The length of lipids bound to human CD1d molecules modulates the affinity of NKT cell TCR and the threshold of NKT cell activation.

CD1d-restricted lymphocytes recognize a broad lipid range. However, how CD1d-restricted lymphocytes translate T cell receptor (TCR) recognition of lipids with similar group heads into distinct biological responses remains unclear. Using a soluble invariant NKT (iNKT) TCR and a newly engineered antibody specific for alpha-galactosylceramide (alpha-GalCer)-human CD1d (hCD1d) complexes, we measured the affinity of binding of iNKT TCR to hCD1d molecules loaded with a panel of alpha-GalCer analogues and assessed the rate of dissociation of alpha-GalCer and alpha-GalCer analogues from hCD1d molecules. We extended this analysis by studying iNKT cell synapse formation and iNKT cell activation by the same panel of alpha-GalCer analogues. Our results indicate the unique role of the lipid chain occupying the hCD1d F' channel in modulating TCR binding affinity to hCD1d-lipid complexes, the formation of stable immunological synapse, and cell activation. These data are consistent with previously described conformational changes between empty and loaded hCD1d molecules (Koch, M., V.S. Stronge, D. Shepherd, S.D. Gadola, B. Mathew, G. Ritter, A.R. Fersht, G.S. Besra, R.R. Schmidt, E.Y. Jones, and V. Cerundolo. 2005. Nat. Immunol 6:819-826), suggesting that incomplete occupation of the hCD1d F' channel results in conformational differences at the TCR recognition surface. This indirect effect provides a general mechanism by which lipid-specific lymphocytes are capable of recognizing both the group head and the length of lipid antigens, ensuring greater specificity of antigen recognition.

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.

A closer look at CD1d molecules: new horizons in studying NKT cells.

Recent findings have highlighted the ability of invariant natural killer T (iNKT) cells to recognize microbe-derived glycolipids and have demonstrated the role of these cells in several disease states, from autoimmune disease to cancer. It has also become clear that iNKT cells can rapidly mature dendritic cells and licence them to prime antigen-specific T- and B-cell responses. The use of CD1d tetramers to monitor iNKT cell frequency and phenotype has moved the field forward at a fast pace. To harness iNKT cells for therapeutic purposes and to understand their role in vivo, it is essential to characterize the molecular events that contribute to iNKT cell activation. Here we review new reagents and novel protocols that are facilitating a closer look at lipid presentation by CD1d molecules and their recognition by iNKT cells.

The crystal structure of human CD1d with and without alpha-galactosylceramide.

The glycolipid alpha-galactosylceramide binds with high affinity to CD1d and stimulates natural killer T cells. Here we report the crystal structure of human CD1d in complex with synthetic alpha-galactosylceramide at a resolution of 3.0 A. The structure shows a tightly fit lipid in the CD1d binding groove, with the sphingosine chain bound in the C' pocket and the longer acyl chain anchored in the A' pocket. We also present the CD1d structure without lipid, which has a more open conformation of the binding groove, suggesting a dual conformation of CD1d in which the 'open' conformation is more able to load lipids. These structures provide clues as to how CD1 molecules load glycolipids as well as data to guide the design of new therapeutic agents.