The Role of Regulatory T cells in the Immune Modulation of Disease Activity in Murine Lupus

紅斑性狼瘡(systemic lupus erythematosus, SLE)是一種多重器官受損的自體免疫疾病，其致病因素很多，包括調節一些可以對自體抗原產生免疫反應淋巴細胞的功能有缺陷。在我的第一部份的研究，我們想要探討是否調節性T細胞可以減緩病徵。 Foxp3是一個專一表現在CD4+CD25+調節性T細胞中的轉錄調節因子，在調節性T細胞的發育和功能上是一個重要的調節基因。若將Foxp3送入CD4+CD25-非調節性T細胞中，會使其具有像自然產生的調節性T細胞一樣具有免疫抑制的功能。目前我們已知調節性T細胞的免疫抑制功能調控許多有自體或非自體抗原所引起的免疫反應。在此研究中，我們想要探究如果將Foxp3過度表現的T細胞送入紅斑性狼瘡小鼠中(NZB/NZW F1)，是否具有減緩降低病情的效用。 我們首先從正常老鼠(DBA2/NZW F1)分離純化出CD4+CD25+ T細胞當作正常的調性T細胞，並且利用尾靜脈注射移植到紅斑性狼瘡小鼠中。藉由觀察抗自體抗體的產生程度，我們發現有移植調節性T細胞的小鼠，其抗自體抗體的濃度在大約一個月後開始下降，且抑制功能可以維持三個月之久。接下來我們想要在體外大量產生調節性T細胞，我們利用反轉錄病毒(lentivirus)將Foxp3帶入CD4+CD25- T細胞中使其具有抑制功能，這部份的實驗仍在進行中。 在第二部份的實驗中，我們想知道常被用來作為抗癌藥劑的砒霜(As2O3)，是否也能減緩紅斑性狼瘡小鼠的病徵。我們利用腹腔注射給予不同濃度的砒霜，一個星期給三次，總共給予兩個月。我們發現，在第一次注射一個月之後，給予高劑量砒霜的小鼠，其抗自體抗體的濃度明顯地比給予對照緩衝液的組別來的低。而且有給予砒霜的小鼠其腎絲球發炎(GN)的情形比較輕微，並且能活的比較久。 由上述實驗可知，不管是異體移植調節性T細胞或是給予砒霜，都有助於紅斑性狼瘡小鼠的病情，能減緩其病徵。這些方法未來可以應用在臨床上的治療。Systemic lupus erythematosus (SLE) is a multiple organic autoimmune disease, which caused by has many pathogenic factors including defect in regulation of autoreactive lymphocytes. In the first part of the study, we aimed to study whether regulatory T cells could alleviate disease severity of lupus. Foxp3, a forkhead transcription factor, specifically expressed in CD4+CD25+ regulatory T cells (Treg) is a key regulatory gene for development and function of Treg. Foxp3-transfected CD4+CD25- T cells have immunosuppressive function as nature occurring CD4+CD25+ regulatory T cells. It has been well known that Treg have suppressive function and can control several immune responses caused by self or non-self antigens. In this study, we like to investigate if we can deliver the Foxp3-overexpressed T cells for the alleviation of disease severity in murine lupus. We first isolated CD4+CD25+ T cells isolated from nonautoimmune DBA2/NZW F1 mice and adoptively transferred to NZB/NZW F1 mice, which spontaneously develop lupus. By assaying antibodies against dsDNA and ssDNA, we found that these cells could decrease the level of autoantibodies comparing to control mice. The therapeutic effects were seen one month after transfer, and could sustain for three months. We further wanted to induce regulatory T cells by infected CD4+CD25- T cells with Foxp3-expressing lentivirus. We have concentrated the lentivirus vector expressing Foxp3 gene, however, the in vivo study is yet to be finished. In the second part of the study, we would like to know whether arsenic trioxide (As2O3), a well known anticancer agent, could alleviate disease severity of SLE. We intraperitoneally injected different dose of arsenic trioxide to NZB/NZW F1 mice three days a week for two months. It was found that, one month after injection of arsenic trioxide the level of autoantibodies was lowered comparing to mice of control group. And the percentage of severe glomerulonephritis (GN) was lower in treatment groups. In addition, mice injected with arsenic trioxide were lived longer than mice injected with PBS only. Both adoptive transfers of CD4+CD25+ regulatory T cells and treatment of arsenic trioxide could alleviate the disease severity in lupus mice. In the future, these therapeutic approaches may be applied to clinical purposes.標題………………………………………………………..….……... i 口試委員會審定書……………………………………….…..…….. ii 授權書………………………………………………………...……... iii 誌謝………………………………………………………………….. iv 中文摘要……………………………………………….......………... vi ABSTRACT …...……………………………………...….………..... viii CONTENTS…………………………………………………………. x PART I………………………………………………………………. 1 CHAPTER I General Introduction………………….……………... 2 1.1 Background…………………………………………………. 3 1.2 Hypothesis………………………………. …………………. 15 1.3 Specific aim……………………………………………..…... 15 1.4 Siginificance………………………………………………... 15 CHAPTER II Materials and Methods……………………………... 16 2.1 Reagents…………….…………………………….………… 17 2.2 Mice………………………….……………………………... 22 2.3 Cell lines……………………………………………………. 23 2.4 Exploration of immunotherapy……………………………... 23 2.5 Transfection of primary T cells by lentiviral vector with Foxp3………………………………………...… 29 CHAPTER III Results………………….……………………….…. 35 3.1 immunotherapy analysis……………………………………. 36 3.2 Transfection of primary T cells by lentiviral vector……….. 39 CHAPTER IV Discussion and Perspectives…………….………… 43 PART II……………………………………………………………… 63 CHAPTER I General Introduction………………….……………... 64 1.1 Background…………………………………………………. 65 1.2 Specific aim……………………………………………..…... 67 CHAPTER II Materials and Methods……………………………... 68 2.1 Reagents…………….…………………………….…………. 69 2.2 Mice…………………………………………………………. 70 2.3 Methods……………………………………………………… 70 CHAPTER III Results………………….……………………….…. 73 REFERENCES……………………………………………….……… 79 Appendix…………………………………………………………….. 8