臺灣博碩士論文加值系統

EB病毒所表現的致癌基因LMP1為好發於台灣的鼻咽癌生成之重要因子之一。而從台灣鼻咽癌檢體中分離出於C端缺少10個氨基酸與數個點突變的變異型LMP1(稱為NLMP1)比原型EB病毒所表現的LMP1(為BLMP1)更具致癌性與弱免疫原性。然而依據本實驗室所建立起的小鼠腫瘤模型也證明了NLMP1是具有免疫原性能控制腫瘤生長，更進一步也鑑定出NLMP1的CD8+ T的抗原決定區之胜肽 (IYLEILWRL)。除此之外，我們也證實了CD4＋ T細胞在抗腫瘤免疫反應中扮演一重要角色。樹突細胞會將epitope表現在細胞表面MHC分子上以活化CD4+ T 細胞。最近許多文獻指出，Toll-like receptor (TLR) ligand可以刺激未成熟之樹突細胞成為能活化具對抗腫瘤效果的Th1反應之第一型樹突細胞，而Th1細胞進而能活化CD8+ T細胞或自然殺手細胞 (natural killer cells)以消滅腫瘤。因此，CD4+ T的抗原決定區的鑑定有助於加強未來CD4 T細胞對抗與毒殺腫瘤的研究。在本研究中，我們純化正常小鼠脾臟中的樹突細胞，經過不同組合的TLR ligand刺激後，發現經由LPS和poly(I:C)同時刺激可誘導未成熟之樹突細胞成為第一型樹突細胞。接下來，我們利用胜肽預測電腦軟體(RANKPEP)所預測出八條可能是NLMP1的CD4+ T細胞之抗原決定位之胜肽鏈。以合成的胜肽鏈添加到純化出來的樹突細胞上，再經TLR ligand刺激使其成為第一型樹突細胞，再引發CD4 T細胞釋放IFN-γ的條件下，找到了一條可能為CD4+ T細胞的抗原決定位，其序列為HGPRHTDEH。因此，本研究顯示，我們已鑑定出NLMP1的CD4+ T細胞之抗原決定位，且其位於NLMP1之C端。

The EBV-encoded oncoprotein LMP1 has been considered as one of the important cofactors in the carcinogenesis of nasopharyngeal carcinoma (NPC), which is an endemic cancer in Taiwan. NLMP1, with 10 amino acid deletion in C-terminus and several point mutations, isolated from Taiwan’s NPC biopsies has been found to be more tumorigenic and less immunogenic in BALB/c mice than BLMP1, the LMP1 originally identified in the prototype B95.8 strain. In the NLMP1 tumor animal model established in our laboratory, we have demonstrated the immunogenicity of NLMP1 to inhibit development of tumors expressing NLMP1 and have identified a CD8+ T epitope of NLMP1 (IYLEILWRL). In addition, we demonstrated that the CD4+ helper T cells play an important role in anti-NLMP1 tumor response. Dendritic cells present epitope on major histocompatibility complex (MHC) to activate naïve CD4+ T cells. Recently, many studies have shown that a Toll-like receptor ligand can stimulate immature DC to become type 1 DC (DC1), which can effectively activate naïve CD4+ helper T cells to become T helper type 1 (Th1) cells, followed by the activation of CD8+ cytotoxic T cells or natural killer (NK) cells, thereby eliminating tumor. Therefore, the identification of CD4+ T epitope of NLMP1 would be equally important to enhance additional anti-tumor effects by CD4+ cytotoxic T cells. In order to identify the CD4 epitope of NLMP1, we isolated normal BALB/c mouse splenic immature DCs, which could uptake exogenous tumor antigens. Subsequently, we stimulated the freshly purified DC with TLR ligands, specifically, a combination of LPS and poly(I:C) (L/P), and found that immature DC could be induced to become DC1. The CD4 epitopes of NLMP1 were predicted by the computer-based prediction algorithms (RANKPEP). Eight candidate peptides were selected and pulsed onto purified DC treated with LPS/poly(I:C) followed by the IFN-γ production assay. Finally, one of the eight candidate peptides was identified to be the CD4 epitope (HGPRHTDEH) located at the C-terminus domain in NLMP1.

中文摘要…………………………………………………………
Abstract…………………………………………………………..
Introduction……………………………………………………..1
Material and Methods…………………………………………..5
Results…………………………………………………………12
Discussion……………………………………………………..17
Reference……………………………………………………...22
Figures and Tables…………………………………………….31

1.Murray, P. G., and L. S. Young. 2001. Epstein-Barr virus infection: basis of malignancy and potential for therapy. Expert Rev Mol Med 2001:1.
2.Thompson, M. P., and R. Kurzrock. 2004. Epstein-Barr virus and cancer. Clin Cancer Res 10:803.
