之前我們的實驗室已經發現在老鼠攝護腺癌模式下以70Gy放射線照射的攝護腺腫瘤細胞(TRAMP C1)為抗原來刺激樹突狀細胞效果比用腫瘤細胞裂解物的效果好。本篇論文的研究目的是想在攝護腺癌所建立的樹突狀細胞疫苗中找出適合的胜肽，並利用來研究蛋白酶體抑制劑(proteasome inhibitor)在抗原交互呈現（cross presentation）過程裡的影響。首先我們利用電腦演算法找出四個較有潛力的抗原決定因子(probasin epitopes)。再藉由細胞毒殺反應實驗和斑點免疫法挑選出兩個較具有潛力可以利用在樹突狀細胞疫苗的胜肽。進一步由MHC-II、CD80和CD86的表現量增加指出被選出的胜肽可以使樹突狀細胞成熟。由腫瘤生長延遲、細胞毒殺反應實驗和斑點免疫法的實驗結果發現使用胜肽及使用腫瘤細胞所製成之樹突細胞狀疫苗具有相同對抗腫瘤的效果。我們的實驗結果也顯示出蛋白酶體抑制劑無法在抗原交互呈現過程裡增加樹突狀細胞的抗原表現。此外從攝護腺腫瘤細胞在經過放射線照射後的實驗結果顯示可以增加ICAM-1、MHC-1和Fas抗原分子的表現。這可以使我們去解釋為什麼在樹突狀細胞所建立的免疫治療裡，以放射線照射的攝護腺腫瘤細胞作為抗原來刺激樹突狀細胞效果會比用腫瘤細胞裂解物還要好。

Early study from our lab had shown that 70 Gy irradiated-TRAMP-C1 cells were better than freeze and thawed-cell lyses in pulsing dendritic cells (DCs) in DC-based tumor vaccine against murine prostate cancers. This study aimed to search a suitable murine prostate specific peptide for DC-based tumor vaccine and use this peptide as a model to study the influence of proteasome inhibitor on the process of DC-mediated antigen cross presentation. To achieve this goal, computer algorithms were first used to predict 4 potential peptide epitopes. Their ability to generate DC-based immunity was first screened by short term CTL and ELISPOT assay. Two of them showed promised potential as the epitopes to be presented by MHC molecules. Their ability to cause DC maturation was indicated by the increase expression of MHC-II, CD80, and CD86 molecules. Further tumor growth delay, CTL, and ELISPOT assays demonstrated that these two peptides were as efficient as irradiated TRAMP-C1 in DC-based immunotherapy for prostate cancer. This study also showed that the administration of protease inhibitor, PS-341, could not enhance the process of antigen cross presentation by DCs. I have also demonstrated the radiation could enhance the expression of ICAM-1, MHC-1, and Fas antigens by TRAMP-C1 cells. This provides an evidence to explain why the irradiated-tumor cells were better antigen sources than freeze and thawed cell lyses in DC-based immunotherapy.

1. Shortman, K. and Liu, Y. J. Mouse and human dendritic cell subtypes. Nat Rev Immunol, 2: 151-161, 2002.
2. Banchereau, J. and Steinman, R. M. Dendritic cells and the control of immunity. Nature, 392: 245-252, 1998.
3. Boes, M. and Ploegh, H. L. Translating cell biology in vitro to immunity in vivo. Nature, 430: 264-271, 2004.
4. Boes, M., Bertho, N., Cerny, J., Op den Brouw, M., Kirchhausen, T., and Ploegh, H. T cells induce extended class II MHC compartments in dendritic cells in a Toll-like receptor-dependent manner. J Immunol, 171: 4081-4088, 2003.
5. Walsh, S. R., Bhardwaj, N., and Gandhil, R. T. Dendritic cells and the promise of therapeutic vaccines for human immunodeficiency virus (HIV)-1. Curr HIV Res, 1: 205-216, 2003.
6. Steinman, R. M. The dendritic cell system and its role in immunogenicity. Annu Rev Immunol, 9: 271-296, 1991.
7. Kagi, D., Ledermann, B., Burki, K., Zinkernagel, R. M., and Hengartner, H. Molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo. Annu Rev Immunol, 14: 207-232, 1996.
8. Jan, Y. H. Establishment of dendritic cell-based tumor vaccine for prostate cancer in mice. MS thesis NTHU, Taiwan., 2004.
9. Wei, C., Willis, R. A., Tilton, B. R., Looney, R. J., Lord, E. M., Barth, R. K., and Frelinger, J. G. Tissue-specific expression of the human prostate-specific antigen gene in transgenic mice: implications for tolerance and immunotherapy. Proc Natl Acad Sci U S A, 94: 6369-6374, 1997.
10. Hussain, M. H., Blumenstein, B., Eisenberger, M., and Crawford, E. D. Southwest Oncology Group studies in hormone-refractory prostate cancer. Semin Oncol, 23: 24-27, 1996.
11. Watts, C. Capture and processing of exogenous antigens for presentation on MHC molecules. Annu Rev Immunol, 15: 821-850, 1997.
12. Watts, C. and Amigorena, S. Antigen traffic pathways in dendritic cells. Traffic, 1: 312-317, 2000.
13. Melief, C. J. Mini-review: Regulation of cytotoxic T lymphocyte responses by dendritic cells: peaceful coexistence of cross-priming and direct priming? Eur J Immunol, 33: 2645-2654, 2003.

