热休克肿瘤细胞上清及EXOSOME中趋化因子的发现及一种非细胞结构性新型高效肿瘤疫苗的研究
|
摘要
恶性肿瘤严重威胁着人类的健康，在各种疾病所致的死亡中，恶性肿瘤现已成为主要病因，并有进一步增高的趋势。目前的传统治疗方法，虽有一定疗效，但却十分有限。生物治疗的研究给肿瘤治疗带来了新的希望，特别是肿瘤疫苗的研制更成为目前肿瘤治疗研究的热点之一。
现有的比较受到关注的制备肿瘤疫苗的方法，包括了如各种细胞因子、共刺激分子、肿瘤抗原肽等基因修饰的肿瘤细胞疫苗；体外抗原致敏的或肿瘤抗原肽基因修饰的树突状细胞疫苗；肿瘤细胞与抗原提呈细胞融合疫苗等。这些肿瘤疫苗各有特色，但总体来说，制备工艺复杂，效果相对不够理想。
我们在研究热休克蛋白与肿瘤免疫之间的关系时，发现了肿瘤细胞经热休克后，其培养上清对树突状细胞(Dendritic cell，DC)及T淋巴细胞有明显的趋化作用，继而证实了肿瘤细胞热休克后上清中含有多种趋化因子。由于这种趋化作用在热休克后很快就表现出来，因而我们推测可能是肿瘤细胞通过某种特殊的分泌形式，将趋化因子排出胞外。进一步的实验证实这种外排形式就是EXOSOME，因为我们发现肿瘤细胞热休克上清中的EXOSOME含有多种高浓度的趋化因子，而分离出EXOSOME后的上清中却几乎检测不到趋化因子。同时，我们还发现了在热休克后EXOSOME中含有高水平的MHC Ⅰ类和Ⅱ类分子以及多种共刺激分子、粘附分子、肿瘤抗原提呈的伴侣分子HSP70等。由于这种EXOSOME来自于肿瘤细胞，携带有肿瘤抗原，又有几乎所有目前研制的高效肿瘤疫苗所必须的公刺激信号的特性，强烈提示这是一种非细胞性的新型疫苗。接着进行的动物体内实验证实这种新型疫苗不仅能够保护小鼠免受同系以及不同系肿瘤细胞的攻击，而且对小鼠已形成的肿瘤亦有明显的治疗作用。这种新型高效的肿瘤疫苗，由于其为非细胞
卜
【
g
博士论文（（热休克肿周细胞上消及＊＊＊S＊N田中趋化因子的发现及一种非细胞结构性新型高效肿瘤疫苗的研究。
＿s
g
性结构，且未经基因修饰和外源基因导入，因此对机体潜在危害作用小，没有伦
理的难题，且制备简单易行，因而有可能成为一种极有临床应用前景的新型生物
制剂和疫苗。
g
一．热休克及HSP70诱导肿瘤细胞合成和分泌多种趋化因子，体外能够趋化树突状g
细胞和T淋巴细胞
】
我们发现热休克后的细胞培养上清可以趋化DC和 T细胞，而且上清中含有大
且
量的HSp70。用人和小鼠的多种细胞系（人肺癌细胞系A549、人结肠癌细胞系LW趴
5
人宫颈癌细胞系He la、人乳腺癌细胞系MCF7、小鼠Le。is肺癌细胞系3LL、小鼠
结肠癌细胞系CT26、小鼠黑色素瘤细胞系B16和正常表型的小鼠成纤维细胞系
3‘r3、小鼠骨髓基质细胞系K－1和小鼠骨髓基质内皮细胞系m7B）43℃热休克lh
后，在 37℃孵箱内恢复 4h，1，0009离心 10min，收取上清，蛋白定量后进行体外
趋化实验。实验结果发现热休克后的人和小鼠的细胞培养上清对DC和 T细胞有明
显的趋化作用，可以使 DC和 T细胞穿过基质内皮细胞迁移到下层趋化小室中，提
示上清中可能含有具有趋化活性的物质。同时由于多种肿瘤细胞系及正常细胞系
均显示出在热休克后上清的趋化活性，因此我们认为，热休克后趋化活性增加是
一种普遍的现象，而在本文中我们仅针对肿瘤细胞细胞进行了深入的研究。由于
有研究发现HSP70在热休克或热疗后显著升高，因此我们用Western blot检测了
肿瘤细
Malignant tumor is one of the threats of human health. Among the diseases causing death, tumor has climbed to be the one of the major killer and is still becoming more and more imposing. At present, the conventional treatments are always inefficient. Biotherapy, especially tumor vaccine, has brought new hopes for human beings who have suffered a lot from tumors and have become one of the focuses of tumor treatment and studies. Methods employed to prepare vaccine at present include tumor cells modified with cytokines, costimulatory molecules and tumor antigenic peptides, dendritic cells (DC) primed with tumor antigens in vitro or genetically modified with tumor antigens and the fusion of tumor cells with antigen presenting cells (APC). Though with various advantages, these vaccines are always complicated to prepare and the effects are relatively not promising.