3.Babcock, G. J., L. L. Decker, M. Volk, and D. A. Thorley-Lawson. 1998. EBV persistence in memory B cells in vivo. Immunity 9:395.
4.Cohen, J. I. 2000. Epstein-Barr virus infection. N Engl J Med 343:481.
5.Schultheiss, U., S. Puschner, E. Kremmer, T. W. Mak, H. Engelmann, W. Hammerschmidt, and A. Kieser. 2001. TRAF6 is a critical mediator of signal transduction by the viral oncogene latent membrane protein 1. Embo J 20:5678.
6.Gires, O., U. Zimber-Strobl, R. Gonnella, M. Ueffing, G. Marschall, R. Zeidler, D. Pich, and W. Hammerschmidt. 1997. Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule. Embo J 16:6131.
7.Izumi, K. M., and E. D. Kieff. 1997. The Epstein-Barr virus oncogene product latent membrane protein 1 engages the tumor necrosis factor receptor-associated death domain protein to mediate B lymphocyte growth transformation and activate NF-kappaB. Proc Natl Acad Sci U S A 94:12592.
8.Li, H. P., and Y. S. Chang. 2003. Epstein-Barr virus latent membrane protein 1: structure and functions. J Biomed Sci 10:490.
9.Aviel, S., G. Winberg, M. Massucci, and A. Ciechanover. 2000. Degradation of the epstein-barr virus latent membrane protein 1 (LMP1) by the ubiquitin-proteasome pathway. Targeting via ubiquitination of the N-terminal residue. J Biol Chem 275:23491.
10.Sylla, B. S., S. C. Hung, D. M. Davidson, E. Hatzivassiliou, N. L. Malinin, D. Wallach, T. D. Gilmore, E. Kieff, and G. Mosialos. 1998. Epstein-Barr virus-transforming protein latent infection membrane protein 1 activates transcription factor NF-kappaB through a pathway that includes the NF-kappaB-inducing kinase and the IkappaB kinases IKKalpha and IKKbeta. Proc Natl Acad Sci U S A 95:10106.
11.Zhang, X. S., K. H. Song, H. Q. Mai, W. H. Jia, B. J. Feng, J. C. Xia, R. H. Zhang, L. X. Huang, X. J. Yu, Q. S. Feng, P. Huang, J. J. Chen, and Y. X. Zeng. 2002. The 30-bp deletion variant: a polymorphism of latent membrane protein 1 prevalent in endemic and non-endemic areas of nasopharyngeal carcinomas in China. Cancer Lett 176:65.
12.Nicholls, J., P. Hahn, E. Kremmer, T. Frohlich, G. J. Arnold, J. Sham, D. Kwong, and F. A. Grasser. 2004. Detection of wild type and deleted latent membrane protein 1 (LMP1) of Epstein-Barr virus in clinical biopsy material. J Virol Methods 116:79.
13.Hu, L. F., E. R. Zabarovsky, F. Chen, S. L. Cao, I. Ernberg, G. Klein, and G. Winberg. 1991. Isolation and sequencing of the Epstein-Barr virus BNLF-1 gene (LMP1) from a Chinese nasopharyngeal carcinoma. J Gen Virol 72 ( Pt 10):2399.
14.Chen, M. L., C. N. Tsai, C. L. Liang, C. H. Shu, C. R. Huang, D. Sulitzeanu, S. T. Liu, and Y. S. Chang. 1992. Cloning and characterization of the latent membrane protein (LMP) of a specific Epstein-Barr virus variant derived from the nasopharyngeal carcinoma in the Taiwanese population. Oncogene 7:2131.
15.Li, S. N., Y. S. Chang, and S. T. Liu. 1996. Effect of a 10-amino acid deletion on the oncogenic activity of latent membrane protein 1 of Epstein-Barr virus. Oncogene 12:2129.
16.Trivedi, P., L. F. Hu, F. Chen, B. Christensson, M. G. Masucci, G. Klein, and G. Winberg. 1994. Epstein-Barr virus (EBV)-encoded membrane protein LMP1 from a nasopharyngeal carcinoma is non-immunogenic in a murine model system, in contrast to a B cell-derived homologue. Eur J Cancer 30A:84.
17.Edwards, R. H., D. Sitki-Green, D. T. Moore, and N. Raab-Traub. 2004. Potential selection of LMP1 variants in nasopharyngeal carcinoma. J Virol 78:868.
18.Lin, J. C., J. M. Cherng, H. J. Lin, C. W. Tsang, Y. X. Liu, and S. P. Lee. 2004. Amino acid changes in functional domains of latent membrane protein 1 of Epstein-Barr virus in nasopharyngeal carcinoma of southern China and Taiwan: prevalence of an HLA A2-restricted 'epitope-loss variant'. J Gen Virol 85:2023.
