Natural killer T cells (NKT) are a unique subset of lymphocytes that bridge the gap between innate and adaptive immunity by recognizing lipid antigen presented by CD1d. While type I NKT cells have been extensively studied by using CD1d multimers loaded with α-galactosylceramide (α-GalCer), a type I specific antigen, studying type II NKT cells has lagged behind, since loading sulfatide, a type II-specific lipid antigen, to the tetramer has been difficult to accomplish reliably. In this study, we have developed sulfatide loaded CD1d dimers that accurately identify type II NKT cells by flow cytometry. Using saposin C, a lipid loading chaperone protein, and acidic conditions, as found in the natural loading environment, we could create sulfatide-loaded CD1d dimers to identify this subset. Sulfatide-loaded CD1d dimers specifically identified type II NKT cells in both WT and type I NKT deficient mice, and were not observed in NKT cell-deficient CD1d-/- mice. The specificity of the binding through TCRβ was confirmed by TCRβ blockade. Consistent with previous findings, in the liver, the sulfatide-reactive type II NKT cells were a distinct population that encompasses about 2% of liver lymphocytes. These data suggest that the method we developed can create sulfatide-loaded CD1d dimers to identify type II NKT cells, and has enabled us to identify other markers of this subset. This method can be used to load other lipids onto CD1d to identify other NKT subsets across multiple models.
- Copyright © 2013 by The American Association of Immunologists, Inc.