過繼單一pMHC特異性的同種CTL清除體內(nèi)特定性狀的靶細(xì)胞
發(fā)布時(shí)間：2018-07-26 08:27
【摘要】： 由于移植排斥反應(yīng)的存在,T細(xì)胞對(duì)于同種細(xì)胞的應(yīng)答通常被認(rèn)為是有害的。然而伴隨移植物抗宿主病(GVHD)發(fā)生的移植物抗白血病反應(yīng)/移植物抗腫瘤反應(yīng),表明了同種反應(yīng)性T細(xì)胞中存在一群有利的細(xì)胞,在清除惡性腫瘤中具有巨大作用。我們的前期工作提示同種T細(xì)胞與一般抗原反應(yīng)性T細(xì)胞一樣,識(shí)別的表位均為抗原肽/MHC復(fù)合物(pMHC),且均具有pMHC特異性,故在實(shí)際應(yīng)用中有可能根據(jù)同種反應(yīng)性T細(xì)胞的不同特異性將有利和不利的同種T細(xì)胞區(qū)分開。本研究擬在單個(gè)pMHC水平上,提供實(shí)驗(yàn)依據(jù)證實(shí)過繼單一表位特異性的同種CTL可在體內(nèi)清除特定性狀的靶細(xì)胞,同時(shí)避免多組織損傷的發(fā)生。不僅有利于進(jìn)一步認(rèn)識(shí)同種反應(yīng)發(fā)生的機(jī)制及其規(guī)律,而且為利用同種T細(xì)胞打破病理性耐受(慢性感染與腫瘤等)提供了新手段。 然而由于細(xì)胞表面存在極其繁多的pMHC,用傳統(tǒng)的方法制備獲得的同種反應(yīng)性T細(xì)胞為針對(duì)多種表位的細(xì)胞群體。因此,本研究通過建立“單一表位特異性同種T細(xì)胞誘導(dǎo)體系”制備單一表位特異性同種CTL(胰島素肽/H-2Kb特異性同種CTL),并過繼輸入動(dòng)物體內(nèi),攻擊表達(dá)該pMHC的靶細(xì)胞(H-2b小鼠的胰島β細(xì)胞),觀察其特異性殺傷效應(yīng)。論文共分為三部分,其主要內(nèi)容與結(jié)果如下： 一、制備H-2Kb/IgG2aFc分子,其能有效結(jié)合到C3H小鼠巨噬細(xì)胞表面 首先通過融合C57BL/6小鼠H-2Kb重鏈胞外段和IgG2aFc段獲得H-2Kb/IgG2aFc融合基因,并與小鼠小鼠β2m基因分別插入桿狀病毒雙表達(dá)載體pFastBacTMDual的PP10和PPH啟動(dòng)子下游,利用昆蟲桿狀病毒表達(dá)系統(tǒng)表達(dá)以二聚體形式存在的β2m/H-2Kb/IgG2aFc分子(簡稱H-2Kb二聚體)。融合分子中IgG2aFc為高親和力的Fc,可通過與FcR的相互作用加載H-2Kb二聚體到C3H小鼠巨噬細(xì)胞(H-2Kk)表面,使其能夠“提呈”單一同種表位(特異性肽/H-2Kb),從而為誘導(dǎo)單一pMHC特異性的同種CTL奠定了基礎(chǔ)。 二、體內(nèi)外聯(lián)合誘導(dǎo)自身/同系淋巴細(xì)胞產(chǎn)生胰島素肽/H-2Kb特異性的同種CTL 利用加載了胰島素肽/H-2Kb二聚體的自身巨噬細(xì)胞作為抗原刺激細(xì)胞,經(jīng)過腹腔注射C3H小鼠(H-2Kk),誘導(dǎo)其產(chǎn)生針對(duì)胰島素肽/H-2Kb表位的特異性同種T細(xì)胞,收集并對(duì)其進(jìn)行特異性四聚體染色和細(xì)胞毒效應(yīng)檢測。結(jié)果顯示誘導(dǎo)產(chǎn)生的CD8+T細(xì)胞特異性四聚體染色頻率顯著高于非特異性四聚體染色頻率；同時(shí),該CTL對(duì)具有相應(yīng)表位(胰島素肽/H-2Kb)的靶細(xì)胞具有明顯的殺傷作用,而對(duì)無關(guān)表位的靶細(xì)胞殺傷率極低。 三、尾靜脈過繼誘生的單一pMHC特異性的同種T細(xì)胞定向損傷胰島β細(xì)胞 將胰島素肽/H-2Kb特異性CTL由尾靜脈分別過繼受者C57BL/6小鼠(H-2b)、B6C3F1小鼠(H-2b×k)和SCID beige小鼠(H-2d),過繼后監(jiān)測血糖、胰島素及淋巴細(xì)胞浸潤情況。結(jié)果顯示接受過繼后,受者C57BL/6和B6C3F1小鼠第3天便出現(xiàn)明顯的血糖升高,胰島素水平降低并且胰島出現(xiàn)較明顯的β細(xì)胞缺如和T細(xì)胞浸潤；而受者SCID beige小鼠血糖和胰島素水平未見明顯變化,組織未見明顯損傷。結(jié)果提示過繼的單一pMHC特異性CTL能夠定向殺傷特定性狀的靶細(xì)胞。 本研究的創(chuàng)新點(diǎn)和意義： 1.提供直接的動(dòng)物體內(nèi)實(shí)驗(yàn)證據(jù)顯示同種反應(yīng)的發(fā)生機(jī)制,即任何抗原肽,只要能夠與MHC分子結(jié)合,都能夠形成同種T細(xì)胞的識(shí)別表位；同種反應(yīng)性T細(xì)胞與一般抗原反應(yīng)性T細(xì)胞一樣,都具有pMHC特異性。 2.選用小鼠體內(nèi)胰島β細(xì)胞損傷作為觀察同種CTL效應(yīng)的指標(biāo),研究結(jié)果將顯示過繼特異性同種T細(xì)胞是干預(yù)病理性免疫耐受的新途徑,同時(shí)為研究同種反應(yīng)提供新的技術(shù)平臺(tái)。
[Abstract]:Due to the existence of graft rejection, the response of T cells to the same cell is usually considered harmful. However, with graft versus host disease (GVHD), the graft anti leukemic reaction / graft antitumor response shows that there are a group of beneficial cells in the homologous T cells, which have a huge role in the elimination of malignant tumors. Our previous work suggests that the homologous T cells, like the general antigen reactive T cells, are all epitopes of the antigen peptide /MHC complex (pMHC), and all have pMHC specificity. Therefore, it is possible to separate the favorable and unfavorable homologous T cells according to the different specificity of the homologous T cells in practical applications. On the pMHC level, we provide experimental evidence that adoptive single epitopes specific homologous CTL can remove specific target cells in the body and avoid multiple tissue damage. It is not only conducive to further understanding the mechanism and regularity of the homologous reaction, but also for the use of the same T cells to break the pathological tolerance (chronic infection and tumor). And so on) provided a new means.
