Abstract
Maturation of conventional dendritic cells (cDCs) is crucial for maintaining tolerogenic safeguards against auto-immunity and for promoting immunogenic responses to pathogens and cancer. The subcellular mechanism for cDC maturation remains poorly defined. We show that cDCs mature by leveraging an internal reservoir of cholesterol (harnessed from extracellular cell debris and generated by de novo synthesis) to assemble lipid nanodomains on cell surfaces of maturing cDCs, enhance expression of maturation markers and stabilize immune receptor signaling. This process is dependent on cholesterol transport through Niemann–Pick disease type C1 (NPC1) and mediates homeostatic and Toll-like receptor (TLR)-induced maturation. Importantly, we identified the receptor tyrosine kinase AXL as a regulator of the NPC1-dependent construction of lipid nanodomains. Deleting AXL from cDCs enhances their maturation, thus improving anti-tumor immunity. Altogether, our study presents new insights into cholesterol mobilization as a fundamental basis for cDC maturation and highlights AXL as a therapeutic target for modulating cDCs.
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Data availability
The scRNA-seq data generated for this study are available using GEO accession number GSE282849.
Code availability
No new code was developed for the completion of this study.
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Acknowledgements
We sincerely thank members of the Merad laboratory for helpful discussions; P. Suri and R. Samstein in the Department of Immunology and Immunotherapy for providing the 4T1 cells; S.D. Kumar and the Advanced Microscopy and Bioimaging Core; and the Mount Sinai Flow Cytometry Core and Human Immune Monitoring Center for technical support. Finally, we thank BerGenBio for generously providing the AXL inhibitor for our in vitro and in vivo studies.
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Contributions
M.M. conceived the project. M.B., M.D.P. and M.M. designed the experiments. M.B., M.D.P. and M.M. wrote the manuscript. M.B. performed experiments with support from M.D.P., J.L.B., R.M. and C.M.W. K.J.R., S.B. and A.P. performed experiments with human cDCs, including RNA sequencing. M.D.P. performed computational analyses. C.M.B. generated the D4-GFP cholesterol probe. S.T.C. and N.M.L. contributed to the conceptual development of the project. D.J.P., S.G., C.V.R., C.M.B. and C.L. advised on study design. N.M.L. was supported by the Cancer Research Institute and Bristol Myers Squibb Irvington Postdoctoral Research Fellowship to Promote Racial Diversity (award CRI3931). R.M. was supported by the 2021 AACR–AstraZeneca Immuno-oncology Research Fellowship (grant 21-40-12-MATT). K.J.R. was supported by a Fulbright Future Scholarship and the Mater Foundation. C.M.B. and C.L. were supported by institutional grants from the Curie Institute, INSERM and the Centre National de la Recherche Scientifique and by grants from the Agence Nationale de la Recherche (ANR NanoGammaR-17-CE15-0032) to C.L. and from the Ligue Nationale contre le Cancer and ARC to C.M.B. C.M.B. and C.L. are members of Labex CelTisPhyBio (ANR-10-LBX-0038) and are part of the IDEX PSL (ANR-10-IDEX-0001-02 18796).
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M.M. serves on the scientific advisory board of and holds stock from Compugen, Dynavax, Innate Pharma, Morphic Therapeutics, Asher Bio, Dren Bio, Nirogy, Genenta, OncoResponse and Owkin. M.M. serves on the ad hoc scientific advisory board of DBV and Genentech and on the foundation advisory board of Breakthrough Cancer. M.M. receives funding for contracted research from Genentech, Regeneron and Boehringer Ingelheim. M.M. is listed as an inventor on a patent application (16/092576) submitted by the Icahn School of Medicine at Mount Sinai that covers the use of multiplex immunohistochemistry to characterize tumors and treatment responses. The technology is filed through the Icahn School of Medicine at Mount Sinai and is currently unlicensed. This technology was used to evaluate tissue in this study, and the results could impact the value of this technology. T.U.M. has served on advisory and/or data safety monitoring boards for Rockefeller University, Regeneron Pharmaceuticals, AbbVie, Bristol Meyers Squibb, Boehringer Ingelheim, Atara, AstraZeneca, Genentech, Celldex, Chimeric, Glenmark, Simcere, Surface, G1 Therapeutics, NGM Bio, DBV Technologies, Arcus and Astellas and has research grants from Regeneron, Bristol Myers Squibb, Merck and Boehringer Ingelheim. The remaining authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Disrupting cholesterol mobilization impairs cDC maturation.
Flow cytometry-based measurement of cDC maturation markers (that is, IFN-ɣR, CD40, CD80, CD86, PD-L1, PD-L2 or MHC-I, MHC-II) on splenic mature cDCs either treated with (a) the LXRα/β agonist GW3965, (b) supplemental mevalonate, (c) simvastatin, (d) the NPC1 inhibitor U18666A, and (e) wild-type (WT) and Npc1 haploinsufficient (Npc1+/-) mature cDCs. Across all panels, mean values of technical replicates ± SEM of a representative experiment are shown. P-values computed by unpaired, two-tailed t-test.
Extended Data Fig. 2 Quantification of cell surface cholesterol.