19.Meij, P., A. Leen, A. B. Rickinson, S. Verkoeijen, M. B. Vervoort, E. Bloemena, and J. M. Middeldorp. 2002. Identification and prevalence of CD8(+) T-cell responses directed against Epstein-Barr virus-encoded latent membrane protein 1 and latent membrane protein 2. Int J Cancer 99:93.
20.Constant, S. L., and K. Bottomly. 1997. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu Rev Immunol 15:297.
21.Moser, M., and K. M. Murphy. 2000. Dendritic cell regulation of TH1-TH2 development. Nat Immunol 1:199.
22.Maldonado-Lopez, R., and M. Moser. 2001. Dendritic cell subsets and the regulation of Th1/Th2 responses. Semin Immunol 13:275.
23.Kalinski, P., C. M. Hilkens, E. A. Wierenga, and M. L. Kapsenberg. 1999. T-cell priming by type-1 and type-2 polarized dendritic cells: the concept of a third signal. Immunol Today 20:561.
24.Maldonado-Lopez, R., T. De Smedt, P. Michel, J. Godfroid, B. Pajak, C. Heirman, K. Thielemans, O. Leo, J. Urbain, and M. Moser. 1999. CD8alpha+ and CD8alpha- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. J Exp Med 189:587.
25.Hochrein, H., K. Shortman, D. Vremec, B. Scott, P. Hertzog, and M. O'Keeffe. 2001. Differential production of IL-12, IFN-alpha, and IFN-gamma by mouse dendritic cell subsets. J Immunol 166:5448.
26.Ikeda, H., K. Chamoto, T. Tsuji, Y. Suzuki, D. Wakita, T. Takeshima, and T. Nishimura. 2004. The critical role of type-1 innate and acquired immunity in tumor immunotherapy. Cancer Sci 95:697.
27.Hokey, D. A., A. T. Larregina, G. Erdos, S. C. Watkins, and L. D. Falo, Jr. 2005. Tumor cell loaded type-1 polarized dendritic cells induce Th1-mediated tumor immunity. Cancer Res 65:10059.
28.Pulendran, B., P. Kumar, C. W. Cutler, M. Mohamadzadeh, T. Van Dyke, and J. Banchereau. 2001. Lipopolysaccharides from distinct pathogens induce different classes of immune responses in vivo. J Immunol 167:5067.
29.Hirschfeld, M., J. J. Weis, V. Toshchakov, C. A. Salkowski, M. J. Cody, D. C. Ward, N. Qureshi, S. M. Michalek, and S. N. Vogel. 2001. Signaling by toll-like receptor 2 and 4 agonists results in differential gene expression in murine macrophages. Infect Immun 69:1477.
30.Schlecht, G., J. Mouries, M. Poitrasson-Riviere, C. Leclerc, and G. Dadaglio. 2006. Purification of splenic dendritic cells induces maturation and capacity to stimulate Th1 response in vivo. Int Immunol 18:445.
31.Chen, L., M. Arora, M. Yarlagadda, T. B. Oriss, N. Krishnamoorthy, A. Ray, and P. Ray. 2006. Distinct responses of lung and spleen dendritic cells to the TLR9 agonist CpG oligodeoxynucleotide. J Immunol 177:2373.
32.Boonstra, A., C. Asselin-Paturel, M. Gilliet, C. Crain, G. Trinchieri, Y. J. Liu, and A. O'Garra. 2003. Flexibility of mouse classical and plasmacytoid-derived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation. J Exp Med 197:101.
33.Adams, M., H. Navabi, B. Jasani, S. Man, A. Fiander, A. S. Evans, C. Donninger, and M. Mason. 2003. Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R). Vaccine 21:787.
34.de Jong, E. C., P. L. Vieira, P. Kalinski, J. H. Schuitemaker, Y. Tanaka, E. A. Wierenga, M. Yazdanbakhsh, and M. L. Kapsenberg. 2002. Microbial compounds selectively induce Th1 cell-promoting or Th2 cell-promoting dendritic cells in vitro with diverse th cell-polarizing signals. J Immunol 168:1704.
35.Cella, M., M. Salio, Y. Sakakibara, H. Langen, I. Julkunen, and A. Lanzavecchia. 1999. Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J Exp Med 189:821.
36.Tsujimoto, H., P. A. Efron, T. Matsumoto, R. F. Ungaro, A. Abouhamze, S. Ono, H. Mochizuki, and L. L. Moldawer. 2006. Maturation of murine bone marrow-derived dendritic cells with poly(I:C) produces altered TLR-9 expression and response to CpG DNA. Immunol Lett 107:155.
37.Napolitani, G., A. Rinaldi, F. Bertoni, F. Sallusto, and A. Lanzavecchia. 2005. Selected Toll-like receptor agonist combinations synergistically trigger a T helper type 1-polarizing program in dendritic cells. Nat Immunol 6:769.
