacetamide warhead at the C16:0-equivalent acyl chain, exhibited the most substantial NKT cell activation and a Th2-skewed cytokine profile. Mass spectrometry analysis confirmed the covalent binding potential of analog 9 to CD1d.
In conclusion, we developed highly Th2-selective NKT cell ligands inspired by the structure of microbiota-derived glycolipids. These ligands feature a terminal-branched sphinganine backbone and covalent warhead-incorporated acyl chain, representing a promising design strategy for therapeutic agents targeting immunomodulation.
Results and discussion
Chemical synthesis of α-GalCer analogs with B. fragilis-derived sphinganine backbone
To synthesize the designed α-GalCer analogs, we began with Garner’s aldehyde (16) as the starting material (Scheme 1). Diastereoselective addition of a vinyl group to 1642, followed by alcohol protection, yielded intermediate 18. Subsequent cleavage of the isopropylidene ring enabled the formation of a terminal *iso-*branched sphinganine backbone via olefin cross-metathesis of intermediate 19 with a branched terminal alkene. The resulting glycosyl acceptor (20) was then coupled with galactosyl iodide43, and subsequent debenzoylation afforded the α-galactosyl sphingoid intermediate (22).
Scheme 1: General synthetic scheme for α-GalCer analogs (7–9**)a.**
scheme 1
aReagents, conditions, and yields; (i) ref. [39]; (ii) benzoic acid, DCC, DMAP, DCM, 0 °C to r.t., 16 h, 97% as a crude; (iii) FeCl3.6H2O, DCM, r.t., 1 h, 80% over 2 steps; (iv) 13-methyltetradec-1-ene, Grubbs catalyst 2nd generation, DCM, 40 °C, 8 h, 58%; (v) 2,3,4,6-tetra-O-benzyl-α-d-galactopyranosyl iodide, TBAI, DIPEA, 4 Å molecular sieves, benzene, 70 °C, 5 h; (vi) NaOMe, THF, 0 °C to r.t., 3 h, 41% over 2 steps; (vii) HCl, EtOH, 70 °C, 3 h; (viii) fatty acid (hexadecenoic acid, S13, or S14), EDC.HCl, HOBt.H2O, DIPEA, DCM, 0 °C to r.t., 16–18 h, yields over 2 steps (76% for 23a; 45% for 24a; 83% for 24b); (ix) H2 (g, 1 atm), Pd(OH)2/C, DCM/MeOH (1:3, v/v), r.t., 18 h, 73%; (x) H2 (g, 10 atm), Pd(OH)2/C, DCM/MeOH (1:3, v/v), r.t., 17–23 h; and (xi) N-hydroxysuccinimide ester (S20), DMF, r.t., 4–5 h, yields over 2 steps (50% for 7; 43% for 9).
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Using intermediate 22 as the key precursor, we pursued divergent N-acylations with various fatty acids after removing the tert-butyloxycarbonyl (Boc) protecting group on the sphinganine chain. For analog 8, which features a linear C16:0 acyl chain without the C3′ hydroxyl group, amide coupling with hexadecanoic acid was performed. For other analogs (7 and 9) with functionalized acyl chains, fatty acids containing protected terminal amines were used to facilitate further modifications. Final deprotection via catalytic hydrogenation and incorporation of an α-chloroacetamide moiety yielded α-GalCer analogs 7 and 9, which include a covalent anchoring moiety, with and without the C3′ hydroxyl group, respectively. These synthesized α-GalCer analogs (5, 7–9) provide a platform to investigate the individual contribution of the C3′ hydroxyl group and the covalent anchoring moiety to NKT cell activation and cytokine modulation.
α-GalCer analog with a C3′-deoxy and chloroacetamide-containing fatty acyl chain elicits enhanced NKT cell activation
To evaluate the activity of the synthesized α-GalCer analogs, we assessed IL-2 production in primary murine hepatic mononuclear cells (HMNCs) upon stimulation. HMNCs were incubated with α-GalCer analogs for 48 h, and IL-2 secretion levels were quantified by enzyme-linked immunosorbent assay (ELISA) (Fig. 2a and Supplementary Fig. 1). For comparison, the α-GalCer KRN7000 (1) and the natural BfaGC ligand (5) were also tested. As shown in Fig. 2a, α-GalCer analogs 8 and 9 exhibited significantly higher IL-2 induction than their counterparts containing the C3′ hydroxyl group, 5 and 7, respectively. Because IL-2 secretion depends on antigen presentation by CD1d5, these results suggest that the removal of the C3′ hydroxyl group facilitated CD1d-loading. Notably, analog 9, which contains an α-chloroacetamide moiety, showed the highest IL-2 secretion, indicating the most substantial interaction with CD1d.