During the study of the relationship between heat shock protein (HSP) and tumor, we found that after heat shock, culture supernatants of tumor cells could chemoattract DC and T lymphocytes. And we went further verify that chemokines were present in the culture supernatants of tumor cells after heat shock. Because of the rapid presence of this activity, we hypothesis that chemokines be released out of tumor cells through a particular secretory form. Our further studies showed that this kind of exocytosis form is exosome as we detected various chemokines in the exosomes derived from tumor cells after heat shock and no chemokines were detected in the supernatants after isolation of exosomes. We also found that abundant MHC molecules, costimulatory molecules, adhesion molecules and chaparones carrying tumor antigens. As the exosomes are derived from tumor cells, carry tumor antigens and share
characters with most of the vaccines prepared previously, they are a kind of non-cellular novel vaccine. In vivo experiments showed that this kind of novel vaccine could not only protect mice from syngeneic and allogeneic tumor challenge but inhibit the growth of established tumors. This novel and efficient vaccine, with the non-cellular structure and contained no exogenous gene modification, is potentially no harm to the recipients. Together with the characters that it cause no ethical problems and it is easy to prepare, this non-cellular vaccine is promising and economic both in clinic and in market.
Heat shock and hsp70 can stimulate tumor cell produce chemokines that can chemoattract DC and T lymphocytes in vitro.
Tumor cell culture supernatant after heat shock could chemoattract DC and T cells, and hsp70 was abundantly present in the supernatants. After heat shock of human and mouse cell lines A549, LoVo, HeLa, MCF7, 3LL, CT26, B16, NIH3T3, TC-1 and 107B for Ih at 43癈 and recovery in 37癈 incubator for 4h, the culture supernatants can chemoattract DC and T cells in an in vitro chemotactic assay, indicating that chemotactic products are present in the supernatants. Considering that various cell lines demonstrate the same kind of chemotactic activity, it can be concluded that this effect is a kind of universal phenomenon. As it has been reported that HSP70 can stimulate CD8+ T cells to produce chemokines, we stimulate tumor cells 3LL with HSP70 (1 u g/ml) for 4 h and collect the culture supernatants to perform in vitro chemotaxis assay. We found that the supernatants can also chemoattract DC and T cells. To exclude the possibility that HSP70 can chemoattract DC and T cells directly, we add HSP70 to the lower well and we found no chemoattraction happened. Thus HSP70 can stimulate the chemoattracting product to produce. As chemokines are the major proteins inducing cell migration, we examined the mRNA expression in 3LL cells after heat shock or HSP70 stimulation and protein
