Detection and activation of human Valpha24+ natural killer T cells using alpha-galactosyl ceramide-pulsed dendritic...

Human CD1d-restricted natural killer T (NKT) cells, which are postulated to regulate the immune response in several clinical settings, can be activated by alpha-galactosylceramide (alpha-GalCer) presented by CD1d molecules on antigen presenting cells (APCs). Simple methods to quantify NKT function in fresh blood will greatly benefit studies targeting NKT cells in humans. Here we show that freshly isolated human NKT cells can be readily quantified by an enzyme-linked immunospot (ELISPOT) assay and have a Th1 profile (secreting interferon-gamma, but not IL-4), after stimulation using alpha-GalCer loaded APCs. Using this assay, we also evaluated APC requirements for human NKT cell activation in fresh blood. Monocyte-derived dendritic cells (DCs) are more effective than monocytes/macrophages for detecting and activating NKT cells in fresh blood, with mature alpha-GalCer pulsed DCs being optimal. DCs are also efficient APCs for expanding NKT cells in culture and generating NKT cell lines. NKT cells expanded with DCs were functional, secreting both IFN-gamma and IL-4, and killing NKT-sensitive targets. Optimal activation of these lines was seen using mature DCs loaded with 10-100 ng/ml of alpha-GalCer. DCs matured with several different stimuli were effective. These data help to establish the conditions for loading DCs with alpha-GalCer for immune therapeutic targeting of NKT cells, and provide a new simple assay to monitor NKT function in humans.