14. Heath, W. R. and Carbone, F. R. Cross-presentation, dendritic cells, tolerance and immunity. Annu Rev Immunol, 19: 47-64, 2001.
15. Yewdell, J. W., Norbury, C. C., and Bennink, J. R. Mechanisms of exogenous antigen presentation by MHC class I molecules in vitro and in vivo: implications for generating CD8+ T cell responses to infectious agents, tumors, transplants, and vaccines. Adv Immunol, 73: 1-77, 1999.
16. Norbury, C. C., Hewlett, L. J., Prescott, A. R., Shastri, N., and Watts, C. Class I MHC presentation of exogenous soluble antigen via macropinocytosis in bone marrow macrophages. Immunity, 3: 783-791, 1995.
17. Sunwoo, J. B., Chen, Z., Dong, G., Yeh, N., Crowl Bancroft, C., Sausville, E., Adams, J., Elliott, P., and Van Waes, C. Novel proteasome inhibitor PS-341 inhibits activation of nuclear factor-kappa B, cell survival, tumor growth, and angiogenesis in squamous cell carcinoma. Clin Cancer Res, 7: 1419-1428, 2001.
18. LeBlanc, R., Catley, L. P., Hideshima, T., Lentzsch, S., Mitsiades, C. S., Mitsiades, N., Neuberg, D., Goloubeva, O., Pien, C. S., Adams, J., Gupta, D., Richardson, P. G., Munshi, N. C., and Anderson, K. C. Proteasome inhibitor PS-341 inhibits human myeloma cell growth in vivo and prolongs survival in a murine model. Cancer Res, 62: 4996-5000, 2002.
19. Adams, J. Preclinical and clinical evaluation of proteasome inhibitor PS-341 for the treatment of cancer. Curr Opin Chem Biol, 6: 493-500, 2002.
20. Arrigo, A. P., Tanaka, K., Goldberg, A. L., and Welch, W. J. Identity of the 19S 'prosome' particle with the large multifunctional protease complex of mammalian cells (the proteasome). Nature, 331: 192-194, 1988.
21. Pajonk, F. and McBride, W. H. Ionizing radiation affects 26s proteasome function and associated molecular responses, even at low doses. Radiother Oncol, 59: 203-212, 2001.
22. Kurts, C., Heath, W. R., Carbone, F. R., Allison, J., Miller, J. F., and Kosaka, H. Constitutive class I-restricted exogenous presentation of self antigens in vivo. J Exp Med, 184: 923-930, 1996.
23. Sigal, L. J., Crotty, S., Andino, R., and Rock, K. L. Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen. Nature, 398: 77-80, 1999.
24. Lizee, G., Basha, G., and Jefferies, W. A. Tails of wonder: endocytic-sorting motifs key for exogenous antigen presentation. Trends Immunol, 26: 141-149, 2005.
25. Wiertz, E. J., Jones, T. R., Sun, L., Bogyo, M., Geuze, H. J., and Ploegh, H. L. The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol. Cell, 84: 769-779, 1996.
26. Meidenbauer, N., Andreesen, R., and Mackensen, A. Dendritic cells for specific cancer immunotherapy. Biol Chem, 382: 507-520, 2001.
27. Liao, Y. P., Wang, C. C., Butterfield, L. H., Economou, J. S., Ribas, A., Meng, W. S., Iwamoto, K. S., and McBride, W. H. Ionizing radiation affects human MART-1 melanoma antigen processing and presentation by dendritic cells. J Immunol, 173: 2462-2469, 2004.
28. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S., and Steinman, R. M. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med, 176: 1693-1702, 1992.
29. Foster, B. A., Gingrich, J. R., Kwon, E. D., Madias, C., and Greenberg, N. M. Characterization of prostatic epithelial cell lines derived from transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Cancer Res, 57: 3325-3330, 1997.
30. Parker, K. C., Bednarek, M. A., and Coligan, J. E. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J Immunol, 152: 163-175, 1994.
31. Rammensee, H., Bachmann, J., Emmerich, N. P., Bachor, O. A., and Stevanovic, S. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics, 50: 213-219, 1999.
32. Lu, J. and Celis, E. Recognition of prostate tumor cells by cytotoxic T lymphocytes specific for prostate-specific membrane antigen. Cancer Res, 62: 5807-5812, 2002.
33. Garnett, C. T., Palena, C., Chakraborty, M., Tsang, K. Y., Schlom, J., and Hodge, J. W. Sublethal irradiation of human tumor cells modulates phenotype resulting in enhanced killing by cytotoxic T lymphocytes. Cancer Res, 64: 7985-7994, 2004.
34. Steinman, R. M., Inaba, K., Turley, S., Pierre, P., and Mellman, I. Antigen capture, processing, and presentation by dendritic cells: recent cell biological studies. Hum Immunol, 60: 562-567, 1999.
35. Jonuleit, H., Giesecke-Tuettenberg, A., Tuting, T., Thurner-Schuler, B., Stuge, T. B., Paragnik, L., Kandemir, A., Lee, P. P., Schuler, G., Knop, J., and Enk, A. H. A comparison of two types of dendritic cell as adjuvants for the induction of melanoma-specific T-cell responses in humans following intranodal injection. Int J Cancer, 93: 243-251, 2001.

36 Degl'Innocenti E, Grioni M, Boni A, Camporeale A, Bertilaccio MT, Freschi M, Monno A, Arcelloni C, Greenberg NM, Bellone M. Peripheral T cell tolerance occurs early during spontaneous prostate cancer development and can be rescued by dendritic cell immunization. Eur J Immunol., 35:66-75, 2005.