expression in 3LL culture supernatants by RT-PCR and Western blot respectively. We found that, after heat shock and HSP70 stimulation, various chemokines are upregulated at mRNA level in 3LL cells and at protein level in the culture supernatants. HSP70 alone can't completely substitute for h
现有的比较受到关注的制备肿瘤疫苗的方法，包括了如各种细胞因子、共刺激分子、肿瘤抗原肽等基因修饰的肿瘤细胞疫苗；体外抗原致敏的或肿瘤抗原肽基因修饰的树突状细胞疫苗；肿瘤细胞与抗原提呈细胞融合疫苗等。这些肿瘤疫苗各有特色，但总体来说，制备工艺复杂，效果相对不够理想。
我们在研究热休克蛋白与肿瘤免疫之间的关系时，发现了肿瘤细胞经热休克后，其培养上清对树突状细胞(Dendritic cell，DC)及T淋巴细胞有明显的趋化作用，继而证实了肿瘤细胞热休克后上清中含有多种趋化因子。由于这种趋化作用在热休克后很快就表现出来，因而我们推测可能是肿瘤细胞通过某种特殊的分泌形式，将趋化因子排出胞外。进一步的实验证实这种外排形式就是EXOSOME，因为我们发现肿瘤细胞热休克上清中的EXOSOME含有多种高浓度的趋化因子，而分离出EXOSOME后的上清中却几乎检测不到趋化因子。同时，我们还发现了在热休克后EXOSOME中含有高水平的MHC Ⅰ类和Ⅱ类分子以及多种共刺激分子、粘附分子、肿瘤抗原提呈的伴侣分子HSP70等。由于这种EXOSOME来自于肿瘤细胞，携带有肿瘤抗原，又有几乎所有目前研制的高效肿瘤疫苗所必须的公刺激信号的特性，强烈提示这是一种非细胞性的新型疫苗。接着进行的动物体内实验证实这种新型疫苗不仅能够保护小鼠免受同系以及不同系肿瘤细胞的攻击，而且对小鼠已形成的肿瘤亦有明显的治疗作用。这种新型高效的肿瘤疫苗，由于其为非细胞
卜
【
g
博士论文（（热休克肿周细胞上消及＊＊＊S＊N田中趋化因子的发现及一种非细胞结构性新型高效肿瘤疫苗的研究。
＿s
g
性结构，且未经基因修饰和外源基因导入，因此对机体潜在危害作用小，没有伦
理的难题，且制备简单易行，因而有可能成为一种极有临床应用前景的新型生物
制剂和疫苗。
g
一．热休克及HSP70诱导肿瘤细胞合成和分泌多种趋化因子，体外能够趋化树突状g
细胞和T淋巴细胞
】
我们发现热休克后的细胞培养上清可以趋化DC和 T细胞，而且上清中含有大
且
量的HSp70。用人和小鼠的多种细胞系（人肺癌细胞系A549、人结肠癌细胞系LW趴
5
人宫颈癌细胞系He la、人乳腺癌细胞系MCF7、小鼠Le。is肺癌细胞系3LL、小鼠
结肠癌细胞系CT26、小鼠黑色素瘤细胞系B16和正常表型的小鼠成纤维细胞系
3‘r3、小鼠骨髓基质细胞系K－1和小鼠骨髓基质内皮细胞系m7B）43℃热休克lh
后，在 37℃孵箱内恢复 4h，1，0009离心 10min，收取上清，蛋白定量后进行体外
趋化实验。实验结果发现热休克后的人和小鼠的细胞培养上清对DC和 T细胞有明
显的趋化作用，可以使 DC和 T细胞穿过基质内皮细胞迁移到下层趋化小室中，提
示上清中可能含有具有趋化活性的物质。同时由于多种肿瘤细胞系及正常细胞系
均显示出在热休克后上清的趋化活性，因此我们认为，热休克后趋化活性增加是
一种普遍的现象，而在本文中我们仅针对肿瘤细胞细胞进行了深入的研究。由于
有研究发现HSP70在热休克或热疗后显著升高，因此我们用Western blot检测了
肿瘤细
Malignant tumor is one of the threats of human health. Among the diseases causing death, tumor has climbed to be the one of the major killer and is still becoming more and more imposing. At present, the conventional treatments are always inefficient. Biotherapy, especially tumor vaccine, has brought new hopes for human beings who have suffered a lot from tumors and have become one of the focuses of tumor treatment and studies. Methods employed to prepare vaccine at present include tumor cells modified with cytokines, costimulatory molecules and tumor antigenic peptides, dendritic cells (DC) primed with tumor antigens in vitro or genetically modified with tumor antigens and the fusion of tumor cells with antigen presenting cells (APC). Though with various advantages, these vaccines are always complicated to prepare and the effects are relatively not promising.
