Abstract
CD1d-restricted natural killer T (NKT) cells are innate-like lymphocytes that express a conserved T-cell receptor and contribute to host defence against various microbial pathogens1,2. However, their target lipid antigens have remained elusive. Here we report evidence for microbial, antigen-specific activation of NKT cells against Gram-negative, lipopolysaccharide (LPS)-negative alpha-Proteobacteria such as Ehrlichia muris and Sphingomonas capsulata. We have identified glycosylceramides from the cell wall of Sphingomonas that serve as direct targets for mouse and human NKT cells, controlling both septic shock reaction and bacterial clearance in infected mice. In contrast, Gram-negative, LPS-positive Salmonella typhimurium activates NKT cells through the recognition of an endogenous lysosomal glycosphingolipid, iGb3, presented by LPS-activated dendritic cells. These findings identify two novel antigenic targets of NKT cells in antimicrobial defence, and show that glycosylceramides are an alternative to LPS for innate recognition of the Gram-negative, LPS-negative bacterial cell wall.
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Acknowledgements
We thank D. Wei for reading the manuscript, K. Thompson for help with biochemical characterization of Sphingomonas and growth of bacteria, S. Porcelli for the gift of anti-human CD1d, R. Duggan, J. Marvin and B. Eisfelder for cell sorting, L. Taylor for managing the mouse colonies, and the University of Chicago Digestive Disease Research center for equipment. Supported by NIH grants to A.B., P.S.B. and L.T., an NIH award to K.L.D. and O.S., an NIH grant to B.B., a Cancer Research Institute fellowship to J.M. and D.Z., and a fellowship from the Fondation pour la Recherche Medicale to P.S.-M.Authors' contributions P.B.S. and A.B. are co-senior authors.
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Supplementary Figure S1
Cell divisions of NKT cells in response to bacterial stimuli or 100ng/ml αGalCer, as indicated, six days after stimulation (NS, no stimulus). Upper row, CD1d- αGalCer/B220 staining of spleen cells with NKT cell gate and percentage as indicated; lower row, CFSE dilution profile of 5 × 103 gated NKT cells. (PDF 283 kb)
Supplementary Figure S2
IFN-γ released by whole spleen cells of indicated genotypes cultured with heat killed Salmonella typhimurium, Sphingomonas capsulata and Ehrlichia muris for 48 hours. Data shown as percentage of control wild type. Mean and SD of two to three separate experiments. (PDF 78 kb)
Supplementary Figure S3
Bacterial burden in the lungs of CD1d+/- and CD1d-/- mice at different days after infection with 1 × 106 CFU of Sphingomonas (each bar represents mean and SD of four to five mice). Fold increase and p values are indicated. (PDF 124 kb)
Supplementary Figure S4
Acute lethality after inoculation of 5 × 108 Sphingomonas capsulata to Jα18+/- and Jα18-/- mice (n=12 each, p= 0.034). (PDF 54 kb)
Supplementary Figure S5
Acute serum release of p40 after infection with 1 × 107 Sphingomonas capsulata in heterozygous and homozygous Jα18 mutant mice. Similar results were obtained in two independent experiments. (PDF 75 kb)
Supplementary Figure S6
Ehrlichia PCR counts in lungs and livers of CD1+/- and CD1-/- mice recovered at day 2 and day 7 post-infection (each bar shows mean and SD of three mice). Fold increase and p values are indicated. One experiment representative of two is shown. (PDF 203 kb)
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Mattner, J., DeBord, K., Ismail, N. et al. Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434, 525–529 (2005). https://doi.org/10.1038/nature03408
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DOI: https://doi.org/10.1038/nature03408
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