However, due to the extremely large number of pMHC on the surface of the cells, the homologous T cells obtained by the traditional methods are the cell populations of various epitopes. Therefore, the single epitope specific homologous T cell induction system was established to prepare the single epitopes specific homologous CTL (insulin peptide /H-2Kb specific homologous CTL) in this study. The target cells expressing the pMHC (islet beta cells of H-2b mice) were introduced and the specific killing effects were observed. The paper was divided into three parts. The main contents and results were as follows:
First, the preparation of H-2Kb/IgG2aFc molecules can effectively bind to the surface of C3H macrophages.
The H-2Kb/IgG2aFc fusion gene was obtained by fusion of the H-2Kb heavy chain and IgG2aFc segment of the C57BL/6 mice, and the beta 2m gene of mice was inserted into the downstream of PP10 and PPH promoter of the baculovirus double expression vector pFastBacTMDual, respectively. The beta 2m/H-2Kb/IgG2aFc molecule in the form of two polymer was expressed by the insect baculovirus expression system. (H-2Kb two polymer). The fusion molecule IgG2aFc is a high affinity Fc, and can be loaded with H-2Kb two polymer to C3H mouse macrophage (H-2Kk) surface by interaction with FcR, so that it can "present" a single homohomoepitope (specific peptide /H-2Kb), thus laying the foundation for the induction of single pMHC specific CTL.
Two, in vivo and in vitro, it induces self / homologous lymphocytes to produce insulin peptide /H-2Kb specific CTL.
The autologous macrophages loaded with the insulin peptide /H-2Kb two polymer were used as antigen stimulating cells, and the specific homologous T cells were induced by intraperitoneal injection of C3H mice (H-2Kk) to produce specific homologous T cells against the insulin peptide /H-2Kb epitopes. The specific four polymer staining and the detection of the cytotoxic effect were collected and detected. The results showed that the induced CD8+T was fine. The frequency of cell specific four polymer staining was significantly higher than that of non specific four polymer staining; at the same time, the CTL had an obvious killing effect on target cells with corresponding epitopes (insulin peptide /H-2Kb), and the target cells of unrelated epitopes were very low in killing rate.
Three, the single pMHC specific allogeneic T cells induced by caudal vein were targeted to damage islet beta cells.
The insulin peptide /H-2Kb specific CTL was adoptive from the tail vein to the recipient C57BL/6 mice (H-2b), the B6C3F1 mice (H-2b x k) and SCID beige mice (H-2d). The blood glucose, insulin and lymphocyte infiltration were monitored after adoptive follow-up. The results showed that the recipients of C57BL/6 and B6C3F1 mice showed significant increase in blood glucose and insulin levels in third days. Lower beta cells and T cell infiltration were found in the pancreatic islets, while the blood glucose and insulin levels in SCID beige mice were not significantly changed, and no obvious damage was found in the tissues. The result suggested that the adoptive single pMHC specific CTL could target specific target cells.