Fig. 2: Immunological evaluations of α-GalCer analogs (7–**9).**
figure 2
a IL-2 secretion from murine hepatic mononuclear cells (HMNCs) was measured after 48 h upon treatment of α-GalCer analogs (n = 2, biological replicates. *p < 0.05, **p < 0.01, ***p < 0.005, and ns: not significant by one-way ANOVA). Data are shown as mean ± SD; **b** IL-2 production of NKT cells was modulated by *α*-GalCer analogs. Each analog was pre-treated to HMNCs before 30 min of KRN7000 (**1**) treatment, and IL-2 secretion was measured after 72 h (*n* = 3, biological replicates. *p < 0.05, **p < 0.01, ****p < 0.0001, and ns: not significant by two-way ANOVA). Data are shown as mean ± SD; c overview of the binding mode of 7 and its location of the A′ pocket and F′ pocket in mCD1d (PDB: 7M72) using computational docking analysis. The structure of 7 is shown as green sticks. Thr159 and Cys12 in the A′ pocket of mCD1d are shown in pink.
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To further confirm CD1d-mediated NKT cell activation by the α-GalCer analogs, we performed competition studies using co-treatment with the potent CD1d ligand 1 and the designed analogs in murine HMNCs for 72 h (Fig. 2b). If the α-GalCer analogs were efficiently presented by CD1d, they would compete with 1, reducing its ability to activate NKT cells. The C3′-deoxy analogs 8 and 9 effectively inhibited 1-induced IL-2 secretion, whereas ligands 5 and 7 showed minimal competitive effects. Remarkably, analog 9 reduced more than half of the IL-2 secretion induced by 1, further supporting the role of the C3′-deoxy fatty acyl chain and the α-chloroacetamide moiety in optimal CD1d loading and subsequent TCR interaction.
To explore the molecular basis of CD1d binding of the α-GalCer analogs, we conducted computational docking analyses within the binding pocket of the murine CD1d (mCD1d) protein (PDB: 7M72, Fig. 2c). The docking result for 7 indicated that its C3′ hydroxyl group forms a hydrogen bond with Thr159 in the A′ pocket in contrast to the binding mode of C3′ deoxy analog KRN7000 (1) (Supplementary Fig. 2). Interestingly, analogs with C3′ hydroxyl group (7 and 9) showed reduced NKT cell activation, as reflected by the lower IL-2 secretion level. This result implies that the binding mode of glycolipid and CD1d Thr159 may affect the ternary interaction between CD1d–antigen complex and TCR. The docking analysis for 9 revealed that its α-chloroacetamide moiety is positioned near Cys12 in the A′ pocket (Supplementary Fig. 3), suggesting the potential for covalent bond formation between CD1d and the α-chloroacetamide group. This interaction likely underpins the strong NKT cell activation observed for analog 9.
Design and synthesis of structural variants of α-GalCers with C3′-deoxy fatty acyl chains
Building on the initial results from the first set of NKT cell ligands, we designed additional α-GalCer analogs 10–13 with C3′-deoxy fatty acyl chains (Fig. 3a). To investigate the impact of different functional groups, a propionamide moiety was introduced in analog 10 to compare its effect with the covalent α-chloroacetamide warhead. Shorter acyl chains were also incorporated in analogs 11–13 to explore the optimal position of terminal functional groups. Computational docking analyses were conducted to examine the binding modes of the designed analogs within the CD1d pocket (Fig. 3b, c and Supplementary Fig. 4). For analog 10, the propionamide-containing acyl chain fits into the A′ pocket, forming interactions with polar residues such as Gln14, Ser28, and Cys12 (Fig. 3b). Analog 12, featuring a C14:0-equivalent acyl chain, showed similar pocket occupancy to C16:0-length analog, indicating their effective binding to CD1d (Fig. 3c).
Fig. 3: Structures of C3′-deoxy α-GalCer analogs with B. fragilis-derived sphinganine backbone and functionalized acyl chains*.*
figure 3
a Chemical structures of *α-*GalCer analogs (8–13) with acyl chains equivalent to C16:0 and C14:0; b docking mode of 10 in the binding pocket of mCD1d (PDB: 7M72). The structure of 10 is shown as green sticks. Hydrophilic residues (Cys12, Gln14, Ser28) are shown in pink; c docking mode of 12 in the binding pocket of mCD1d (PDB: 7M72). The structure of 12 is shown as green sticks, while Cys12 is demonstrated in pink.
