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

Abstract

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

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

“…To ensure a single iNKT-TCR per QD, the iNKT-TCR-QD conjugate was prepared in an excess of free biotin to occlude SAV-QD excess binding sites. As another control to rule out any potential cross-linking effects induced by the SAV-QD multivalency, we also used the α-GalCer-hCD1d-specific Fab fragment (Fab9b) (9,28,29) covalently attached to the small dye Atto647N. As a third control for the iNKT-TCR probe, α-GalCer-loaded hCD1d molecules were labeled using a monovalent anti-CD1d Ab (CD1d42) conjugated to a QD under excess of free biotin.…”

Lysosomal recycling terminates CD1d-mediated presentation of short and polyunsaturated variants of the NKT cell lipid antigen αGalCer

“…Some studies suggested that short or polyunsaturated lipids failed to efficiently traffic to the lysosome (14,15) but this has not been tested for ␣GC variants. While the variants could directly load CD1d molecules at the cell surface without trafficking to endosomal compartments (5,12,13), lysosomal loading might be required for access to membrane rafts and efficient T-cell stimulation (16).…”

Structural Basis for the Recognition of C20:2-αGalCer by the Invariant Natural Killer T Cell Receptor-like Antibody L363

“…Antibodies with TCR-like binding properties exist for all MHC molecules that present antigens to T cells, including MHC class I (44 -47), MHC class II (48), as well as CD1d (36,49), and are widely used to study or modulate T cell function by specifically blocking antigen-mediated TCR activation. High affinity antibodies that exhibit superior binding affinity are especially useful to block T cell activation and as such have therapeutic potential in various T cell-mediated diseases (46).…”