Abstract:
: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.
journal_name
Eur J Immunoljournal_title
European journal of immunologyauthors
Denkberg G,Stronge VS,Zahavi E,Pittoni P,Oren R,Shepherd D,Salio M,McCarthy C,Illarionov PA,van der Merwe A,Besra GS,Dellabona P,Casorati G,Cerundolo V,Reiter Ydoi
10.1002/eji.200737518subject
Has Abstractpub_date
2008-03-01 00:00:00pages
829-40issue
3eissn
0014-2980issn
1521-4141journal_volume
38pub_type
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