Antigen handling and storage by dendritic cells for improved CTL effector function

To achieve effective cancer immunotherapy, it is crucial to understand the molecular events leading to efficient and optimal induction of tumor-specific T-cell immunity. Detailed knowledge of antigen processing by dendritic cells (DC) is essential for rational design of well-defined therapeutic vaccine formulations. We have demonstrated that long antigenic peptides can be used to induce a strong T-cell mediated anti-tumor immune response. Synthetic long peptides-based vaccination trials in patients with Human Papilloma Virus Type 16 (HPV16) -induced premalignant lesions resulted in complete clinical regression in 50% of the patients. This very promising clinical study shows that therapeutic vaccination works. The mechanisms behind the synthetic long peptides (SLP) vaccine induced tumor regression are only partially understood. SLP were significantly more potent in inducing a CD8+ T-cell response in mice than minimal cytotoxic T-lymphocyte (CTL) peptides. In these mouse models SLP require an obligatory dendritic cell processing step for efficient presentations to the T-cell immune system. For this reason we have studied several different clinically relevant targeting systems for efficient antigen (Ag) delivery into DCs for optimal induction of specific T-cells resulting in an effective anti-cancer immune response. We found that targeting of peptides or protein antigens via FcRs or Toll-like receptors (TLRs) are both highly potent methods of achieving optimal DC activation, efficient and sustained antigen presentation and effective tumor control in vivo. We have recently demonstrated that DCs accumulate exogenously derived antigens into intracellular compartments (antigen depots) thereby aiding sustained MHC class I cross-presentation several days after the DCs has encountered the Ags. DCs with intracellular storage depots facilitates: 1) long-term MHC class I cross-presentation, 2) strong cytotoxic T-lymphocyte cross-priming in vivo several days after antigen challenge, 3) full effectiveness in CTL-mediated tumor control. We found this storage depot to have the character of a lysosome-like compartment with low degradative conditions rather than a MHC class I loading compartment, with distinct features compared to a recently identified early endosomal compartment involved in acute MHC class I antigen presentation. Our findings suggest that DCs require the important function of intracellular storage of exogenous Ags to ensure continuous generation of MHC class I ligands for presentation to T-cells, by facilitating slow release of antigenic peptides. Ag depot formation is crucial in view of the fact that surface MHC class I molecules are very unstable due to a rapid turnover. The intracellular Ag depot enables the DCs to refill the unstable MHC class I molecules with peptides and thereby favors cross-priming and induces long lasting CTL effector function. In this project we will use innovative strategies combining novel tools from cell biology and chemistry to gain detailed insight into antigen handling and storage in dendritic cells. Therefore we will: 1. Characterize our recently identified antigen storage compartment biochemically to better understand its role in MHC class I and MHC class II antigen presentation. 2. Study the subcellular trafficking and handling of endocytosed antigen in dendritic cells upon TLR and FcR mediated targeting. 3. Explore the contribution of proteases involved in MHC class I cross-presentation and MHC class II antigen presentation. 4. Investigate the involvement of autophagy in MHC class I cross-presentation in DCs. 5. Use the knowledge from objective 1-4 for optimal induction of tumor-specific immunity in vivo.