During the study of the relationship between heat shock protein (HSP) and tumor, we found that after heat shock, culture supernatants of tumor cells could chemoattract DC and T lymphocytes. And we went further verify that chemokines were present in the culture supernatants of tumor cells after heat shock. Because of the rapid presence of this activity, we hypothesis that chemokines be released out of tumor cells through a particular secretory form. Our further studies showed that this kind of exocytosis form is exosome as we detected various chemokines in the exosomes derived from tumor cells after heat shock and no chemokines were detected in the supernatants after isolation of exosomes. We also found that abundant MHC molecules, costimulatory molecules, adhesion molecules and chaparones carrying tumor antigens. As the exosomes are derived from tumor cells, carry tumor antigens and share
characters with most of the vaccines prepared previously, they are a kind of non-cellular novel vaccine. In vivo experiments showed that this kind of novel vaccine could not only protect mice from syngeneic and allogeneic tumor challenge but inhibit the growth of established tumors. This novel and efficient vaccine, with the non-cellular structure and contained no exogenous gene modification, is potentially no harm to the recipients. Together with the characters that it cause no ethical problems and it is easy to prepare, this non-cellular vaccine is promising and economic both in clinic and in market.
Heat shock and hsp70 can stimulate tumor cell produce chemokines that can chemoattract DC and T lymphocytes in vitro.
Tumor cell culture supernatant after heat shock could chemoattract DC and T cells, and hsp70 was abundantly present in the supernatants. After heat shock of human and mouse cell lines A549, LoVo, HeLa, MCF7, 3LL, CT26, B16, NIH3T3, TC-1 and 107B for Ih at 43癈 and recovery in 37癈 incubator for 4h, the culture supernatants can chemoattract DC and T cells in an in vitro chemotactic assay, indicating that chemotactic products are present in the supernatants. Considering that various cell lines demonstrate the same kind of chemotactic activity, it can be concluded that this effect is a kind of universal phenomenon. As it has been reported that HSP70 can stimulate CD8+ T cells to produce chemokines, we stimulate tumor cells 3LL with HSP70 (1 u g/ml) for 4 h and collect the culture supernatants to perform in vitro chemotaxis assay. We found that the supernatants can also chemoattract DC and T cells. To exclude the possibility that HSP70 can chemoattract DC and T cells directly, we add HSP70 to the lower well and we found no chemoattraction happened. Thus HSP70 can stimulate the chemoattracting product to produce. As chemokines are the major proteins inducing cell migration, we examined the mRNA expression in 3LL cells after heat shock or HSP70 stimulation and protein
expression in 3LL culture supernatants by RT-PCR and Western blot respectively. We found that, after heat shock and HSP70 stimulation, various chemokines are upregulated at mRNA level in 3LL cells and at protein level in the culture supernatants. HSP70 alone can't completely substitute for h
引文
1. Needham, D, Anyarambhatla, G, Kong G, Dewhirst MW. 2000. A new temperature-sensitive liposome for use with mild hyperthermia: characterization and testing in a human tumor xenograft model. Cancer Res 60(5): 1197-201
2. Oleson, J.R. 1995. Eugene Robertson Special Lecture. Hyperthermia from the clinic to the laboratory: a hypothesis. Int. J. Hyperthermial 1:315-22.
3. Lee CK, Song CW, Rhee JG, Foy JA, Levitt SH. 1995. Clinical experience using 8 MHz radiofrequency capacitive hyperthermia in combination with radiotherapy: results of a phase Ⅰ/Ⅱ study. Int J Radiat Oncol Biol Phys 15:733-45
4. Burd R, Dziedzic TS, Xu Y, Caligiuri MA, Subjeck JR, Repasky EA. 1998. Tumor cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (fever-like) whole body hyperthermia. J. Cell. Physiol 177:137-47
5. Bull JM. 1984. An update on the anticancer effects of a combination of chemotherapy and hyperthermia. Cancer Res 44:4853s-4856s
6. Frank J, Kelleher DK, Pompella A, Thews O, Biesalski HK, Vaupel P. 1998. Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase. Cancer Res 58:2693-8
7. Muralidharan V, Malcontenti-Wilson C, Christophi C. 2002. Effect of blood flow occlusion on laser hyperthermia for liver metastases. J. Surg. Res 103:165-74
8. Murata R, Overgaard J, Horsman MR. 2001. Potentiation of the anti-tumour effect of hyperthermia by combining with the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid. Int J Hyperthermia 17:508-19
9. Kakehi, M., Ueda, K., Mukojima, T., Hiraoka, M., Seto, O., Akanuma, A., and Nakatsugawa, S. 1990. Multi-institutional clinical studies on hyperthermia combined with radiotherapy or chemotherapy in advanced cancer of deep-seated organs. Int. J. Hyperthermia 6:719-40.
