Phage display-derived recombinant antibodies with TCR-like specificity against alpha-galactosylceramide and its analogues in complex with human CD1d molecules

Eur J Immunol. 2008 Mar;38(3):829-40. doi: 10.1002/eji.200737518.


The glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent activator of invariant natural killer T (iNKT) cells and has been shown to be an effective agent against cancer, infections and autoimmune diseases. The effectiveness of alpha-GalCer and its alkyl chain analogues depends on efficient loading and presentation by the antigen-presenting molecule CD1d. To monitor the ability of CD1d to present the glycolipids, we have used a phage display strategy to generate recombinant antibodies with T cell receptor-like (TCRL) specificity against the human CD1d (hCD1d)-alpha-GalCer complex. These Fab fragments were able to detect specifically hCD1d-alpha-GalCer complexes in cell-free systems such as surface plasmon resonance and ELISA, as well as on the surface of hCD1d(+) antigen-presenting cells (APC) by flow cytometry and immunofluorescence microscopy, the latter of which could also detect intracellular complexes. We show that our TCRL antibodies can stain dendritic cells from CD11c-hCD1d-transgenic mice administered in vivo with alpha-GalCer and its analogues. Furthermore, the antibody was also able to detect the presentation by hCD1d molecules of analogues of alpha-GalCer with the same polar head structure. Using this reagent, we were able to confirm directly that the alpha-GalCer analogue C20:2 preferentially loads onto cell surface CD1d rapidly without the need for internalization, while the loading of alpha-GalCer is improved with longer incubation times on professional APC. This reagent will be essential for assessing the loading and presenting capabilities of hCD1d of alpha-GalCer and its analogues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology
  • Antigen-Presenting Cells / immunology
  • Antigen-Presenting Cells / metabolism
  • Antigens, CD1 / genetics
  • Antigens, CD1 / immunology*
  • Antigens, CD1d
  • Cell Line
  • Cytotoxicity, Immunologic / drug effects
  • Cytotoxicity, Immunologic / immunology
  • Dendritic Cells / immunology
  • Dendritic Cells / metabolism
  • Enzyme-Linked Immunosorbent Assay
  • Galactosylceramides / immunology*
  • Glycolipids / immunology
  • Humans
  • Immunoglobulin Fab Fragments / biosynthesis
  • Immunoglobulin Fab Fragments / immunology*
  • Immunoglobulin Fab Fragments / pharmacology
  • Interferon-gamma / metabolism
  • Killer Cells, Natural / immunology
  • Killer Cells, Natural / metabolism
  • Mice
  • Mice, Inbred Strains
  • Mice, Transgenic
  • Peptide Library*
  • Receptors, Antigen, T-Cell / immunology*
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / immunology
  • Spleen / cytology
  • Spleen / immunology
  • Surface Plasmon Resonance
  • Transfection


  • Antibodies, Monoclonal
  • Antigens, CD1
  • Antigens, CD1d
  • CD1D protein, human
  • Galactosylceramides
  • Glycolipids
  • Immunoglobulin Fab Fragments
  • Peptide Library
  • Receptors, Antigen, T-Cell
  • Recombinant Proteins
  • alpha-galactosylceramide
  • tetracosanoic acid 1-galactopyranosyloxy-3,4-dihydroxydec-2-yl amide
  • Interferon-gamma