Flow cytometric quantification of (a) D4-GFP and (b) CTxB staining of wild-type (WT) and Npc1+/- mature cDCs. (c) Immunofluorescence staining for free cholesterol on the cell surface of 1) cDCs, 2) cDCs supplemented with mevalonate, 3) mature cDCs, 4) mature cDCs treated with the NPC1 inhibitor U18666A, 5) mature cDCs treated with simvastatin, and 6) mature cDCs treated with the LXR agonist GW3965, 7) mature cDCs treated with methyl-β-cyclodextrin, 8) mature cDCs treated with sphingomyelinase. (d) Fluorescence quantification of D4-GFP staining on cDCs and cDCs supplemented with mevalonate, scale 5μM. (e) Fluorescence quantification of D4-GFP staining on groups 3-8 in (c). Across all panels, data represent mean ± SEM of at least three independent replicates or biologically distinct experiments (c, d, e). P-values computed by unpaired, two-tailed t-test.
Extended Data Fig. 3 The phagocytic cell sensor AXL controls cDC maturation.
Flow cytometric measurement (a) of free cholesterol using the D4-GFP probe on WT and KO mature cDCs or (b) of free cholesterol acquired extracellularly by mature cDCs exposed to D4-GFP-stained apoptotic cell debris. (c) Immuno-fluorescence staining and quantification for free cholesterol on the surface of CD11c+ mature cDCs that were fed D4-GFP-stained cell debris, scale 5 μM. (d)-(h) Flow cytometric measurement of cDC maturation markers (that is, CCR7, IFN-ɣR, MHC-I, MHC-II, CD40, CD80, CD86, PD-L1, PD-L2). Across all panels, mean values of technical replicates ± SEM of a representative experiment are shown. P-values computed by ordinary one-way ANOVA t-test.
Extended Data Fig. 4 De novo cholesterol synthesis and transport via NPC1 are regulated by AXL during cDC maturation.
Flow cytometric measurement of cDC maturation markers (that is, CCR7, IFN-ɣR, CD40, CD80, MHC-I, MHC-II, PD-L1, and PD-L2) on WT and AXL KO mature cDCs either left untreated or treated with (a) the NPC1 inhibitor U18666A or (b) simvastatin. Across all panels, mean values of technical replicates ± SEM of a representative experiment are shown. P-values computed by either ordinary one-way ANOVA t-test.
Extended Data Fig. 5 NPC1 haploinsufficiency impairs cDC maturation and T cell activation.
(a) Proliferation and activation of OT-I and OT-II cells following co-culture with wild-type (WT) and Npc1 haploinsufficient (Npc1+/-) mature cDCs. (b)-(e) Flow cytometric measurement of cDC maturation markers on mature cDCs isolated from tumor-bearing lungs of WT and Npc1 haploinsufficient (Npc1+/-) mice. Across all panels, mean values of technical replicates ± SEM of a representative experiment are shown. P-values computed by unpaired, two-tailed t-test.
Extended Data Fig. 6 The therapeutic efficacy of the cDC checkpoint AXL is a T cell-dependent response.
(a) mRNA expression of AXL by KrasG12D/+Trp53-/- tumor cells, according to single-cell RNA-sequencing of tumor cells from the KP GEMM model of lung adenocarcinoma (Marjanovic et al., 2020). (b) Ex vivo flow cytometric staining of CTxB on Axl+/+ (WT) and Axl-/- (KO) mature cDCs from tumor-bearing lungs. (c) Flow cytometric measurement of intracellular IL-12p40 produced by mature cDCs from the tumor-bearing lungs of (left) WT and BCT-treated mice or (right) WT, KO and Zbtb46Cre-Axlfl/fl (AxlΔDC) mice. (d) Frequency of NK cells in the (left) lungs and (right) tumor-draining lymph nodes of WT, KO, and AxlΔDC mice. (e) (Left) histology and (right) quantification of tumor burden in the lungs of WT, KO, and KO mice depleted of CD8 T cells at 21 days post-tumor cell inoculation. Same scale bar as panel (f). Scale bar, 1 mm. (f) (Left) histology and (right) quantification of tumor burden in the lungs of WT, KO, and KO mice depleted of CD4 T cells at 21 days post-tumor cell inoculation. Same scale bar as panel (e). Scale bar, 1 mm. (g) Frequency of total CD8 T cells in the (left) blood and (right) lungs of WT, KO, and KO mice depleted of CD8 T cells. (h) Frequency of total CD4 T cells in the (left) blood and (right) lungs of WT, KO, and KO mice depleted of CD4 T cells. Across all panels, data represent mean ± SEM of at least three biologically distinct experiments. All p-values computed by ordinary one-way ANOVA t-test.
Supplementary information
Supplementary Tables 1–3
Supplementary Table 1. Submodules defined by the identification of highly correlated variable genes across DCs integrated from different scRNA-seq datasets. Supplementary Table 2. Metadata per cell barcode of integrated scRNA-seq analysis, including cell subtype annotation and dataset of origin. Supplementary Table 3. Active metabolic pathways inferred from gene set enrichment analysis of genes enriched in mature cDCs.
Supplementary Video 1
Z-stack recording of pSTAT1 and DAPI staining of WT BMDCs fed with KP-GFP cells.
Supplementary Video 2
Z-stack recording of pSTAT1 and DAPI staining of Axl-KO BMDCs fed with KP-GFP cells.
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Belabed, M., Park, M.D., Blouin, C.M. et al. Cholesterol mobilization regulates dendritic cell maturation and the immunogenic response to cancer. Nat Immunol 26, 188–199 (2025). https://doi.org/10.1038/s41590-024-02065-8
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DOI: https://doi.org/10.1038/s41590-024-02065-8
