过继单一pMHC特异性的同种CTL清除体内特定性状的靶细胞
发布时间：2018-07-26 08:27
【摘要】： 由于移植排斥反应的存在,T细胞对于同种细胞的应答通常被认为是有害的。然而伴随移植物抗宿主病(GVHD)发生的移植物抗白血病反应/移植物抗肿瘤反应,表明了同种反应性T细胞中存在一群有利的细胞,在清除恶性肿瘤中具有巨大作用。我们的前期工作提示同种T细胞与一般抗原反应性T细胞一样,识别的表位均为抗原肽/MHC复合物(pMHC),且均具有pMHC特异性,故在实际应用中有可能根据同种反应性T细胞的不同特异性将有利和不利的同种T细胞区分开。本研究拟在单个pMHC水平上,提供实验依据证实过继单一表位特异性的同种CTL可在体内清除特定性状的靶细胞,同时避免多组织损伤的发生。不仅有利于进一步认识同种反应发生的机制及其规律,而且为利用同种T细胞打破病理性耐受(慢性感染与肿瘤等)提供了新手段。 然而由于细胞表面存在极其繁多的pMHC,用传统的方法制备获得的同种反应性T细胞为针对多种表位的细胞群体。因此,本研究通过建立“单一表位特异性同种T细胞诱导体系”制备单一表位特异性同种CTL(胰岛素肽/H-2Kb特异性同种CTL),并过继输入动物体内,攻击表达该pMHC的靶细胞(H-2b小鼠的胰岛β细胞),观察其特异性杀伤效应。论文共分为三部分,其主要内容与结果如下： 一、制备H-2Kb/IgG2aFc分子,其能有效结合到C3H小鼠巨噬细胞表面 首先通过融合C57BL/6小鼠H-2Kb重链胞外段和IgG2aFc段获得H-2Kb/IgG2aFc融合基因,并与小鼠小鼠β2m基因分别插入杆状病毒双表达载体pFastBacTMDual的PP10和PPH启动子下游,利用昆虫杆状病毒表达系统表达以二聚体形式存在的β2m/H-2Kb/IgG2aFc分子(简称H-2Kb二聚体)。融合分子中IgG2aFc为高亲和力的Fc,可通过与FcR的相互作用加载H-2Kb二聚体到C3H小鼠巨噬细胞(H-2Kk)表面,使其能够“提呈”单一同种表位(特异性肽/H-2Kb),从而为诱导单一pMHC特异性的同种CTL奠定了基础。 二、体内外联合诱导自身/同系淋巴细胞产生胰岛素肽/H-2Kb特异性的同种CTL 利用加载了胰岛素肽/H-2Kb二聚体的自身巨噬细胞作为抗原刺激细胞,经过腹腔注射C3H小鼠(H-2Kk),诱导其产生针对胰岛素肽/H-2Kb表位的特异性同种T细胞,收集并对其进行特异性四聚体染色和细胞毒效应检测。结果显示诱导产生的CD8+T细胞特异性四聚体染色频率显著高于非特异性四聚体染色频率；同时,该CTL对具有相应表位(胰岛素肽/H-2Kb)的靶细胞具有明显的杀伤作用,而对无关表位的靶细胞杀伤率极低。 三、尾静脉过继诱生的单一pMHC特异性的同种T细胞定向损伤胰岛β细胞 将胰岛素肽/H-2Kb特异性CTL由尾静脉分别过继受者C57BL/6小鼠(H-2b)、B6C3F1小鼠(H-2b×k)和SCID beige小鼠(H-2d),过继后监测血糖、胰岛素及淋巴细胞浸润情况。结果显示接受过继后,受者C57BL/6和B6C3F1小鼠第3天便出现明显的血糖升高,胰岛素水平降低并且胰岛出现较明显的β细胞缺如和T细胞浸润；而受者SCID beige小鼠血糖和胰岛素水平未见明显变化,组织未见明显损伤。结果提示过继的单一pMHC特异性CTL能够定向杀伤特定性状的靶细胞。 本研究的创新点和意义： 1.提供直接的动物体内实验证据显示同种反应的发生机制,即任何抗原肽,只要能够与MHC分子结合,都能够形成同种T细胞的识别表位；同种反应性T细胞与一般抗原反应性T细胞一样,都具有pMHC特异性。 2.选用小鼠体内胰岛β细胞损伤作为观察同种CTL效应的指标,研究结果将显示过继特异性同种T细胞是干预病理性免疫耐受的新途径,同时为研究同种反应提供新的技术平台。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
本文编号：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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
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