Abstract 1262: Identification of sulfatide reactive type II NKT cells using CD1d dimers.

Natural killer T cells (NKT) are a unique subset of lymphocytes that bridge the gap between innate and adaptive immunity. Unlike conventional T cells that recognize MHC-presented antigen, NKT cells recognize lipid antigen presented by CD1d, and two types of NKT cells recognize different sets of antigens. Type I NKT cells have been extensively studied by using CD1d tetramers or dimers loaded with α-galactosylceramide (α-GalCer), a type I specific lipid antigen. In contrast, studying type II NKT cells has remained elusive because it has been difficult in many labs to make CD1d tetramers to identify this subset with the best characterized antigen known to stimulate specifically a large fraction of type II NKT cells, sulfatide. The methodology used to load α-GalCer onto CD1d has failed to provide reproducible outcomes when used to load sulfatide. In this study, we have developed sulfatide loaded CD1d dimers that can accurately identify type II NKT cells. Using the knowledge of the endogenous lipid loading conditions onto CD1d, we developed a method to load lipid antigens onto CD1d dimers for flow cytometry. By using both saposin C, a chaperone protein necessary for lipid loading, and acidic conditions, as found in the endosome in which lipid loading onto CD1d occurs, we can reliably create sulfatide-loaded CD1d dimers to identify type II NKT cells. Sulfatide-loaded CD1d dimers specifically identify type II NKT cells in both WT and type I NKT deficient mice, but do not detect cells in CD1d -/- mice that lack both types of NKT cells, as expected. The specificity of the binding of dimers through TCRβ is also confirmed by TCRβ blockade. Consistent with previous findings in the liver, the sulfatide-reactive type II NKT cell population encompasses about 2% of all lymphocytes, and is completely distinct from its type I counterpart, which represents approximately 10% of liver lymphocytes; no double positive cells were detected. In addition, although the predominant Vβ usage for both subsets is Vβ8.1/8.2, the type II cells include cells expressing Vβ9 and 13, but not Vβ 2 and 7, in contrast to type I NKT cells. Additionally, we observed a sulfatide reactive NKT population in human PBMCs that is Vα24-Vβ11- and comparable in numbers to type I NKT cells, which are Vα24+Vβ11+. All these data suggest that the method we developed can create sulfatide-loaded CD1d dimers in a highly reproducible manner that can identify type II NKT cells. This method to load glycolipids antigens onto recombinant CD1d molecules has enabled us to not only identify a type II NKT cell subset based on previous knowledge of these cells, but also expand on that by potentially identifying other markers. Ultimately, this method of loading previously problematic lipids onto CD1d dimers may allow for their use to identify many more subsets of NKT cells across multiple models. Citation Format: Stanley Parish, Liat Izhak, Zheng Xia, Motoshi Suzuli, Masaki Terabe, Jay A. Berzofsky. Identification of sulfatide reactive type II NKT cells using CD1d dimers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1262. doi:10.1158/1538-7445.AM2013-1262