Phage display-derived recombinant antibodies with TCR-like specificity against alpha-galactosylceramide and its analogues in complex with human CD1d molecules.
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.
Denkberg
Galit
G
Faculty of Biology, Technion-Israel Institute of Technology, Haifa, Israel.
Stronge
Victoria S
VS
Zahavi
Efrat
E
Pittoni
Paola
P
Oren
Ravit
R
Shepherd
Dawn
D
Salio
Mariolina
M
McCarthy
Corinna
C
Illarionov
Petr A
PA
van der Merwe
Anton
A
Besra
Gurdyal S
GS
Dellabona
Paolo
P
Casorati
Giulia
G
Cerundolo
Vincenzo
V
Reiter
Yoram
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000938:Antigen-Presenting Cells / Q000276:immunology / Q000378:metabolism; D018949:Antigens, CD1 / Q000235:genetics / Q000276:immunology*; D055610:Antigens, CD1d; D002460:Cell Line; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000276:immunology; D003713:Dendritic Cells / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005699:Galactosylceramides / Q000276:immunology*; D006017:Glycolipids / Q000276:immunology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis / Q000276:immunology* / Q000494:pharmacology; D007371:Interferon-gamma / Q000378:metabolism; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D019151:Peptide Library; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D013154:Spleen / Q000166:cytology / Q000276:immunology; D020349:Surface Plasmon Resonance; D014162:Transfection
D000911:Antibodies, Monoclonal; D018949:Antigens, CD1; D055610:Antigens, CD1d; C526877:CD1D protein, human; D005699:Galactosylceramides; D006017:Glycolipids; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins; C493154:alpha-galactosylceramide; C501055:tetracosanoic acid 1-galactopyranosyloxy-3,4-dihydroxydec-2-yl amide; D007371:Interferon-gamma