Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer
Kumiko Nishio, … , Amy R. Howell, Jay A. Berzofsky
Kumiko Nishio, … , Amy R. Howell, Jay A. Berzofsky
Published December 21, 2023
Citation Information: J Clin Invest. 2024;134(4):e165281. https://doi.org/10.1172/JCI165281.
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Research Article Cell biology Immunology

Lysosomal processing of sulfatide analogs alters target NKT cell specificity and immune responses in cancer

Abstract

In a structure-function study of sulfatides that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogs with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by the CD1d monomer on plastic stimulated type II, not type I, NKT cell hybridomas, as expected. Unexpectedly, when presented by bone marrow–derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT cell hybridomas, mimicking the corresponding β-galactosylceramide (βGalCer) without sulfate. C24:2 induced IFN-γ–dependent immunoprotection against CT26 colon cancer lung metastases, skewed the cytokine profile, and activated conventional DC subset 1 cells (cDC1s). This was abrogated by blocking lysosomal processing with bafilomycin A1, or by sulfite blocking of arylsulfatase or deletion of this enyzme that cleaves off sulfate. Thus, C24:2 was unexpectedly processed in BMDCs from a type II to a type I NKT cell–stimulating ligand, promoting tumor immunity. We believe this is the first discovery showing that antigen processing of glycosylceramides alters the specificity for the target cell, reversing the glycolipid’s function from stimulating type II NKT cells to stimulating type I NKT cells, thereby introducing protective functional activity in cancer. We also believe our study uncovers a new role for antigen processing that does not involve MHC loading but rather alteration of which type of cell is responding.

Authors

Kumiko Nishio, Lise Pasquet, Kaddy Camara, Julia DiSapio, Kevin S. Hsu, Shingo Kato, Anja Bloom, Stewart K. Richardson, Joshua A. Welsh, Tianbo Jiang, Jennifer C. Jones, Susanna Cardell, Hiroshi Watarai, Masaki Terabe, Purevdorj B. Olkhanud, Amy R. Howell, Jay A. Berzofsky

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Figure 8

Inhibition of arylsulfatase A prevents the processing of sulfatides, and their antitumor activity is dependent on type I NKT cells.

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Inhibition of arylsulfatase A prevents the processing of sulfatides, and...
(A) Type I NKT hybridoma clone DN32 cells were stimulated with BMDCs prepulsed with either sulfatide C24:2 (10 μM), βGalCer C24:2 (10 μM), or KRN7000 (25 nM), or with 0.25 μg plate-bound anti-CD3 in the presence of titrated 0–12.5 μM NaSulfite (left) or titrated 0–50 nM bafilomycin A1 (BAF-A1) (right) for 16 hours. IL-2 levels in the culture media were measured by ELISA and the percentage and stimulation of each treatment were calculated relative to IL-2 levels in DN32 cells cocultured with PBS-treated BMDCs. Data represent at least 2 experiments and indicate the mean ± SD of duplicate cultures. (B) DN32 cells were stimulated for 24 hours with 50,000 BMDCs at a 1:1 ratio. Arsa-KO and WT BMDCs were prepulsed with sulfatide analogs, KRN7000, or C24:1. Results are representative data from 2 experiments and indicate the mean ± SD. LOD, limit of detection. (C) Mice were injected i.p. with the vehicle used to dissolve the sulfatide analogs, 500 pmol KRN7000, or 30 nmol sulfatide analogs, and spleens were harvested 12 hours later to stain type I NKT cells (CD45+, TCRβ+, PBS57-loaded CD1d tetramer+) and their activation (CD69, MFI) by flow cytometry. Each symbol represents an individual mouse. Data represent at least 2 experiments and the mean ± SD. Data were assessed by the Mann-Whitney U test with Holm-Sidak corrections. (D) Traj18-KO mice were injected i.v. via the tail vein with 5 × 105 CT26 cells and were subsequently injected i.p. with the vehicle used to dissolve the sulfatide or βGalCer analogs, 500 pmol KRN7000, or 30 nmol sulfatide or βGalCer analogs. Mice were sacrificed 12 days after tumor challenge, and lung metastases were enumerated. The mean nodule number for each group is indicated by a horizontal bar, and each symbol represents an individual mouse. (E) Healthy human PBMCs (1 × 106) were cultured with 10 μg/mL glycolipid (C24:2 with and without BAF 50 nM) for 15 hours and then for 1 hour with brefeldin A. Additionally, human PBMCs were cultured with cell activation (act.) cocktail in the presence of BAF-A1. (E) Quantification of IFN-γ+ type I NKT cells after glycolipid treatment. Data were pooled from 3 experiments and represent the mean ± SD. *P < 0.05 and **P < 0.01, by 2-way ANOVA with Dunnett’s multiple comparisons for batch effects.