The innovation and significance of this study:
1. the experimental evidence of direct animal body shows that the mechanism of the homologous reaction, that is, any antigen peptide, can form the identification epitopes of the homologous T cells as long as they can be combined with MHC molecules; the same reactive T cells, like the general antigen reactive T cells, are all pMHC specific.
2. the pancreatic islet beta cell damage in mice is used as an indicator of the CTL effect. The results will show that adoptive specific homologous T cells are new ways to intervene in the pathological immune tolerance, and provide a new technical platform for the study of the same reaction.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
本文編號(hào)：2145433
[Abstract]:Due to the existence of graft rejection, the response of T cells to the same cell is usually considered harmful. However, with graft versus host disease (GVHD), the graft anti leukemic reaction / graft antitumor response shows that there are a group of beneficial cells in the homologous T cells, which have a huge role in the elimination of malignant tumors. Our previous work suggests that the homologous T cells, like the general antigen reactive T cells, are all epitopes of the antigen peptide /MHC complex (pMHC), and all have pMHC specificity. Therefore, it is possible to separate the favorable and unfavorable homologous T cells according to the different specificity of the homologous T cells in practical applications. On the pMHC level, we provide experimental evidence that adoptive single epitopes specific homologous CTL can remove specific target cells in the body and avoid multiple tissue damage. It is not only conducive to further understanding the mechanism and regularity of the homologous reaction, but also for the use of the same T cells to break the pathological tolerance (chronic infection and tumor). And so on) provided a new means.
However, due to the extremely large number of pMHC on the surface of the cells, the homologous T cells obtained by the traditional methods are the cell populations of various epitopes. Therefore, the single epitope specific homologous T cell induction system was established to prepare the single epitopes specific homologous CTL (insulin peptide /H-2Kb specific homologous CTL) in this study. The target cells expressing the pMHC (islet beta cells of H-2b mice) were introduced and the specific killing effects were observed. The paper was divided into three parts. The main contents and results were as follows:
First, the preparation of H-2Kb/IgG2aFc molecules can effectively bind to the surface of C3H macrophages.
The H-2Kb/IgG2aFc fusion gene was obtained by fusion of the H-2Kb heavy chain and IgG2aFc segment of the C57BL/6 mice, and the beta 2m gene of mice was inserted into the downstream of PP10 and PPH promoter of the baculovirus double expression vector pFastBacTMDual, respectively. The beta 2m/H-2Kb/IgG2aFc molecule in the form of two polymer was expressed by the insect baculovirus expression system. (H-2Kb two polymer). The fusion molecule IgG2aFc is a high affinity Fc, and can be loaded with H-2Kb two polymer to C3H mouse macrophage (H-2Kk) surface by interaction with FcR, so that it can "present" a single homohomoepitope (specific peptide /H-2Kb), thus laying the foundation for the induction of single pMHC specific CTL.
Two, in vivo and in vitro, it induces self / homologous lymphocytes to produce insulin peptide /H-2Kb specific CTL.
The autologous macrophages loaded with the insulin peptide /H-2Kb two polymer were used as antigen stimulating cells, and the specific homologous T cells were induced by intraperitoneal injection of C3H mice (H-2Kk) to produce specific homologous T cells against the insulin peptide /H-2Kb epitopes. The specific four polymer staining and the detection of the cytotoxic effect were collected and detected. The results showed that the induced CD8+T was fine. The frequency of cell specific four polymer staining was significantly higher than that of non specific four polymer staining; at the same time, the CTL had an obvious killing effect on target cells with corresponding epitopes (insulin peptide /H-2Kb), and the target cells of unrelated epitopes were very low in killing rate.
Three, the single pMHC specific allogeneic T cells induced by caudal vein were targeted to damage islet beta cells.
The insulin peptide /H-2Kb specific CTL was adoptive from the tail vein to the recipient C57BL/6 mice (H-2b), the B6C3F1 mice (H-2b x k) and SCID beige mice (H-2d). The blood glucose, insulin and lymphocyte infiltration were monitored after adoptive follow-up. The results showed that the recipients of C57BL/6 and B6C3F1 mice showed significant increase in blood glucose and insulin levels in third days. Lower beta cells and T cell infiltration were found in the pancreatic islets, while the blood glucose and insulin levels in SCID beige mice were not significantly changed, and no obvious damage was found in the tissues. The result suggested that the adoptive single pMHC specific CTL could target specific target cells.
The innovation and significance of this study:
1. the experimental evidence of direct animal body shows that the mechanism of the homologous reaction, that is, any antigen peptide, can form the identification epitopes of the homologous T cells as long as they can be combined with MHC molecules; the same reactive T cells, like the general antigen reactive T cells, are all pMHC specific.
2. the pancreatic islet beta cell damage in mice is used as an indicator of the CTL effect. The results will show that adoptive specific homologous T cells are new ways to intervene in the pathological immune tolerance, and provide a new technical platform for the study of the same reaction.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392
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