【作者】 卢小玲；

【导师】 吴雄文；

【作者基本信息】 华中科技大学 ， 免疫学， 2007， 博士

【摘要】 抗原特异性T细胞的活化是机体启动适应性免疫应答的关键事件。在此过程中,T细胞的活化需要抗原呈递细胞(antigen-presenting cell,APC)提供的双信号,第一信号由APC上的抗原肽/MHC复合物(peptide/MHC, pMHC)和T细胞上的T细胞抗原受体(TCR/CD3)结合提供,该信号决定T细胞识别抗原的特异性;第二信号即共刺激信号,由APC上的共刺激分子(B7、ICAM-1和LFA-3等)与T细胞上的相应受体(CD28、LFA-1、CD2等)结合后产生,该信号为T细胞活化不可或缺的。TCR以及其他T细胞表面分子和APC表面相应配体结合,通过介导信号转导事件和提高T细胞-APC相互作用的总体亲和力而触发T细胞活化。T细胞表面的CD28分子在功能上是作为一个重要的共刺激受体,其结合B7分子或者抗CD28抗体后,可以提供共刺激信号。据此,在细胞样大小(4-5um)的聚苯乙烯乳胶微粒上吸附pMHC和抗CD28抗体分子,使之成为能够为T细胞活化提供双信号的人工抗原呈递细胞(artificial antigen-presenting cell,aAPC)。显然aAPC表面呈递给T细胞的pMHC易于控制,克服了传统抗原呈递细胞因表面pMHC分子复杂,而不利于制备抗原特异性T细胞的缺点。因此,本研究在构建aAPC的基础上,将其用于同种T细胞识别的研究和肿瘤实验治疗的研究。我们的研究结果表明同种T细胞是pMHC特异性的,根据同种T细胞具有pMHC特异性的特点,用aAPC制备单一pMHC特异性的同种T细胞,过继治疗小鼠黑色素瘤,取得一定效果。本研究的主要内容及结果如下:1.重组的抗原肽/MHC-I复合物分子制备技术的建立背景:重组的抗原肽/MHC-I复合物分子对构建人工抗原呈递细胞和研究T细胞识别机制是十分有用的工具。目的:构建一种有功能的HLA-A*2402/BRLF1198-206四聚体,同时建立一套可用于制备各种重组的抗原肽/MHC-I复合物分子的制备技术。由于HLA-A*2402是亚洲人群中最常见型别之一,我们选择HLA-A*2402分子与HLA-A*2402限制性的EB病毒抗原肽BRLF1198-206来构建HLA-A*2402/BRLF1198-206四聚体。方法:克隆HLA-A*2402-BSP-pET21d重组质粒,经IPTG诱导原核表达目的蛋白,提取包涵体并初步纯化后溶于8M尿素中。在HLA-A*2402限制性的EB病毒抗原肽BRLF1198-206存在条件下,将得到的HLA-A*2402-BSP融合蛋白和β2m进行稀释复性折叠,形成折叠产物HLA-A*2402/BRLF1198-206复合物单体。通过一定的缓冲液更换与产物浓缩,在BirA酶的作用下,将折叠产物进行生物素化。用Western blot与ELISA方法来鉴定折叠和生物素化的产物。通过生物素与亲合素4:1的特异性结合关系,4个生物素化的HLA-A*2402/BRLF1198-206单体与1个荧光标记的亲合素结合而四聚化形成HLA-A*2402/BRLF1198-206四聚体。最后,用HLA-A*2402/BRLF1198-206四聚体来检测aAPCs体外诱导的特异性细胞毒T淋巴细胞(CTLs)。结果:HLA-A*2402-BSP-pET21d重组质粒被成功克隆,HLA-A*2402-BSP融合蛋白以包涵体的形式在细菌内实现高效表达(12L饱和菌液共得300 mg蛋白,纯度为67%) , Western blot与ELISA证实产物的折叠和生物素化成功。制备的HLA-A*2402/BRLF1198-206四聚体能特异有效地结合相应T细胞(10.7% vs 0.11%)。结论:成功地制备了HLA-A*2402/BRLF1198-206四聚体,所摸索出的技术路线和实验方法同样适用于制备其他型别MHC I类分子及其相应抗原肽形成的复合物分子。从而为下面构建人工抗原呈递细胞和研究同种T细胞识别机制奠定了基础。2.人工抗原呈递细胞制备技术的建立背景:依据T细胞活化的原理,人工抗原呈递细胞被设计用来刺激抗原特异性T细胞。人工抗原呈递细胞表面呈递给T细胞抗原分子可以严格被控制,克服了传统抗原呈递细胞因表面分子复杂不利于研究抗原特异性的T细胞识别的缺点。因此,构建aAPC利于同种T细胞识别的研究。目的:构建人工抗原呈递细胞HLA-A2/LMP2A426~434-aAPCs同时建立一套可用于制备各种人工抗原呈递细胞的制备技术。方法:将HLA-A2/LMP2A426~434复合物分子和抗CD28抗体分子吸附在细胞大小的聚苯乙烯乳胶微球表面制成HLA-A2/LMP2A426~434-aAPCs。采用流式细胞仪表型分析。然后,通过HLA-A2/LMP2A426~434-aAPCs和HLA-A2阳性个体人外周血淋巴细胞(PBLs)混合培养,LMP2A426~434特异性T细胞被诱导和扩增。