38.Iwasaki, A., and R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat Immunol 5:987.
39.Mazzoni, A., and D. M. Segal. 2004. Controlling the Toll road to dendritic cell polarization. J Leukoc Biol 75:721.
40.Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu Rev Immunol 21:335.
41.Yoshizaki, T., T. Horikawa, R. Qing-Chun, N. Wakisaka, H. Takeshita, T. S. Sheen, S. Y. Lee, H. Sato, and M. Furukawa. 2001. Induction of interleukin-8 by Epstein-Barr virus latent membrane protein-1 and its correlation to angiogenesis in nasopharyngeal carcinoma. Clin Cancer Res 7:1946.
42.Tang, K. F., S. Y. Tan, S. H. Chan, S. M. Chong, K. S. Loh, L. K. Tan, and H. Hu. 2001. A distinct expression of CC chemokines by macrophages in nasopharyngeal carcinoma: implication for the intense tumor infiltration by T lymphocytes and macrophages. Hum Pathol 32:42.
43.Nakajima, H., M. Kobayashi, R. B. Pollard, and F. Suzuki. 2001. Monocyte chemoattractant protein-1 enhances HSV-induced encephalomyelitis by stimulating Th2 responses. J Leukoc Biol 70:374.
44.Gu, L., S. Tseng, R. M. Horner, C. Tam, M. Loda, and B. J. Rollins. 2000. Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404:407.
45.Matsukawa, A., N. W. Lukacs, T. J. Standiford, S. W. Chensue, and S. L. Kunkel. 2000. Adenoviral-mediated overexpression of monocyte chemoattractant protein-1 differentially alters the development of Th1 and Th2 type responses in vivo. J Immunol 164:1699.
46.Huang, D. R., J. Wang, P. Kivisakk, B. J. Rollins, and R. M. Ransohoff. 2001. Absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific T helper cell type 1 immune response in experimental autoimmune encephalomyelitis. J Exp Med 193:713.
47.Wu, C. C. 1998. The study of the immunogenicity of NPC-LMP1-expressing tumor cells in immunocompetent mice. Master thesis.
48.Chung, Y. C. 2003. The immunogenicity of Epstein-Barr virus (EBV)-encoded deletion variant of latent membrane protein (LMP)-1 in mouse model. Master thesis.
49.Shen, G. Y. 2004. Dendritic cell (DC)-based generation of cytotoxic T lymphocytes (CTL) specific for Epstein-Barr Virus (EBV)-encoded Latent Membrane Protein 1 (LMP1) isolated from nasopharyngeal carcinoma (NPC) in Taiwan. Master thesis.
50.Chen, J. R. 2005. The enhanced production of chemokines and immune down-modulation by the formation of solid tumor expressing EBV-del-LMP1. Master thesis.
51.Hu, L., B. Troyanovsky, X. Zhang, P. Trivedi, I. Ernberg, and G. Klein. 2000. Differences in the immunogenicity of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus genomes derived from LMP1-positive and -negative nasopharyngeal carcinoma. Cancer Res 60:5589.
52.Liu, H. P. 1999. The peotective immunity in BALB/c mice induced by irradiated EBV-N-LMP1 tumor cells. Master thesis.
53.Pasare, C., and R. Medzhitov. 2003. Toll pathway-dependent blockade of CD4+CD25+ T cell-mediated suppression by dendritic cells. Science 299:1033.
54.Qian, C., H. An, Y. Yu, S. Liu, and X. Cao. 2006. TLR agonists induce regulatory dendritic cells to recruit Th1 cells via preferential IP-10 secretion and inhibit Th1 proliferation. Blood.
55.Eisenbarth, S. C., D. A. Piggott, J. W. Huleatt, I. Visintin, C. A. Herrick, and K. Bottomly. 2002. Lipopolysaccharide-enhanced, toll-like receptor 4-dependent T helper cell type 2 responses to inhaled antigen. J Exp Med 196:1645.
56.Hiura, T., H. Kagamu, S. Miura, A. Ishida, H. Tanaka, J. Tanaka, F. Gejyo, and H. Yoshizawa. 2005. Both regulatory T cells and antitumor effector T cells are primed in the same draining lymph nodes during tumor progression. J Immunol 175:5058.
57.Feng, I. C., L. B. Koay, M. J. Sheu, H. T. Kuo, C. S. Sun, C. Lee, W. L. Chuang, S. K. Liao, S. L. Wang, L. Y. Tang, C. J. Cheng, and S. L. Tsai. 2006. HBcAg-specific CD4(+)CD25 (+)regulatory T cells modulate immune tolerance and acute exacerbation on the natural history of chronic hepatitis B virus infection. J Biomed Sci.