Vincenzo Cerundolo

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

The glycolipid α‐galactosylceramide (α‐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 α‐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)‐α‐GalCer complex. These Fab fragments were able to detect specifically hCD1d‐α‐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 α‐GalCer and its analogues. Furthermore, the antibody was also able to detect the presentation by hCD1d molecules of analogues of α‐GalCer with the same polar head structure. Using this reagent, we were able to confirm directly that the α‐GalCer analogue C20:2 preferentially loads onto cell surface CD1d rapidly without the need for internalization, while the loading of α‐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 α‐GalCer and its analogues.