10. Hei, T.K., Hall, E. J., Kushner, S., and Osmak, R.S. 1986. Hyperthermia, chemotherapeutic agents and oncogenic transformation. Int. J. Hyperthermia 2:311-20.
11. Somersan, S., Larsson, M., Fonteneau, J.F., Basu, S., Srivastava, P., and Bhardwaj, N. 2001. Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells. J. Immunol. 167:4844-52.
12. Kuppner, M.C., Gastpar, R., Gelwer, S., Nossner, E., Ochmann, O., Scharner, A., and Issels, RD. 2001. The role of heat shock protein (hsp70) in dendritic cell maturation: hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors. Eur. J. Immunol. 31:1602-9.
13. Blachere, N.E., Li, Z., Chandawarkar, R. Y., Suto, R., Jaikaria, N.S., Basu, S., Udono, H., and Srivastava, P.K. 1997. Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J. Exp. Med. 186:1315-22.
14. Singh-Jasuja, H., Toes, R.E., Spee, P., Munz, C., Hilf, N., Schoenberger, S.P., Ricciardi-Castagnoli, P., Neefjes, J., Rammensee, H.G., Arnold-Schild, D., Schild, H. 2000. Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis. J. Exp. Med. 191:1965-74.
15. Zheng H, Dai J, Stoilova D, Li Z. 2001. Cell surface targeting of heat shock protein gp96 induces dendritic cell maturation and antitumor immunity. J Immunol 167:6731-5
16. Steinman RM. 1991. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9:271-296
17. Banchereau J, Steinman RM. 1998.Dendritic cells and the control of immunity. Nature;392(6673):245-252
18. Sallusto F, Lanzavecchia A. 1999. Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J Exp Med 189:611-614
19. Luster AD. 2002. The role of chemokines in linking innate and adaptive immunity. Curr Opin Immunol 14:129-135
20. Todryk S, Melcher AA, Hardwick N, Linardakis E, Bateman A, Colombo MP, Stoppacciaro A, Vile RG. 1999. Heat shock protein 70 induced during tumor cell killing induces Th1 cytokines and targets immature dendritic cell precursors to enhance antigen uptake. J Immunol 163:1398-408
21. Lehner T, Bergmeier LA, Wang Y, Tao L, Sing M, Spallek R, van der Zee R. 2000. Heat shock proteins generate beta-chemokines which function as innate adjuvants enhancing adaptive immunity. Eur J Immunol30:594-603
22. Ju, D.W., Tao, Q., Lou, G., Bai, M., He, L., Yang, Y., and Cao, X. 2001. Interleukin 18 transfection enhances antitumor immunity induced by dendritic cell-tumor cell conjugates. Cancer Res. 61:3735-40.
23. Cao, X., Zhang, W., Wan, T., He, L., Chen, T., Yuan, Z., Ma, S., Yu, Y., and Chen, G. 2000. Molecular cloning and characterization of a novel CXC chemokine macrophage inflammatory protein-2 gamma chemoattractant for human neutrophils and dendritic cells. J. Immunol. 165:2588-95.
24. Chantry, D., Romagnani, P., Raport, C. J., Wood, C.L., Epp, A., Romagnani, S., and Gray, P.W. 1999. Macrophage-derived chemokine is localized to thymic medullary epithelial cells and is a chemoattractant for CD3(+), CD4(+), CD8(low) thymocytes. Blood 94:1890-1898.
25. Zhang, W., Chen, T., Wan, T., He, L., Li, N., Yuan, Z., and Cao, X. 2000. Cloning of DPK, a novel dendritic cell-derived protein kinase activating the ERK1/ERK2 and JNK/SAPK pathways. Biochem. Biophys. Res. Commun. 274:872-9.
