孔祥智臺灣大學：免疫學研究所葉子菱Yeh, Tzu-LinTzu-LinYeh2007-11-292018-07-092007-11-292018-07-092005http://ntur.lib.ntu.edu.tw//handle/246246/63342記憶型T淋巴球可抵抗對外來抗原，在免疫系統當中扮演重要的角色。 然而，對於控制記憶型CD4+ T淋巴球產生的詳細機制卻還不甚清楚。 在本研究當中，我們嘗試著去研究輔助刺激因子（costimulatory molecule）是否參與在記憶型CD4+ T淋巴球的生成。 經由cDNA的轉染（transfection）製造出表現在細胞表面帶有CD24、CD40、CD70、CD86、CD153、OX40L、4-1BBL和IcosL的EL4腫瘤細胞株。 另外，為了使這些轉染細胞株（transfectant）可以當作抗原呈現細胞（antigen-presenting cell）以活化CD4+ T淋巴球，因此在每種轉染細胞株的細胞表面上再加以轉染FcgRI，使轉染細胞株可以結合上anti-CD3單株抗體，並將之呈現給CD4+ T淋巴球以刺激之。 在活體外實驗的結果發現，除了CD28:CD86，在其他輔助型因子的刺激之下，CD4+ T淋巴球的活化狀況都不是很好。 因此在利用轉染細胞株EL4活化CD4+ T淋巴球時，我們同時加入anti-CD3和anti-CD28的刺激。 CD4+ T淋巴球在帶有各種不同輔助型刺激因子轉染細胞株EL4的刺激之後，再打入宿主老鼠體內，我們發現細胞表面表現CD40的轉染細胞株，可提供CD40:CD40L的刺激，造成大量長期存活的CD4+ T淋巴球。 另外，我們還發現CD4+ T淋巴球在B淋巴球的刺激之下再外加第四介白素也可以幫助生成長期存活的CD4+ T淋巴球。 至於其他的輔助刺激因子，包括CD28:CD86、CD27:CD70、CD30:CD153、OX40:OX40L、Icos:IcosL和4-1BB:4-1BBL，則對於記憶型CD4 T 淋巴球的生成沒有正向的影響。以上的研究發現對於腫瘤的治療與疫苗的開發皆有所幫助。Long-lived memory T cells play important roles in the defense against foreign invaders, although the mechanisms involved in memory T cell generation are not fully understood. In this study, I studied the effects costimulatory molecules play on memory CD4+ T cell memory generation. EL4 tumor cell lines that had through transfection been made to stably co-express FcgRI and one or combinations of CD24, CD40, CD70, CD86, CD153, OX40L, 4-1BBL, and IcosL costimulators were used as antigen-presenting cells to activate CD4+ T cells. Such transfectants delivered signal 1 through anti-CD3 mAbs presented by its FcgRI and signal 2 through the given costimulatory molecules it expresses. Except CD28:CD86, all other costimulatory molecules were ineffective at causing CD4+ T cell proliferation. To insure proper activation of CD4 T cells, anti-CD3 and anti-CD28 were both added and were presented via the FcgRI on the various costimulator-expressing EL4 transfectants. Activated CD4+ T cells were adoptively transferred into histocompatible, congenic hosts and their presence was monitored over time. CD4+ T cells activated by anti-CD3/CD28 in the context of CD40:CD154 developed long-term survival potential. In addition, CD4+ T cells activated by anti-CD3/CD28 in the context of B cells + IL-4 also developed long-term survival potential. All other costimulators, CD28:CD86, CD27:CD70, CD30:CD153, OX40:OX40L, Icos:IcosL, and 4-1BB:4-1BBL, did not have positive effects on long-term CD4 memory generation. These results may have implications in tumor therapy and vaccine development.Table of Contents Abstract………………………………………………………………. v Abstract (Chinese)…………………………………………………... vi CHAPTER I Introduction………………………………………… 1 CHAPTER II Experimental Setup………………………..….…... 8 2.1 Mice…………….………………………………….………… 9 2.2 Isolation of spleen CD4+ T cells………………………….….. 10 2.3 RNA extraction and reverse transcription……………....……. 12 2.4 Cloning of CD24, CD40, CD153……………………………. 14 2.5 Transfection…………………………………………….……. 16 2.6 Preparation of Antigen-presenting cells (B cell blasts)…..….. 18 2.7 Activation of B10 CD4+ T cells………………………...……. 20 2.8 Adoptive transfer……………………………………….……. 21 2.9 Donor cell persistence traced by flow cytometry………..…... 22 CHAPTER III Experiment Results………………….…………... 23 3.1 Generation of EL4 lines that co-express FcgRI and different costimulatory molecules……………………………….…... 24 3.2 Using EL4 transfectants as APC to induce CD4+ T cells proliferation…………………………………………..……. 25 3.3 The effect of 4-1BBL, IcosL, CD70, and OX40L on the generation of memory CD4 T cells…………………….….. 26 3.4 The effect of IL-2 on the generation of memory CD4+ T cells ………………………………………………..…….. 27 3.5 The effect of IL-4 on the generation of long-lived T cells…… 28 3.6 The effect of combined costimulation signaling on the generation of memory CD4 T cells………………………… 29 3.7 The effect of CD40, CD153, OX40L on the generation of memory CD4+ T cells……………………………….……... 30 3.8 Rapid decline of donor T cells might be caused by rejecrion …………………………………………………. 30 3.9 Summary……………………………………………………... 31 CHAPTER IV Discussion………………….………………..……. 32 CHAPTER V Experiment Figures……………………………….. 39 REFERENCES……………………………………………………… 49578924 bytesapplication/pdfen-US輔助刺激因子第四介白素記憶型 CD4+ T 淋巴球co-stimulatory moleculesIL-4memory CD4 T cell[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63342/1/ntu-94-R92449004-1.pdf