Dendritic cells (DCs) have multiple functions in regulating immune responses, including the initiation of immune responses to pathogens and the maintenance of peripheral tolerance by regulating the numbers and states of self-reactive T cells. Strategies to selectively access these different DC functions could greatly advance immunotherapy and facilitate the design of more rational, mechanistic vaccines. In this proposal, we describe experiments that utilize chemical synthesis technologies to target DC-specific lectins. Carbohydrate-binding C-type lectins comprise a class of DC receptors with unique family members expressed on each DC subtype in mouse and human. Recent experimental evidence1 demonstrates that targeting antigen to CD11c+CD8+ dendritic cells (DCs) via an endocytic cell-surface lectin (DEC-205) results in antigen-specific T cell anergy, deletion and, more recently, the formation of regulatory T cells2. Furthermore, engagement of DC-SIGN, BDCA-2, and DCIR, three other DC-specific lectins, has been shown to inhibit aspects of DC maturation and may also lead to T cell tolerance. In preliminary experiments, we have synthesized five structures derived from (Man)9G1cNAc2, coupled them to a model antigen (ovalbumin), and demonstrated binding and internalization by the human DC-specific lectin DC-SIGN in cells expressing this receptor. Extending these studies to murine DCs, we have observed enhanced (Man)9-ovalbumin presentation to antigen-specific CD4+ and CD8+ T cells in vitro and in vivo, which is enhanced further by the presence of excess mannose-BSA which blocks non-specific binding of conjugates to abundant monosaccharide-binding lectins). In this proposal, we would like to address the mechanism of action and immunological consequences of (Man)9-ovalbumin enhancement of CD4 and CD8 T cell activation. To characterize the molecular basis for enhanced presentation, we will: 1) characterize the binding of (Man)9-ovalbumin to DC and non-DC lectins in order to increase binding to DC lectins and decrease binding to non-DC lectins; 2) identify the targeted antigen presenting cell that mediates enhanced presentation; 3) assess the immunological consequences of (Man)9-ovalbumin immunization in mice by determining the fate of ovalbumin-specific T cells. The synthesis and biological studies of these conjugates will support the development of therapeutics for tolerance and immunity and advance our understanding of carbohydrate-lectin interactions at both structural and physiological levels.
| Adams, Eddie W; Ratner, Daniel M; Seeberger, Peter H et al. (2008) Carbohydrate-mediated targeting of antigen to dendritic cells leads to enhanced presentation of antigen to T cells. Chembiochem 9:294-303 |