26. Terashima, H., Ishino, Y., Nakata, H., Norimura, T., and Tsuchiya, T. 1987. Combined radiotherapy and local external hyperthermia in advanced cancer-animal experiment and clinical study. J. UOEH 9:171-80.
27. Wong, H.R., Ryan, M., and Wispe, J.R. 1997. Stress response decreases NF-kappaB nuclear translocation and increases I-kappaBalpha expression in A549 cells. J. Clin. Invest. 99:2423-8.
28. DeMeester, S.L., Buchman, T.G, and Cobb, J.P. 2001. The heat shock paradox: does NF-kappaB determine cell fate? FASEB J. 15:270-274.
29. Ito, A., Shinkai, M., Honda, H., Wakabayashi, T., Yoshida, J., and Kobayashi, T. 2001. Augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia. Cancer Immunol. Immunother. 50:515-22.
30. Huang, Q., Hu, J.K, Lohr, F., Zhang, L, Braun, R., Lanzen, J., Little, J.B., Dewhirst, M.W., and Li, C.Y. 2000. Heat-induced gene expression as a novel targeted cancer gene therapy strategy. Cancer Res. 60:3435-9.
31. Wissing, D., and Jaattela, M. 1996. HSP27 and HSP70 increase the survival of WEHI-S cells exposed to hyperthermia. Int. J. Hyperthermia. 12:125-38.
32. Robert, J., Gantress, J., Rau, L., Bell, A., and Cohen, N. 2002. Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins. J. Immunol. 168:1697-703.
33. Blachere, N.E., Li, Z., Chandawarkar, R.Y., Suto, R., Jaikaria, N.S., Basu, S., Udono, H., and Srivastava, P.K. 1997. Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J. Exp. Med. 186:1315-22.
34. Somersan, S., Larsson, M., Fonteneau, J. F., Basu, S., Srivastava, P., and Bhardwaj, N. 2001. Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells. J. Immunol. 167:4844-52.
35. Rafiee M, Kanwar JR, Berg RW, Lehnert K, Lisowska K, Krissansen GW. 2001. Induction of systemic antitumor immunity by gene transfer of mammalian heat shock protein 70.1 into tumors in situ. Cancer Gene Ther 8(12):974-81
36.Breloer M, Fleischer B, von Bonin A. 1999. In vivo and in vitro activation of T cells after administration of Ag-negative heat shock proteins. J Immunol 162:3141-7
37.Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK. 2000. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat. Med. 6:435-42
38.Binder RJ, Han DK, Srivastava PK. 2000. CD91: a receptor for heat shock protein gp96. Nat Immunol. 1(2): 151-5
39.Seliger B, Maeurer MJ, Ferrone S. 2000. Antigen-processing machinery breakdown and tumor growth. Immunol Today 21:455-64
40.Gilboa E. 1999. How tumors escape immune destruction and what we can do about it. Cancer Immunol Immunother 48:382-5
41.Malhotra V, Eaves-Pyles T, Odoms K, Quaid G, Shanley TP, Wong HR. 2002. Heat shock inhibits activation of NF-kappaB in the absence of heat shock factor-1. Biochem Biophys Res Commun 291:453-7
2. Oleson, J.R. 1995. Eugene Robertson Special Lecture. Hyperthermia from the clinic to the laboratory: a hypothesis. Int. J. Hyperthermial 1:315-22.
