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- Materials and methods
Vaccination with heat shock proteins (HSP) protects mice from challenge with the tumor from which the HSP were isolated. The antigenicity of HSP vaccination is thought to result from HSP-associated endogenous major histocompatibility complex class I peptides or their precursors. The vaccination effect can be achieved in an adjuvant-free manner and is mediated by CD8+ T cells, indicating that HSP can act as a natural adjuvant and cross-prime T cells in vivo. We previously devised a recombinant vaccine composed of a CD8+ T cell epitope fused to the carboxyl-terminus of hsc70 and demonstrated efficient generation of antigen-specific cytotoxic T lymphocyte (CTL) after vaccination with a few micrograms of the hsc70-CTL epitope fusion protein. The present study aimed to determine if the fusion protein vaccine could control tumor growth in vivo and whether simultaneous fusion of a CD4+ T cell epitope to the amino terminus of the hsc70-CTL epitope would be a more potent vaccine compared to the CTL epitope alone. Ovalbumin (OVA)–derived 8 mer peptide, OVA257-264, and 16mer peptide, OVA265-280, were used as CD8+ and CD4+ T cell epitopes, respectively. Vaccination with hsc70-OVA257-264 generated peptide specific CTL more effectively than a peptide plus incomplete Freund's adjuvant combination, and suppressed growth of OVA expressing EL4 (E.G7) and B16 melanoma tumor cells. Addition of OVA265-280 to the amino-terminus of hsc70-OVA257-264 (OVA265-280-hsc70-OVA257-264) enhanced the generation of the OVA257-264-specific CTL population, leading to better eradication of MO5 lung metastasis compared to hsc70-OVA257-264. Our results suggest that fusion of both CD4+ and CD8+ T cell epitopes to hsc70 enhances tumor immunity beyond the effect of the CD8+ T cell epitope alone. (Cancer Sci 2008; 99: 1008–1015)
Heat shock proteins (HSP) have been implicated as tumor rejection antigens in vivo, for example vaccination with HSP gp96, hsp90, and hsc70, even without adjuvants, protects mice from challenge by the tumors from which the HSP were originally isolated.(1–3) The immunogenicity of HSP vaccines has been suggested to result from carry-over of endogenous peptides that were associated with HSP in cells, and not due to HSP per se.(1) Indeed, we previously identified major histocompatibility complex (MHC) class I ligands or their precursors that were acid-eluted from gp96, hsp90, and hsc70 derived from the radiation leukemia, RL♂1.(4) An important observation from those studies was that the quantity of tumor antigen peptides associated with HSP was no more abundant than we expected. Thus, it was surprising that such a tiny amount of peptide was sufficient to prime CD8+ T cells that enabled tumor rejection in vivo. These findings raise the fascinating possibility that HSP may serve as potent and safe natural adjuvants, better than any now available.
The recent observation that toll-like receptors (TLR) 4 and 2 interact with gp96 or hsp70, resulting in production of pro-inflammatory cytokines by antigen presenting cells (APC)(5,6) may provide the rationale behind the strong HSP adjuvant effect, since activation of APC is essential for the full-priming of T cells. In addition, HSP seemed to target a number of receptors such as CD91,(7) LOX-1,(8) and SRA(9) on dendritic cells (DC) or macrophages in vivo. HSP are efficiently captured by these receptors on DC and internalized into endosome/lysosome compartments in which peptides associated with HSP are channeled into the cytosol, probably through a retro-translocation mechanism via a translocon consisting of Sec61 and p97.(10) The peptides released into the cytosol are on a track to a classical MHC class I antigen-processing pathway to be presented to CD8+ T cells. This conceptual mechanism for HSP-induced immunity encouraged us to construct a novel recombinant vaccine in which MHC class I ligands are covalently fused to either the carboxyl- or amino-terminus of hsc70. In these fusion proteins, each hsc70 molecule contained at least one T cell epitope, thus the number of specific peptides associated with HSP was dramatically increased, in comparison with the natural HSP purified from tumor cells. Moreover, presentation of HSP-bound self-peptides that might evoke autoimmunity could in principle be avoided. We previously showed that specific CTL were easily generated against five distinct CD8+ T cell epitopes by vaccination with a few micrograms of those hsc70-peptide complexes,(11) but the protective efficacy of this approach against in vivo tumor growth has not been investigated. Another issue to be addressed is how CD4+ T cells modulate HSP-mediated immunity. HSP-induced cross-priming of CD8+ T cell does not require CD4+ T cells.(11,12) Nonetheless, it is also true that antigen-specific CD4+ T cells are activated by vaccination with antigen-HSP complexes.(13)
In the present study, we investigated whether vaccination with CTL epitopes fused to hsc70 could protect against tumor growth in vivo. We also examined whether simultaneous fusion of a helper epitope in addition to CTL epitope to hsc70 gives rise to the enhanced protection over the effect of CTL epitope alone.
