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Cell-based assays are widely used in all aspects of research ranging from understanding basic biological function to identifying compounds for disease intervention. Immortalized cell lines have been ideal components of these assays due to the low cost of growth, easy maintenance, and the ability to obtain homogenous cell populations. Like other molecular assays, cell-based systems have been automated to reduce experimental error. Complex lipids are now recognized as important components of the allergic response, the study of the interaction between NKTs and lipid-activated DCs opens a new perspective into the intrinsic allergenicity of proteins. Here, we describe a semi-automated method to measure IL-2 release upon activation of mouse NKT cells (DN32.D3) by various lipids in a 384-well plate using the Biomek® 3000 laboratory automated workstation (Beckman Coulter).

Activation of NKT cells leads to the maturation of dendritic cells and efficiently assists priming of antigen-specific immune responses. The lack of polymorphism of CDld molecules and the evolutionary conservation of NKT cell responses highlight the important role of these cells in bridging innate and adaptive immune responses and advocate the value of harnessing this system in clinical settings. Compounds capable of fine tuning NKT cell activation should be actively exploited as potent adjuvants in vaccination strategies or as immunomodulators of autoimmune diseases.

α-Galactosyl-ceramide ( 1 ) has been identified as a powerful modulator of immunological processes through its capacity to bind CD1d molecules and specifically activate invariant natural killer (NK)-like T cells ( i NKT cells). This paper describes the synthesis of 1 , the analogous α-galactosyl-ceramide 3 , and its short chain analogue ‘OCH’ ( 2 ), by use of the 4,6-di- O - tert -butylsilylene (DTBS) protecting group to produce a powerful α-galactosylating agent. In vivo experiments confirmed these compounds to be potent and selective activators of i NKT cells in a CD1d-dependent manner, each inducing a unique profile of cytokine release. This synthesis strategy will permit the generation of novel derivatives for use in the study of the mechanism of i NKT cell activation.