It is well-established that heat shock proteins (HSPs)-peptides complexes elicit antitumor responses in prophylactic and therapeutic immunization protocols. HSPs such as gp96 and Hsp70 have been demonstrated to undergo receptor-mediated uptake by APCs with subsequent representation of the HSP-associated peptides to MHC class I molecules on APCs, facilitating efficient cross-presentation. On the contrary, despite its abundant expression among HSPs in the cytosol, the role of Hsp90 for the cross-presentation remains unknown. We show here that exogenous Hsp90-peptide complexes can gain access to the MHC class I presentation pathway and cause cross-presentation by bone marrow-derived dendritic cells. Interestingly, this presentation is TAP independent, and followed chloroquine, leupeptin-sensitive, as well as cathepsin S-dependent endosomal pathways. In addition, we show that Hsp90-chaperoned precursor peptides are processed and transferred onto MHC class I molecules in the endosomal compartment. Furthermore, we demonstrate that immunization with Hsp90-peptide complexes induce Ag-specific CD8+ T cell responses and strong antitumor immunity in vivo. These findings have significant implications for the design of T cell-based cancer immunotherapy.

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eat shock proteins (Hsps) Hsp70, Hsp90 and gp96 are associated with a broad range of peptides derived from cells in such a manner that the Hsps chaperone the antigenic repertoire of the cells from which are purified. 1) Therefore, vaccination with autologous tumor-derived Hsps has been demonstrated to elicit specific immune responses against tumors from which Hsps were isolated.2-6) Immunization of mice with tumor-derived Hsp-peptide complex is exclusively dependent on the presence of functional APCs in the immunized host, since depletion of such cells renders the host incapable of mounting immune responses after injection of Hsp-peptide complex preparations.2) These phenomena are tumor-specific, peptide-specific and T cell-mediated, but not Hsp-specific per se.On the other hand, priming of CD8 + T cells requires recognition through the T cell receptor (TCR) of peptide-major histocompatibility complex (MHC) class I complexes on the surface of appropriate antigen-presenting cells (APCs), together with expression of adhesion and costimulatory molecules. In general, MHC class I-associated peptides are derived solely from proteins synthesized within the cell. However, bone marrow-derived dendritic cells (DCs) can also translocate antigens from the endocytic to the cytosolic compartment of the cells and thereby direct endocytosed proteins into the MHC class I presentation pathway, a process known as cross-presentation.