Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T cells
Suzanne E. Engelen, … , Claudia Monaco, Henk S. Schipper
Suzanne E. Engelen, … , Claudia Monaco, Henk S. Schipper
Published March 28, 2023
Citation Information: JCI Insight. 2023;8(9):e158089. https://doi.org/10.1172/jci.insight.158089.
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Research Article Immunology

Lipoproteins act as vehicles for lipid antigen delivery and activation of invariant natural killer T cells

Abstract

Invariant natural killer T (iNKT) cells act at the interface between lipid metabolism and immunity because of their restriction to lipid antigens presented on CD1d by antigen-presenting cells (APCs). How foreign lipid antigens are delivered to APCs remains elusive. Since lipoproteins routinely bind glycosylceramides structurally similar to lipid antigens, we hypothesized that circulating lipoproteins form complexes with foreign lipid antigens. In this study, we used 2-color fluorescence correlation spectroscopy to show, for the first time to our knowledge, stable complex formation of lipid antigens α-galactosylceramide (αGalCer), isoglobotrihexosylceramide, and OCH, a sphingosine-truncated analog of αGalCer, with VLDL and/or LDL in vitro and in vivo. We demonstrate LDL receptor–mediated (LDLR-mediated) uptake of lipoprotein-αGalCer complexes by APCs, leading to potent complex-mediated activation of iNKT cells in vitro and in vivo. Finally, LDLR-mutant PBMCs of patients with familial hypercholesterolemia showed impaired activation and proliferation of iNKT cells upon stimulation, underscoring the relevance of lipoproteins as a lipid antigen delivery system in humans. Taken together, circulating lipoproteins form complexes with lipid antigens to facilitate their transport and uptake by APCs, leading to enhanced iNKT cell activation. This study thereby reveals a potentially novel mechanism of lipid antigen delivery to APCs and provides further insight into the immunological capacities of circulating lipoproteins.

Authors

Suzanne E. Engelen, Francesca A. Ververs, Angela Markovska, B. Christoffer Lagerholm, Jordan M. Kraaijenhof, Laura I.E. Yousif, Yasemin-Xiomara Zurke, Can M.C. Gulersonmez, Sander Kooijman, Michael Goddard, Robert J. van Eijkeren, Peter J. Jervis, Gurdyal S. Besra, Saskia Haitjema, Folkert W. Asselbergs, Eric Kalkhoven, Hidde L. Ploegh, Marianne Boes, Vincenzo Cerundolo, G.K. Hovingh, Mariolina Salio, Edwin C.A. Stigter, Patrick C.N. Rensen, Claudia Monaco, Henk S. Schipper

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Figure 1

Lipoproteins form stable complexes with the prototypic iNKT cell ligand αGalCer.

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Lipoproteins form stable complexes with the prototypic iNKT cell ligand ...
(A) Representative confocal image of αGalCer-AF488-LDL-DyLight 633 complexes loaded on human monocyte-derived macrophages. Scale bar: 10 μm. (B) Spatial colocalization of the αGalCer and LDL complex components (zoomed box in A). (C) Manders and Pearson colocalization coefficients of αGalCer and LDL (n = 15). (D) Principle of 2-color fluorescence cross-correlation spectroscopy (2c-FCCS). Diffusing particles are observed via their fluorescence. The measured fluorescence fluctuations over time are fitted in an autocorrelation model, which yields a decay time that reflects the average retention time of particles in the observation volume, as well as particle diffusion times. In case of complex formation, decay and diffusion times of the complex components will synchronize, and cross-correlated diffusion of the complex components will occur. (E) Autocorrelation curves of αGalCer singly and in complex with human LDL in vitro and in vivo, showing synchronization of αGalCer diffusion with LDL upon complexation (n = 15). (F) Autocorrelation curve of LDL in complex with αGalCer in vitro and in vivo (n = 15). (G) Cross-correlated diffusion of the αGalCer and LDL complex components (n = 15). (H) Diffusion time of single αGalCer and LDL, compared with complex components in vitro and in vivo (n = 15). (I) Fluorescent counts per molecule of single LDL and αGalCer and complexed components in vitro and in vivo. The αGalCer counts per molecule in vivo were normalized for the lower LDL counts per molecule in vivo (n = 15). (J) Overview of 2c-FCCS studies of lipoprotein-αGalCer complex formation. Statistics: error bars represent mean ± SD (E–H). Unpaired t tests with Bonferroni’s correction for multiple comparisons (H). **P < 0.01. Apo, apolipoprotein.