3. Lee CK, Song CW, Rhee JG, Foy JA, Levitt SH. 1995. Clinical experience using 8 MHz radiofrequency capacitive hyperthermia in combination with radiotherapy: results of a phase Ⅰ/Ⅱ study. Int J Radiat Oncol Biol Phys 15:733-45
4. Burd R, Dziedzic TS, Xu Y, Caligiuri MA, Subjeck JR, Repasky EA. 1998. Tumor cell apoptosis, lymphocyte recruitment and tumor vascular changes are induced by low temperature, long duration (fever-like) whole body hyperthermia. J. Cell. Physiol 177:137-47
5. Bull JM. 1984. An update on the anticancer effects of a combination of chemotherapy and hyperthermia. Cancer Res 44:4853s-4856s
6. Frank J, Kelleher DK, Pompella A, Thews O, Biesalski HK, Vaupel P. 1998. Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase. Cancer Res 58:2693-8
7. Muralidharan V, Malcontenti-Wilson C, Christophi C. 2002. Effect of blood flow occlusion on laser hyperthermia for liver metastases. J. Surg. Res 103:165-74
8. Murata R, Overgaard J, Horsman MR. 2001. Potentiation of the anti-tumour effect of hyperthermia by combining with the vascular targeting agent 5,6-dimethylxanthenone-4-acetic acid. Int J Hyperthermia 17:508-19
9. Kakehi, M., Ueda, K., Mukojima, T., Hiraoka, M., Seto, O., Akanuma, A., and Nakatsugawa, S. 1990. Multi-institutional clinical studies on hyperthermia combined with radiotherapy or chemotherapy in advanced cancer of deep-seated organs. Int. J. Hyperthermia 6:719-40.
10. Hei, T.K., Hall, E. J., Kushner, S., and Osmak, R.S. 1986. Hyperthermia, chemotherapeutic agents and oncogenic transformation. Int. J. Hyperthermia 2:311-20.
11. Somersan, S., Larsson, M., Fonteneau, J.F., Basu, S., Srivastava, P., and Bhardwaj, N. 2001. Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells. J. Immunol. 167:4844-52.
12. Kuppner, M.C., Gastpar, R., Gelwer, S., Nossner, E., Ochmann, O., Scharner, A., and Issels, RD. 2001. The role of heat shock protein (hsp70) in dendritic cell maturation: hsp70 induces the maturation of immature dendritic cells but reduces DC differentiation from monocyte precursors. Eur. J. Immunol. 31:1602-9.
13. Blachere, N.E., Li, Z., Chandawarkar, R. Y., Suto, R., Jaikaria, N.S., Basu, S., Udono, H., and Srivastava, P.K. 1997. Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J. Exp. Med. 186:1315-22.
14. Singh-Jasuja, H., Toes, R.E., Spee, P., Munz, C., Hilf, N., Schoenberger, S.P., Ricciardi-Castagnoli, P., Neefjes, J., Rammensee, H.G., Arnold-Schild, D., Schild, H. 2000. Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis. J. Exp. Med. 191:1965-74.
15. Zheng H, Dai J, Stoilova D, Li Z. 2001. Cell surface targeting of heat shock protein gp96 induces dendritic cell maturation and antitumor immunity. J Immunol 167:6731-5
16. Steinman RM. 1991. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol 9:271-296
17. Banchereau J, Steinman RM. 1998.Dendritic cells and the control of immunity. Nature;392(6673):245-252
18. Sallusto F, Lanzavecchia A. 1999. Mobilizing dendritic cells for tolerance, priming, and chronic inflammation. J Exp Med 189:611-614
19. Luster AD. 2002. The role of chemokines in linking innate and adaptive immunity. Curr Opin Immunol 14:129-135
20. Todryk S, Melcher AA, Hardwick N, Linardakis E, Bateman A, Colombo MP, Stoppacciaro A, Vile RG. 1999. Heat shock protein 70 induced during tumor cell killing induces Th1 cytokines and targets immature dendritic cell precursors to enhance antigen uptake. J Immunol 163:1398-408
21. Lehner T, Bergmeier LA, Wang Y, Tao L, Sing M, Spallek R, van der Zee R. 2000. Heat shock proteins generate beta-chemokines which function as innate adjuvants enhancing adaptive immunity. Eur J Immunol30:594-603
22. Ju, D.W., Tao, Q., Lou, G., Bai, M., He, L., Yang, Y., and Cao, X. 2001. Interleukin 18 transfection enhances antitumor immunity induced by dendritic cell-tumor cell conjugates. Cancer Res. 61:3735-40.
23. Cao, X., Zhang, W., Wan, T., He, L., Chen, T., Yuan, Z., Ma, S., Yu, Y., and Chen, G. 2000. Molecular cloning and characterization of a novel CXC chemokine macrophage inflammatory protein-2 gamma chemoattractant for human neutrophils and dendritic cells. J. Immunol. 165:2588-95.
