Heat shock protein-antigen fusions lose their enhanced immunostimulatory capacity after endotoxin depletion.

Heat shock proteins (HSPs) induce cross-presentation of antigens by dendritic cells (DC) as well as DC maturation. These properties make HSP antigen complexes good candidates to prime CD8 T cell responses against tumor-associated antigens. In this study, we analyzed four different members of the HSP70 family fused to a fragment of ovalbumin (OVA) as a model tumor antigen. E. coli-derived recombinant HSP70-OVA fusion proteins efficiently primed antigen-specific cytotoxic T cells in short-term in vivo immunization assays. Because of concerns that the adjuvant effect of HSPs may be due to endotoxin contamination, we studied this issue in detail. Induction of OVA-specific cytotoxicity was significantly decreased in mice deficient for the LPS receptor, TLR4. After careful removal of endotoxins, immunization with HSP70-OVA failed to prime cytotoxic T cell responses. However, we obtained strong in vivo kill responses when endotoxin-depleted HSP70-OVA was used in combination with the TLR9 ligand CpG oligodeoxynucleotide 1668. Importantly, prophylactic and therapeutic treatment with endotoxin-depleted HSP70-OVA together with CpG significantly delayed the outgrowth of OVA-expressing B16 melanoma cells. However, we were unable to detect significant differences in the magnitudes of immune responses against endotoxin-depleted recombinant OVA vs. endotoxin-depleted HSP70-OVA fusion protein. Thus, immunization with recombinant HSP70-antigen fusion protein does not provide an advantage over recombinant antigen alone when combined with a suitable adjuvant. Altogether, our data suggest that the adjuvant effect of the HSP70 part of the fusion protein is completely lost after endotoxin removal.

Calcium signaling in dendritic cells by human or mycobacterial Hsp70 is caused by contamination and is not required for Hsp70-mediated enhancement of cross-presentation.

Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation.