【作者】 宋银宏；

【导师】 吴雄文；

【作者基本信息】 华中科技大学 ， 免疫学， 2010， 博士

【摘要】 T细胞在同种移植物排斥反应和移植物抗宿主病中起主要作用,CD4+和CD8+T细胞都参与了这些反应。T细胞TCR识别的配体是抗原肽和MHCⅠ或MHCⅡ分子形成的复合物(pMHC),分别被CD8+和CD4+T细胞识别。急性排斥反应中T细胞对同种抗原的直接识别起主要作用。研究表明T细胞识别抗原肽／同种MHC复合物和抗原肽／自身MHC复合物一样通常也是pMHC特异性的,并且TCR和同种MHC分子的相互作用方式与传统的自身MHC分子提呈抗原肽给TCR识别是一样的。抗原肽特异性的的同种反应性T细胞被称为同种限制性T细胞。已有研究证实是CD4+而非CD8+T细胞在同种排斥反应中起本质作用：一方面CD4+T可以辅助其他淋巴细胞参加免疫反应,另一方面它本身也是移植物排斥反应的效应细胞。同时,临床资料显示移植物抗白血病反应(GVLR)使白血病病人的病情得以缓解,并且移植物抗肿瘤反应(GVTR)在许多实体瘤的病例中也得到证实,这是由于供者T淋巴细胞对宿主的白血病细胞及肿瘤细胞发生了免疫反应。尽管许多研究都集中于CD8+T细胞在GVLR或者GVTR中的作用并且发现CD8+T细胞在其中发挥效应作用,但实际上在GVLR中需要CD4+T和CD8+T细胞两者共同参与。由于CD4+T细胞在普通免疫反应和同种反应中均发挥重要作用,我们认为同种反应性CD4+T细胞尤其是同种限制性CD4+T细胞可能在GVLR或者GVTR中起重要作用。本研究目的是要建立一个诱生同种限制性CD4+T细胞的方法并且观察这种CD4+T细胞的肿瘤排斥效应。本研究先制备出肿瘤抗原肽/HLA-DR15/IgG1-Fc二聚体,再将结合了此二聚体的HLA-DR15阴性的单核细胞和自体的外周血淋巴细胞(PBL)共培养诱导出肿瘤肽特异性的同种CD4+T细胞,并将此细胞过继到荷瘤裸鼠体内,发现其可以明显抑制荷瘤裸鼠体内的肿瘤生长。论文共分为三部分,其主要内容与结果如下：一、制备HLA-DR15/IgGl-Fc二聚体,并且该二聚体能有效结合到]HLA-DR15阴性(DR15-ve)个体的单核细胞表面采用RT-PCR方法从人宫颈癌细胞系SiHa(为HLA-DR15基因型)中扩增DRa链和DRp链的信号肽及胞外序列以及转录因子Fos和Jun的亮氨酸拉链序列,将扩增的DRa段和DRβ段分别和转录因子Fos和Jun的亮氨酸拉链区连接形成DRa-Fos和DRP-Jun,再将DRa-Fos和人IgG1的Fc段相连,构建出重组载体pFastBacTM(?)ual+[DR15/IgG1-Fc]。将此载体转化大肠杆菌DH10BacTM,得到重组的穿梭质粒Bacmid+[DR15/IgG1-Fc],将此穿梭质粒转入昆虫细胞Sf9中,收获上清得到成熟病毒颗粒,再用此病毒颗粒大量感染Sf9细胞,收集病毒感染的Sf9细胞培养上清,通过葡萄球菌蛋白A (SPA)柱纯化得到可溶性DR15/IgG1-Fc二聚体融合蛋白(简称DR15二聚体)。ELISA和Western-blotting检测均证实DR15二聚体产物由两部分组成：二聚化的DR15的胞外段提供有效的TCR反应部分,IgG1-Fc段能与单核细胞表面高亲和力受体FcyRI结合。将纯化的DR15二聚体与DR15-ve个体的单核细胞孵育,用流式细胞术检测具有HLA-DR15阳性表型的单核细胞百分数以确定二聚体与DR15-ve单核细胞的结合能力。流式分析结果显示DR15-ve单核细胞结合DR15二聚体后大多数具有HLA-DR15阳性表型,说明DR15二聚体可以有效结合到DR15-ve单核细胞表面。二、结合了抗原肽/HLA-DR15/IgG1-Fc二聚体的单核细胞诱导肽特异性的同种CD4+T细胞的产生为诱导抗原肽特异性的同种CD4+T细胞,本研究建立了一个共培养体系：纯化的DR15二聚体加载其限制性肽后结合到酸洗过的DR15-ve个体的单核细胞表面,形成共培养体系中的刺激细胞；同一个体来源的外周血淋巴细胞(PBLs)作为共培养体系中的效应细胞。共培养7 d后,通过CFSE稀释法由流式细胞仪检测到共培养体系中的CD4+T细胞发生了明显的增殖。再次用同样方法进行二次刺激,7 d后,该体系中淋巴细胞的特异性肽/DR15二聚体染色阳性细胞数显著高于对照,且诱导出的特异性肽／DR15二聚体阳性的CD4+T细胞能特异性的分泌细胞因子IFN-y,这些实验证实本研究所诱导的同种CD4+T细胞具有pMHC特异性。三、肿瘤肽特异性的同种CD4+T细胞能有效抑制荷瘤裸鼠的肿瘤生长人宫颈癌细胞SiHa是HLA-DR15基因型且高表达E7蛋白,可以作为本研究诱导的E7肽特异性的同种CD4+T的靶细胞。将经过二次刺激的淋巴细胞通过磁珠阴性分选得到较纯的肽特异的同种CD4+T细胞,再将其过继到荷SiHa瘤细胞的裸鼠体内,发现过继肿瘤抗原肽特异性的同种CD4+T细胞的裸鼠体内的肿瘤生长受到抑制,其生存期与对照组相比显著延长。免疫组织化学检测发现在特异性组有明显的人CD4+细胞浸润,而对照组不明显。总之,本研究通过将DR15-ve的淋巴细胞和结合了肽／DR15二聚体的自身单核细胞共培养诱导产生了肿瘤肽特异性的同种CD4+T细胞,这种CD4+T细胞在体外具有特异性的生物学功能,在体内可以明显地浸润到肿瘤组织局部,具有抑制肿瘤生长的作用,证实了CD4+T细胞也参与了GVTR (graft versus tumor reaction)并起重要作用。这不仅为诱导同种pMHC限制性CD4+T细胞提供了新的思路,也为肿瘤的过继免疫治疗提供了一种新的策略。本研究的创新点和意义：1.本研究将加载抗原肽的HLA-DR15/IgGl-Fc二聚体(DR15二聚体)结合到DR15-ve单核细胞表面,使之能够对自身PBLs’‘提呈”单一的同种pMHC,这种“提呈”单一pMHC的单核细胞和自身PBLs在体外共培养,能有效刺激自体PBLs产生同种限制性的CD4+T细胞。2.将针对肿瘤抗原的同种限制性CD4+T细胞过继输入到荷瘤裸鼠体内,可以有效抑制肿瘤生长；在肿瘤生长部位,发现有明显的CD4+T细胞浸润,从而证实了同种pMHC限制性CD4+T细胞参与了GVTR。这不仅为诱导同种pMHC限制性CD4+T细胞提供了新的思路,也为肿瘤的过继免疫治疗提供了一种新的策略。更多还原

【Abstract】 T cell plays a major role in allogeneic graft rejection and graft-versus-host disease (GVHD), both CD4+ and CD8+ T cells are involved in the process. The ligands recognized by T cell receptor (TCR) are formed by antigenic peptide associated with MHC class I or classⅡmolecule, which are recognized by CD8+ T and CD4+ T cells, respectively. Direct T-cell allo-recognition is the principal player in the initial vigorous immune response to allogeneic cells that causes acute rejection. Current studies indicate that allogeneic T-cell responses, like syngeneic T-cell responses, are largely peptide-specific, and that TCRs interact with allogeneic MHC in a manner that is almost indistinguishable from conventional recognition of antigenic peptide presented by self-MHC. The allo-reactive T cells of peptide-dependent and allogeneic MHC-restricted properties are referred to as allo-restricted T cells. It has been observed that CD4+ but not CD8+ T cells are essential for allo-rejection, since CD4+ T cells are generally regarded as helper cells by facilitating other lymphoid cells involved in immunity, as well as effector cells of graft rejection. Meanwhile, it is well-established that leukemia patients can benefit from a graft-versus-leukemia reaction (GVLR), and evidence of graft-versus-tumor reaction (GVTR) has also been reported in many solid tumors, in which donor T lymphocytes mount an immune response against recipients’ leukemia or tumor cells. Both CD4+ T cell and CD8+ T cell are required for GVLR, although most studies are focused on the role of CD8+ T cell in GVLR or GVTR and reveal CD8+ T cell is an effective means. Based on the importance of CD4+ T cells in both nominal and allogeneic responses, we suppose that allo-reactive CD4+ T cells, especially allo-restricted CD4+ T cells, would be responsible for GVLR or GVTR. The aim of this study is to set up a strategy to generate allo-restricted CD4+ T cells and observe tumor rejection effects of the CD4+ T cells.The tumor-peptide/HLA-DR15/IgGl-Fc dimer was prepared in this study, then the tumor-peptide specific allogeneic CD4+ T cells were induced by the dimer-loaded monocytes co-culturing with autologous HLA-DR15 negative PBLs. The tumor-peptide specific allogeneic CD4+ T cells adoptively transferred into nude mice challenged by human cervical cancer cells can inhibit the growth of the tumor.1. Generation of HLA-DR15/IgGl-Fc molecule and its binding to HLA-DR15 negative (DR15-ve) monocytesThe cDNA coding for the extracellular domains of DRa and DRβas well as the leucine zipper motifs of Fos and Jun were cloned by RT-PCR from SiHa cell which is HLA-DR15 genotype. The extracellular domains of DRa and DRβwere joined with the leucine zipper motifs of Fos and Junm, then the Fc segment of human IgGl was attached to DRa-Fos through the EcoR I restriction site. The recombinant plasmid named pFastBacTM Dual+[DR15/IgGl-Fc] was constructed and transformed to Escherichia coli DH10BacTM and the recombinant shuttle plamid Bacmid+[DR15/IgG1-Fc] was obtained. The shuttle plamid was transfected into the insect cell line Sf9 by lipofection and the expression of HLA-DR15/IgG1-Fc dimer (DR15 dimer) was detected by sandwich ELISA and Western-blotting. The results of ELISA and