24. Chantry, D., Romagnani, P., Raport, C. J., Wood, C.L., Epp, A., Romagnani, S., and Gray, P.W. 1999. Macrophage-derived chemokine is localized to thymic medullary epithelial cells and is a chemoattractant for CD3(+), CD4(+), CD8(low) thymocytes. Blood 94:1890-1898.
25. Zhang, W., Chen, T., Wan, T., He, L., Li, N., Yuan, Z., and Cao, X. 2000. Cloning of DPK, a novel dendritic cell-derived protein kinase activating the ERK1/ERK2 and JNK/SAPK pathways. Biochem. Biophys. Res. Commun. 274:872-9.
26. Terashima, H., Ishino, Y., Nakata, H., Norimura, T., and Tsuchiya, T. 1987. Combined radiotherapy and local external hyperthermia in advanced cancer-animal experiment and clinical study. J. UOEH 9:171-80.
27. Wong, H.R., Ryan, M., and Wispe, J.R. 1997. Stress response decreases NF-kappaB nuclear translocation and increases I-kappaBalpha expression in A549 cells. J. Clin. Invest. 99:2423-8.
28. DeMeester, S.L., Buchman, T.G, and Cobb, J.P. 2001. The heat shock paradox: does NF-kappaB determine cell fate? FASEB J. 15:270-274.
29. Ito, A., Shinkai, M., Honda, H., Wakabayashi, T., Yoshida, J., and Kobayashi, T. 2001. Augmentation of MHC class I antigen presentation via heat shock protein expression by hyperthermia. Cancer Immunol. Immunother. 50:515-22.
30. Huang, Q., Hu, J.K, Lohr, F., Zhang, L, Braun, R., Lanzen, J., Little, J.B., Dewhirst, M.W., and Li, C.Y. 2000. Heat-induced gene expression as a novel targeted cancer gene therapy strategy. Cancer Res. 60:3435-9.
31. Wissing, D., and Jaattela, M. 1996. HSP27 and HSP70 increase the survival of WEHI-S cells exposed to hyperthermia. Int. J. Hyperthermia. 12:125-38.
32. Robert, J., Gantress, J., Rau, L., Bell, A., and Cohen, N. 2002. Minor histocompatibility antigen-specific MHC-restricted CD8 T cell responses elicited by heat shock proteins. J. Immunol. 168:1697-703.
33. Blachere, N.E., Li, Z., Chandawarkar, R.Y., Suto, R., Jaikaria, N.S., Basu, S., Udono, H., and Srivastava, P.K. 1997. Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity. J. Exp. Med. 186:1315-22.
34. Somersan, S., Larsson, M., Fonteneau, J. F., Basu, S., Srivastava, P., and Bhardwaj, N. 2001. Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells. J. Immunol. 167:4844-52.
35. Rafiee M, Kanwar JR, Berg RW, Lehnert K, Lisowska K, Krissansen GW. 2001. Induction of systemic antitumor immunity by gene transfer of mammalian heat shock protein 70.1 into tumors in situ. Cancer Gene Ther 8(12):974-81
36.Breloer M, Fleischer B, von Bonin A. 1999. In vivo and in vitro activation of T cells after administration of Ag-negative heat shock proteins. J Immunol 162:3141-7
37.Asea A, Kraeft SK, Kurt-Jones EA, Stevenson MA, Chen LB, Finberg RW, Koo GC, Calderwood SK. 2000. HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine. Nat. Med. 6:435-42
38.Binder RJ, Han DK, Srivastava PK. 2000. CD91: a receptor for heat shock protein gp96. Nat Immunol. 1(2): 151-5
39.Seliger B, Maeurer MJ, Ferrone S. 2000. Antigen-processing machinery breakdown and tumor growth. Immunol Today 21:455-64
40.Gilboa E. 1999. How tumors escape immune destruction and what we can do about it. Cancer Immunol Immunother 48:382-5
41.Malhotra V, Eaves-Pyles T, Odoms K, Quaid G, Shanley TP, Wong HR. 2002. Heat shock inhibits activation of NF-kappaB in the absence of heat shock factor-1. Biochem Biophys Res Commun 291:453-7