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 5

Human LDLR mutations are associated with impaired iNKT cell proliferation and activation.

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Human LDLR mutations are associated with impaired iNKT cell proliferatio...
(A) Principle of the iNKT cell proliferation assay. PBMCs of patients with LDLR mutations and controls were cultured in presence of lipoproteins, αGalCer, and IL-2 for 14 days. (B) Baseline percentage of iNKT cells (left) and APCs, including B cells, cDC1s, cDC2s, and monocytes (right), of LDLR-mutated PBMCs and controls. Blue downward triangles represent controls with low circulating LDL-cholesterol levels (≤2.5 mmol/L, n = 5); blue upward triangles represent controls with high circulating LDL-cholesterol levels (>2.5 mmol/L, n = 5); and red circles represent LDLR-mutated patients (n = 4). (C) Representative flow cytometric analysis of LDLR-mutated and control PBMCs on days 0 and 14. Numbers in graphs indicate the percentage of cells in the iNKT cell gate. (D) iNKT cell proliferation on day 14 shown as a percentage of iNKT cells of live cells on days 0 and 14 (left) and fold expansion of the iNKT cells after 14 days (right) (n = 10 controls, 4 patients). (E) Percentage of CD4–CD8– (DN), CD4+, CD8+, CD25+, and CD69+ iNKT cells (n = 10 controls, 4 patients). (F) Principle of the iNKT cell activation assay. Monocytes (CD14+ cells) were isolated from PBMCs of patients and controls, then cultured in presence of MCSF 5 days for differentiation into macrophages. The medium was changed to low-serum medium (2% FCS), and cells were treated with LDL-αGalCer or equivalent amounts of αGalCer (100 ng/mL) or LDL overnight and washed. Human primary iNKT cells from a healthy donor were added for 24 hours, and cytokine production was measured in the supernatant of the cocultures. (G) iNKT cell cytokines in supernatants of LDLR-mutated macrophages and controls in coculture with healthy donor primary iNKT cells. Statistics: error bars represent mean ± SD. Mann-Whitney U tests (D and E), independent t test with Bonferroni’s correction for multiple comparisons (G). *P < 0.05; **P < 0.01; ***P < 0.001.