Natural killer T (NKT) cells are potent, innate-like T cells involved in the regulation of immune responses ranging from reaction to pathogens and tumors to the development or suppression of autoimmunity. Activation of NKT cells may provide new therapies for viral and bacterial infections, for cancer and for a range of autoimmune conditions, such as Type I diabetes, multiple sclerosis and lupus. NKT cells are divided into two major classes, Type I (iNKT) and Type II NKT cells. Both are activated by glycolipids which serve as ligands for an antigen presenting protein, CD1d. Glycolipid activation of iNKT cells has been extensively studied for more than two decades. Much less is known about the Type II cells. Crucial to utilizing NKT cell stimulation therapeutically is the ability to control the nature of the response. Although a number of glycolipids that elicit Th1 (relevant for treatment of cancer and infections) or Th2 (relevant for autoimmune conditions) cytokines from iNKT cells have been identified, the bias often disappears in vivo, or the compounds fail to activate human iNKT cells. Another need in the NKT cell arena is identification of potent activators of sulfatide-reactive Type II NKT cells and development of baseline structural activity relationships in these much less explored, but more abundant in humans, NKT cells. In collaboration with immunologists, structural biologists and physical chemists this proposal has two major thrusts: 1) Design, synthesize and evaluate carbohydrate modified glycolipids that elicit a Th1 biased cytokine response from iNKT cells in contexts relevant for the development of human anti-cancer therapies. To realize this goal three hypotheses will be tested: a) More potent, clinically relevant C4”-analogs can be identified; b) Dual modifications of the ceramide and C6”-position of the sugar can be used to enhance potency and Th1 bias; c) Photochemically cross-linked glycolipid/CD1d conjugates can be used as an avenue for antigen delivery and to further enhance Th1 bias. Each hypothesis is based on solid preliminary studies involving human cell lines and or a humanized mouse model. The studies will elucidate the potential of the carbohydrate moiety for enhancing Th1 bias. The second major thrust is: 2) To continue to build understanding of sulfatide-reactive Type II NKT cells. Most evidence has suggested that the activation of these Type II cells counter-regulates iNKT cells; however results from the current funding period have shown that certain sulfatides elicit responses similar to iNKT cells. Current results and outcomes from new, proposed sulfatides are being/will be used to develop and refine pharmacophoric models for designing potent agonists for Type II NKT cells.