Identification of α-galactosylceramide as an endogenous mammalian antigen for iNKT cells

  • J Exp Med. 2025 Feb 3;222(2):e20240728. doi: 10.1084/jem.20240728.
Yuki Hosono  #  1  2  3 ,  Noriyuki Tomiyasu  #  4 ,  Hayato Kasai  #  1  2 ,  Eri Ishikawa  1  2 ,  Masatomo Takahashi  4  5 ,  Akihiro Imamura  6  7 ,  Hideharu Ishida  6  7 ,  Federica Compostella  8 ,  Hiroshi Kida  9 ,  Atsushi Kumanogoh  3  10  11  12  13 ,  Takeshi Bamba  4  5 ,  Yoshihiro Izumi  4  5 ,  Sho Yamasaki  1  2  12  13
Affiliations
  • 1. Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Japan.
  • 2. Laboratory of Molecular Immunology, Immunology Frontier Research Center, Osaka University , Suita, Japan.
  • 3. Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, Suita, Japan.
  • 4. Department of Systems Life Sciences, Graduate School of Systems Life Sciences, Kyushu University, Fukuoka, Japan.
  • 5. Division of Metabolomics, Medical Research Center for High Depth Omics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
  • 6. Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan.
  • 7. Institute for Glyco-core Research, Gifu University , Gifu, Japan.
  • 8. Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy.
  • 9. Department of Respiratory Medicine, National Hospital Organization Osaka Toneyama Medical Center, Toyonaka, Japan.
  • 10. Department of Immunopathology, World Premier International Research Center Initiative, Immunology Frontier Research Center, Osaka University, Suita, Japan.
  • 11. Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Suita, Japan.
  • 12. Center for Infectious Disease Education and Research, Osaka University , Suita, Japan.
  • 13. Center for Advanced Modalities and DDS, Osaka University , Suita, Japan.
  • # Contributed equally.
PMID: 39704712 DOI: 10.1084/jem.20240728
Abstract

Invariant natural killer T (iNKT) cells are unconventional T cells recognizing lipid antigens in a CD1d-restricted manner. Among these lipid antigens, α-galactosylceramide (α-GalCer), which was originally identified in marine sponges, is the most potent antigen. Although the presence of α-anomeric hexosylceramide and microbiota-derived branched α-GalCer is reported, antigenic α-GalCer has not been identified in mammals. Here, we developed a high-resolution separation and detection system, supercritical fluid chromatography tandem mass spectrometry (SFC/MS/MS), that can discriminate hexosylceramide diastereomers (α-GalCer, α-GlcCer, β-GalCer, or β-GlcCer). The B16 melanoma tumor cell line does not activate iNKT cells; however, ectopic expression of CD1d was sufficient to activate iNKT cells without adding antigens. B16 melanoma was unlikely to generate iNKT cell antigens; instead, antigen activity was detected in Cell Culture serum. Activity-based purification and SFC/MS/MS identified dihydrosphingosine-based saturated α-GalCer as an antigenic component in serum, bile, and lymphoid tissues. These results show the first evidence for the presence of potent antigenic α-GalCer in mammals.

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