Western-blotting showed that DR15 dimer molecule consists of two parts:the dimerization of extracelluar fragment of HLA-DR15 molecule which can bind to specific T cell receptor (TCR) and the Fc fragment of IgG1 which binds to FcyRI on the cell membrane of monocytes with high affinity.DR15-ve monocytes incubated with purified DR15 dimer and the capacity of DR15 dimer binding to monocytes was confirmed by flow cytometry (FCM). The result showed that the percent of DR15 phenotype monocytes which were DR15-ve genotype significantly increased after incubated with purified DR15 dimer, which indicated that DR15 dimer has the capacity of binding to DR15-ve monocytes.2. Raising peptide-specific allogeneic CD4+ T cells from DR15-ve PBLs co-cultured with autologous monocytes loaded with the dimer DR15-ve PBLs were co-cultured with the dimer-loaded autologous monocytes for raising alloreactive CD4+ T cells. The autologous monocytes loaded with dimers (pulsed the specific peptide) were served as stimulating cells. PBLs obtained by density gradient centrifugation were served as stimulated cells. Here an allogeneic MHC classⅡmolecule associated with its restricted peptide is attached to monocytes. The DR15 dimer-loaded monocytes "present" the allogeneic epitope and induce autologous PBLs to generate peptide-specific allogeneic CD4+ T cells.The results of’CFSE dilution assay in vitro show that the dimer-loaded monocyte is able to stimulate autologous CD4+ T-cell proliferation; and specific dimer staining and ELISPOT assays reveal the allogeneic CD4+ T cells are pMHC specific.3. The tumor antigen peptide-specific allogeneic CD4+ T cells can be efficient at tumor rejection in vivoThe DR15-ve PBLs were stimulated by two cycles of tumor peptide/DR15 dimer-loaded autologous monocytes for 14 d, and the CD4+ T cells purified from the bulk were adoptively transferred into nude mice challenged by SiHa cells. The result showed that the percent of tumor free mice was significantly higher in the specific group than in control groups and the percent survival of mice was also significantly higher in the specific group than in control groups. The result of immunohistochemistry showed that there were much more CD4+ cells infiltrating into tumors in the specific group than in control groups.In conclusion, peptide-specific allogeneic CD4+ T cells are induced by cocultruing DR15-ve PBLs and peptide/DR15 dimer-loaded autologous monocytes in this study and the CD4+ T cells have specific biological function in vitro and in vivo. Tumor antigen peptide-specific allogeneic CD4+ T cells infiltrate obviously into the tumor tissue of nude mice and inhibit the growth of the tumor, which verifies alloreactive CD4+ T cells play an important role in GVTR and support the development of clinical strategies focusing on exploiting the function of tumor-reactive CD4+ T cells. This study also affords a new strategy preparing allogenic tumor antigen-specific CD4+ T cells.更多还原