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- Materials and methods
HSP are evolutionally conserved across species and are constitutively expressed in all cells. Their expression level is dramatically increased by heat shock, a phenomenon termed the heat shock response. Heat shock response is caused not only by heat but also by other stresses such as heavy metals, hypoxia, alcohol intoxication, glucose starvation, and even cytokine stimulation.(16) HSP stimulate proper folding of proteins damaged by these stressors and thus constitute a protein salvage system. On the other hand, it was recently suggested that HSP are also linked to the ubiquitin-proteasome pathway to degrade proteins.
Hsp90 and hsc70 recognize unfolded proteins and peptides in the cytosol, where they recruit the E3 ubiquitin ligase CHIP (carboxyl terminus of hsc70 interacting protein) to polyubiquitinylate these substrates.(17,18) The polyubiquitinylated proteins are degraded by the 26S proteasome, and some of the degradation products can potentially serve as peptide ligands for MHC class I molecules.
The proteins and peptides bound to HSP, once released outside cells, are efficiently trapped by antigen-presenting cells via a number of HSP receptors and can then be cross presented to CD8+ T cells in the context of MHC class I molecules.(19) In this context, mild biochemical procedures for the purification of HSP from tumor cells allow the copurification of unknown tumor antigens bound to HSP, which is the rationale behind the use of antigen-HSP complexes for cancer vaccine therapy without knowing the identity of the tumor antigens recognized by T cells. One disadvantage of this vaccine approach may be the quantity of tumor antigens bound to HSP. Thus, many HSP isolated from tumor cells seem to lack associated tumor antigens.(4) Fusion of mini genes encoding T cell epitopes to HSP cDNAs to construct a fusion protein may be a solution to this problem. Indeed, we previously found efficient generation of CTL by vaccination with several epitopes fused to hsc70.(11) A question that remained to be addressed was whether this type of vaccine could eradicate tumors in vivo. In the present study, we have demonstrated that vaccination with a CD8+ T cell epitope fused to hsc70 resulted in a significant protective effect against in vivo tumor growth, and that simultaneous fusion of an epitope recognized by CD4+ T cells to HSP increased the population of primed CD8+ T cells, resulting in enhanced eradication of tumors.
Although antigens are readily cross-presented to CTL in vitro by DC or other antigen presenting cells pulsed with HSP-epitope complexes, these results do not necessarily imply that administration of these HSP-epitope complexes in vivo will generate CTL efficiently enough to eradicate tumors. In order to obtain in vivo protection, T cells need to be fully activated, which can be accomplished by suitable activation of DC with HSP. Hsp70 and Gp96 have been shown to interact with toll-like receptors 4 and 2 (TLR4 and 2),(5,20) an encounter that activates DC to fully prime T cells. However, the idea that DC can be activated with HSP is still controversial.(21) Endotoxin contaminating of the recombinant proteins, even though rigorously removed, might, at least in part, participate in activation of DC.(22,23) On the other hand, Datta et al. showed that among agonists for various TLR, only those for TLR3,7 and 9 but not for TLR2 and 4, enabled DC to cross-prime CD8+ T cells.(24) We also observed that cross-priming by hsp70 fusion proteins occurs equally well in vivo, even in TLR2 and 4 double KO mice (data not shown). These results suggest that the effect of endotoxin contamination is minimum or negligible in cross-priming. It is possible that interaction between CD40 and hsp70 activates DC, as previously demonstrated.(25)
Addition of a CD4+ T cell epitope to the hsc70-CTL epitope augmented the vaccination effect, especially in the MO5 lung metastasis model (Figs 7–9). This effect apparently resulted from an increased population of IFN-γ-producing CD8+ T cells as determined by ELISPOT assay (Fig. 4). Both helper and CTL epitopes should be expressed on the same DC because the two epitopes are within a single hsc70 molecule. Therefore, there may be simultaneous activation of both CD4+ and CD8+ T cells on the surface of a single DC. Activated CD4+ T cells express CD40L, which can interact with CD40 on DC. Thus, conditioning of DCs by CD4+ T cells is a likely possibility, and this might provoke much more efficient activation of CD8+ T cells in vivo.(26,27) Along this line, we attempted to generate OVA265-280 specific CD4+ T cells in vitro by peptide stimulation but were unsuccessful.
From a clinical viewpoint, it is important to successfully produce a fusion protein containing hsc70 together with an epitope from a human cancer antigen. In this context, we recently demonstrated that ESO p157–165 of NY-ESO-1, a cancer/testis antigen, fused to human hsc70, was cross-presented by DC to specific CTL clone, although the hsc70 preparation did not induce maturation of DC.(28) Moreover, repetitive stimulation of CD8+ T cells with human DC pulsed with the fusion protein generated CTL against ESO p157–165. Taken together, the use of HSP-peptide complexes for cancer vaccination is a reasonable approach, not only from theoretical and practical perspectives.