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The synthesis of C3′-deoxy analogs 10–13 followed a similar approach to the first set of α-GalCer analogs (Scheme 2). Starting from the key intermediate 22, the removal of protecting groups and subsequent N-acylations yielded intermediates 23b, 24b, and 24c. Catalytic hydrogenation afforded analog 11, while the incorporation of functional groups using N-hydroxysuccinimide ester provided analogs 10, 12, and 13. Additionally, derivatives 14 and 15, containing a trifluoromethyl propargylamide warhead in their acyl chains, were synthesized to examine the impact of this covalent warhead on NKT cell activation (Supplementary Fig. 5)44,45,46,47,48.
Scheme 2: General synthetic scheme for α-GalCer analogs (10–13**)a.**
scheme 2
aReagents, conditions, and yields; (vii) HCl, EtOH, 70 °C, 5 h; (viii) fatty acid (tetradecanoic acid, S14, or S15), EDC.HCl, HOBt.H2O, DIPEA, DCM, 0 °C to r.t., 16–18 h, yields over 2 steps (92% for 23b; 77% for 24b; and 73% for 24c); (ix) H2 (g, 1 atm), Pd(OH)2/C, DCM/MeOH (1:3, v/v), r.t., 17 h, 77%; (x) H2 (g, 10 atm), Pd(OH)2/C, DCM/MeOH (1:3, v/v), r.t., overnight; and (xi) N-hydroxysuccinimide ester (S20 or S21*), DMF, r.t., 4 h, yields over 2 steps (62% for 10; 50% for 12; 56% for 13). *K2CO3 was used as a base with S21.
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Potent Th2-selective activity of α-GalCer analog 9
The immunomodulatory activity of the α-GalCer analogs was assessed by measuring IL-2 secretion in HMNCs upon stimulation. KRN7000 (1) and OCH (2) were included as potent immunostimulatory and Th2-biasing ligands, respectively. The C3′-deoxy α-GalCer analogs exhibited enhanced IL-2 induction compared to the natural BfaGC 5, with no significant difference observed based on acyl chain length (Fig. 4a and Supplementary Fig. 6). Among these analogs, analog 9, with a C16:0-equivalent acyl chain containing α-chloroacetamide moiety, showed the highest IL-2 secretion, indicating the optimal presentation on CD1d. In contrast, the trifluoromethyl propargylamide analogs (14 and 15) showed no significant improvement over 5 and were excluded from further analysis (Supplementary Fig. 7).
Fig. 4: Immunological evaluations of α-GalCer analogues (8–**13).**
figure 4
a IL-2 secretion from murine hepatic mononuclear cells (HMNCs) was measured after 48 h upon treatment of α-GalCer analogs (n = 2, biological replicates); b IL-4 and c IFN-γ secretion from HMNCs was measured after 72 h upon treatment of α-GalCer analogs (n = 2, biological replicates); d IL-4/IFN-γ ratio of α-GalCer analogs (n = 2, biological replicates. **p < 0.01 and ns: not significant by Student’s *t*-test); **e** IL-2 production of NKT cells were modulated by *α*-GalCer analogs. Each analog was pre-treated to HMNCs before 30 min of KRN7000 (**1**) treatment, and IL-2 secretion was measured after 72 h (*n* = 3, biological replicates. ***p < 0.005 and ****p < 0.0001 by two-way ANOVA). Data are shown as mean ± SD; and f MALDI-TOF mass analysis of mCD1d with and without incubating 9.
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Next, we quantified IL-4 (Th2) and IFN-γ (Th1) cytokine levels to evaluate the immune polarization of each analog. As shown in Fig. 4b–d, IL-4 levels correlated with IL-2 secretion, and all analogs elicited significant increases in IL-4 responses compared to 5, while IFN-γ levels remained unaffected. The natural BfaGC ligand (5) exhibited a similar IL-4/IFN-γ ratio to OCH (2) but had lower IL-2 secretion. In contrast, amide-based analogs (9, 10, 12, and 13) demonstrated enhanced Th2-biased activities, suggesting that acyl chain functionalization with amide groups improved both CD1d-loading and Th2 selectivity. To further validate the Th2 selectivity of α-GalCer analogs, we quantified the induction of pro-inflammatory cytokine IL-17 (Th17), which is known to contribute to inflammatory and autoimmune diseases49. These α-GalCer analogs exhibited similar or lower IL-17 secretion levels compared to OCH (2), supporting their potential immunomodulatory effects (Supplementary Fig. 8).
