The studies described here indicate that HSP–peptide complexes can be reconstituted in vitro and that, by all parameters tested, such complexes show immunological activity similar to the HSP–peptide complexes generated in vivo. The results also show significant differences between gp96 and hsp70 with respect to the conditions in vitro, under which they bind peptides. These differences presumably reflect the fact that although hsp70–ATP interaction plays a crucial role in hsp70–peptide interaction in vivo, the identity of the corresponding ligand for gp96 is presently unknown. In contrast with the situation with hsp70, gp96–ATP interaction does not strip gp96 of its associated peptides (data not shown), even though gp96, like hsp70, is an ATP-binding protein and is an ATPase (11
). Exposure to high temperature and high salt apparently causes the gp96 molecule to assume an open conformation, which permits dissociation from and association with exogenous peptides. The identity of the ligands that catalyze this process in vivo would be of interest in this regard.
The observations reported here have several implications. First, they support the hypothesis that immunogenicity of tumor-derived gp96 preparations results from a physical association of gp96 with antigenic peptides. The HSP–peptide complex elicits immunity under conditions in which the HSP molecules alone, or the peptides alone, do not. Second, these observations show that one does not have to rely on HSP–peptide complexes generated in vivo to elicit immunity; instead, such complexes can be generated reproducibly in vitro, provided the identity of the immunogenic peptides is known. A variety of peptides of different lengths, compositions, and hydrophobicity can bind the HSPs, suggesting that the nature of an epitope is not a limiting factor in its suitability as a vaccine in the form of a HSP–peptide complex. The ability of the gp96 to bind peptides in vitro has also been independently demonstrated recently (20
). The quantity of peptide that is required to be conjugated to the HSPs is extremely small and 1–2 ng of peptides complexed to the HSPs elicit potent cellular immune response. At first sight, this quantity may appear to be unrealistically small; however, when it is considered that the peptides chaperoned by the HSPs are targeted specifically to the professional antigen-presenting cells (15
), 1–2 ng or ~6 × 1011
molecules of specific peptide targeted to the relevant antigen-presenting cells are actually a large number, as argued in more detail elsewhere (22
). This observation has significant implications for vaccination against infectious diseases in which the protective epitopes are known, and for any cancers, such as those of viral etiology, that may share antigenic epitopes.
Essentially, these results show that HSPs are adjuvants. This adjuvanticity has a number of unique characteristics: in contrast with other nonlive adjuvants, the adjuvanticity of HSPs generates MHC class I–restricted T cell responses. No serological antipeptide response has ever been detected among the tens of immunized mice tested (data not shown). The quantitative requirements of antigens administered with HSPs are log scales lower than corresponding requirements for other adjuvants. Finally, HSPs are the first adjuvants of mammalian origin. We have suggested previously that the immunogenicity of HSP–peptide complexes may reflect the role in vivo of such complexes in priming of cellular immune responses (23
). In this view, the observed adjuvanticity of HSPs is simply a reflection of the natural role of HSPs in vivo.
The structural basis of the ability of gp96 molecules to bind a variety of peptides is presently unclear and requires further study. Obviously, there are certain rules for the HSP–peptide interaction as seen in the observation that peptides differ in their ability to compete with a given peptide for binding to gp96 (Fig. ). However, the studies carried out here are not of a broad enough scope to permit elucidation of these rules. Broadly speaking, HSP–peptide interaction is reminiscent of MHC–peptide interaction, which was equally mysterious as to its structural basis until the rules of interaction were identified (24
). The MHC and the HSPs share a number of crucial properties, such as the ability to bind peptides, a ubiquitous tissue distribution, high degree of phylogenetic conservation, inducibility of the respective genes by IFN-γ (25
) and, finally, the ability to prime CTL responses against the peptides chaperoned by them. These considerations led us in the past (26
) to suggest a phylogenetic relationship between the MHC and the HSPs, and a number of recent observations (19
) have not been inconsistent with that suggestion. The association of peptides with HSPs of the cytosol (hsp70 and hsp90) and the endoplasmic reticulum (gp96) had also led us to suggest that HSPs constitute a relay line of molecules that chaperones the peptides and ultimately delivers them to the MHC class I molecules (23
). Therefore, the HSPs were suggested to be accessories to antigen presentation by MHC class I molecules. Our recent results, which show that peptides precursors to the MHC class I–binding epitopes are found in specific association with hsp70, hsp90, and gp96 (Ishii et al., manuscript submitted for publication), are in accord with our suggestion. The recent demonstration by Lammert et al. (29
) that the HSP gp96 acts as a major peptide acceptor for peptides transported into the lumen of the endoplasmic reticulum through transport-associated protein molecules, also supports the relay-line hypothesis.