Marincek Boris-Christian, Kühnle Marie-Cristine, Srokowski Cathy, Schild Hansjörg, Hämmerling Günter, Momburg Frank
Division of Molecular Immunology (D050), German Cancer Research Center (DKFZ), Heidelberg, Germany.
Mol Immunol. 2008 Nov;46(1):181-91. doi: 10.1016/j.molimm.2008.07.039. Epub 2008 Sep 18.
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.
热休克蛋白（HSPs）可诱导树突状细胞（DC）交叉呈递抗原以及DC成熟。这些特性使HSP抗原复合物成为引发针对肿瘤相关抗原的CD8 T细胞应答的良好候选物。在本研究中，我们分析了HSP70家族的四个不同成员与卵清蛋白（OVA）片段融合作为模型肿瘤抗原的情况。在短期体内免疫试验中，大肠杆菌来源的重组HSP70 - OVA融合蛋白能有效引发抗原特异性细胞毒性T细胞。由于担心HSPs的佐剂效应可能是由于内毒素污染，我们对此问题进行了详细研究。在缺乏LPS受体TLR4的小鼠中，OVA特异性细胞毒性的诱导显著降低。在仔细去除内毒素后，用HSP70 - OVA免疫未能引发细胞毒性T细胞应答。然而，当使用去除内毒素的HSP70 - OVA与TLR9配体CpG寡脱氧核苷酸1668联合使用时，我们获得了强烈的体内杀伤应答。重要的是，用去除内毒素的HSP70 - OVA与CpG进行预防性和治疗性治疗可显著延迟表达OVA的B16黑色素瘤细胞的生长。然而，我们未能检测到针对去除内毒素的重组OVA与去除内毒素的HSP70 - OVA融合蛋白的免疫应答强度有显著差异。因此，当与合适的佐剂联合使用时，用重组HSP70 - 抗原融合蛋白免疫并不比单独使用重组抗原有优势。总之，我们的数据表明，融合蛋白中HSP70部分的佐剂效应在内毒素去除后完全丧失。