To validate CD1d binding, competition studies were performed using α-GalCer analogs against the potent ligand KRN7000 (1) in HMNCs. Amide-based α-GalCer analogs (9, 10, 12, and 13) inhibited 1-induced IL-2 secretion in a dose-dependent manner (Fig. 4e). Analogs 9 and 12, containing terminal α-chloroacetamide moieties, showed the highest suppression of CD1d-mediated activation at a ratio of 1:10, suggesting effective competitive binding.
Further investigation into ligand–CD1d interactions was conducted using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Incubation of CD1d with the covalent warhead-containing α-GalCer analog 9 resulted in a peak shift from m/z 44,349 (native CD1d protein) to m/z 44,637, indicating partial covalent binding of 9 to CD1d (Fig. 4f). A similar increase in m/z was observed for 12-treated CD1d (Supplementary Fig. 9). These findings confirmed that the α-chloroacetamide moiety in 9 and 12 enables potential covalent interactions with CD1d, resulting in enhancement of immunomodulatory NKT cell responses.
Conclusions
In this study, we developed potential immunomodulatory ligands targeting NKT cells, inspired by the structure of gut symbiont B. fragilis-derived glycolipids. Through rational acyl chain modifications, we synthesized a series of α-GalCer analogs featuring terminal iso-branched sphinganine backbones. Notably, the C3′ hydroxyl group was found to hinder glycolipid presentation by CD1d protein, as evidenced by reduced IL-2 secretion and weak competition with KRN7000 (1). In contrast, the C3′-deoxy α-GalCer analog with an α-chloroacetamide moiety (9) emerged as the most effective ligand, exhibiting strong NKT cell activation and a pronounced Th2-biased cytokine response. Computational docking studies and mass spectrometry analyses strongly suggested the robust interactions between these glycolipids and CD1d, including covalent bonding. Further work will be needed to assess whether the observed differences in cytokine profiles could translate to stimulation or suppression of the immune system in a disease-relevant animal model. Our findings underscore the potential of leveraging microbiota-derived glycolipid structures to modulate NKT cell function. This approach offers exciting opportunities for the development of novel therapeutic agents aimed at treating immune-related disorders.
Methods
All experimental parts, including chemical synthesis and characterization of *α-*GalCer analogs, in vitro cytokine release assay, computational docking, and mass spectrometry analysis, are given in Supplementary Information.
Data availability
All data generated and analyzed in this study are included in this article and its Supplementary Information. The additional figures, synthetic procedures, NMR data, LRMS, and HRMS have been deposited in the Supplementary Information. NMR spectra of all newly synthesized products are provided as Supplementary Data 1. Source data of biological outcomes are provided as Supplementary Data 2. All data are available from the corresponding author upon request.
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Acknowledgements
This work was supported by the Biomedical Research grant (RS-2024-00438764) and the University Research Institute in Science Technology grant (2019R1A6A1A10073437) through the National Research Foundation of Korea (NRF) funded by the Korean Government (the Ministry of Science and ICT, MSIT). This work was also supported by the Mid-Career Bridging Program through Seoul National University and a SPARK Biopharma research grant (SRnD 0409-20210245).
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These authors contributed equally: Jesang Lee, Sumin Son, Minha Lee.
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Department of Chemistry, Seoul National University, Seoul, Republic of Korea
Jesang Lee, Sumin Son, Minha Lee & Seung Bum Park
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Jesang Lee
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2. Sumin Son
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3. Minha Lee
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4. Seung Bum Park
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S.B.P. conceived and directed all aspects of the project. J.L. and S.S. designed the synthetic experiments, analyzed the characterization data, and performed the synthetic experiments. M.L. designed the biological experiments, analyzed the data, and performed the biological experiments. J.L., S.S., M.L., and S.B.P. prepared the manuscript. All authors discussed the results and critically reviewed the manuscript.
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Correspondence to Seung Bum Park.
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Lee, J., Son, S., Lee, M. et al. Development of potential immunomodulatory ligands targeting natural killer T cells inspired by gut symbiont-derived glycolipids. Commun Chem 8, 98 (2025). https://doi.org/10.1038/s42004-025-01497-z
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Received:21 January 2025
Accepted:20 March 2025
Published:01 April 2025
DOI:https://doi.org/10.1038/s42004-025-01497-z
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