Bystander secretion of hsp70 provides adjuvant effect throughout DC-NK interaction Free

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Tumor derived heat shock proteins (HSP) can induce antigen specific immune response. Studies performed with purified proteins suggest that HSP interact with dendritic cells (DC) providing them with chaperoned antigens and danger signals that allow their maturation and, consequently, proper T cell activation. To dissect the chaperone and adjuvant activity of hsp70, independently from possible contaminations that might occur during protein purification, we modified murine inducible hsp70 into a secreted protein and engineered a transformed fibroblast cell line, F1aFR, to constitutively secrete hsp70 into the culture supernatant. DC incubated with supernatant from hsp70-transfected F1aFR cells bind the protein and, after its internalization, cross-present F1aFR derived peptides to specific T cell lines in vitro. Thus, despite genetic manipulation, secreted hsp70 retains the chaperone activity and enhances tumor immunogenicity in vivo as evaluated by reduced tumor growth and by superior CTL response against F1aFR antigens. The danger activity of secreted hsp70 varies according to the source of DC. Whereas both bone marrow and spleen derived DC show increased uptake of either fluorescent beads or apoptotic cells in the presence of hsp70-containing supernatant, only splenic DC display a small increase in the expression of CD86 costimulatory molecule. The bystander adjuvant activity of hsp70 on DC antigen uptake was confirmed in vivo. Mice were injected with CFSE-labeled C26 cells together with bystander fibroblast secreting or not hsp70. The percentage of CFSE+ DC in the draining lymph node was significantly higher in lymph node from mice receiving fibroblasts secreting hsp70. Accordingly, an enhanced CTL response against C26-derived epitopes was obtained when bystander cells secreting hsp70 where part of the cellular vaccine. However, the almost similar efficacy of hsp70 to induce CTL against either chaperoned or bystander antigens cannot be translated in a therapeutic setting. Mice bearing C26aFR lung metastasis are better cured by a vaccine in which hsp70 is secreted directly by the antigen cell source (F1hsp70aFR) rather than by a bystander cell (F1aFR + F1hsp70). The bystander but not chaperone activity of hsp70 was then lost in mice depleted of NK cells. While uncoupling the danger signal from the chaperone activity of hsp70 slightly reduce vaccine efficacy, the results point to the possibility of using a bystander cells secreting soluble HSP to be admixed with autologous tumor, a practical way to create cellular cancer vaccines.