用HLA-A2/LMP2A426~434四聚体染色法、对特异靶细胞加载抗原肽LMP2A426~434 T2细胞的细胞毒实验和用抗HLA I类分子构象抗体(W6/32)封闭靶细胞的细胞毒实验来检测这aAPCs诱导T细胞的特异性。结果:流式细胞仪表型分析显示HLA-A2/LMP2A426~434-aAPCs表面吸附有HLA-A2/LMP2A426~434复合物分子(85.48% vs 12.02%)和抗CD28抗体分子(72.38% vs 10.54% )。四聚体染色法和细胞毒实验结果表明构建的HLA-A2/LMP2A426~434-aAPCs能有效地诱导LMP2A426~434特异性T细胞的生成。结论:成功地构建了HLA-A2/LMP2A426~434-aAPCs,所摸索出的技术路线和实验方法同样适用于制备其他pMHC人工抗原呈递细胞。从而为下面同种T细胞的研究奠定了基础。3.用重组的抗原肽/MHC-I复合物分子和aAPCs探索肽在T细胞同种识别机制中作用的研究背景: MHC型别不同的供受者移植后会发生强烈的排斥反应(简称同种反应),其机制主要是同种反应性T细胞(简称同种T细胞)针对表达同种异体组织抗原(简称同种抗原)的细胞进行识别和应答。强烈的同种反应是免疫学中尚未完全阐明的现象,对同种抗原的本质和同种识别机制一直存在不同的解释。目的:用重组的抗原肽/MHC-I复合物分子和人工抗原呈递细胞探索同种CD8+ T细胞直接识别同种抗原表位的机制。方法:用H-2Kd限制性的来自BALB/c鼠内源性的五个自身肽段制备五种H-2Kd/自身肽四聚体来检测被BALB/c鼠皮肤致敏的C57BL/6鼠体内的CD8+同种T细胞的肽特异性的同种T细胞的频率。另外, ELISPOT实验和CFSE染色增殖实验中,用表面包被有不同pMHC分子H-2Kd/肽复合物的微球作为人工抗原呈递细胞特异刺激皮肤致敏的同种T细胞来研究CD8+同种T细胞的pMHC特异性。结果:FACS表型分析证实人工抗原呈递细胞H-2Kd/肽-aAPCs表面吸附有一定密度的H-2Kd/肽分子。四聚体染色结果表明五种H-2Kd/自身肽四聚体可以和五种同种反应肽T细胞特异结合,他们并不是重叠的,而是具有pMHC特异性的独立的同种反应T细胞亚群。ELISPOT实验和CFSE染色增殖实验结果显示CD8+同种T细胞反应频率随着aAPCs表面吸附的H-2Kd/肽表位种类的数量增高而增高。结论:同种反应性T细胞特异识别由非自身MHC分子和其结合的肽所形成的特异表位。支持强烈的同种反应是由于众多抗原肽/非自身MHC复合物特异刺激机体T细胞库中数量巨大的同种反应性T细胞克隆的累积效应引起的假说。4.利用由人工抗原呈递细胞诱导肿瘤特异性同种T淋巴细胞治疗肿瘤的研究背景:自身T细胞对自身MHC分子呈递的肿瘤抗原肽的反应通常是微弱和无效应的。同种限制性T细胞库的存在有可能产生针对非自身MHC分子结合的自身肽的T细胞,因为自身耐受是针对自身MHC分子呈递的自身肽无反应。因此,同种T细胞为过继肿瘤特异性T细胞治疗肿瘤提供了很有发展潜力的细胞来源。目的:用人工抗原呈递细胞H-2Kb-Ig/TRP2180-188-aAPCs从BALB/c鼠(H-2Kd)体内诱导黑色素瘤特异性同种T细胞,并过继治疗C57BL/6鼠的黑色素瘤。方法:制备表面包被有H-2Kb-Ig/TRP2180-188复合物分子和抗CD28抗体分子人工抗原呈递细胞H-2Kb-Ig/TRP2180-188-aAPCs。用此aAPCs从BALB/c鼠(H-2Kd)体内同种T细胞库中诱导针对H-2Kb限制性来自酪氨酸酶相关蛋白2(TRP2)黑色素瘤特异性抗原表位TRP2180-188的同种T淋巴细胞。体外用二聚体染色实验、ELISPOT实验、细胞毒实验来检测诱导的同种T淋巴细胞的特异性和杀伤活性。同时体内过继诱导的同种T淋巴细胞治疗C57BL/6鼠黑色素瘤。结果:二聚体染色结果显示诱导的同种T淋巴细胞能特异结合二聚体H-2Kb-Ig/pTRP2而不结合对照肽二聚体。ELISPOT实验结果表明诱导的同种T淋巴细胞对H-2Kb-Ig/pTRP2-aAPCs的抗原特异刺激分泌高的IFN-γ斑点数。细胞毒实验结果说明诱导的同种T淋巴细胞能特异杀伤相应靶细胞。体内过继治疗结果证实诱导的同种T淋巴细能有效地特异抑制黑色素瘤在C57BL/6鼠体内的生长。结论:黑色素瘤特异性H-2Kb限制性的同种T淋巴细胞能够从BALB/c鼠体内用H-2Kb-Ig/TRP2180-188-aAPCs诱导获得,过继治疗黑色素瘤有一定疗效。本研究依据T淋巴细胞活化理论体外构建人工抗原呈递细胞HLA-A2/LMP2A426~434-aAPCs,用其成功制备出HLA-A2/LMP2A426~434特异性T细胞。所摸索出的技术路线和实验方法,同样适用于制备其他类别人工抗原呈递细胞。构建的人工抗原呈递细胞具有表位易于控制的特点,是研究抗原特异性T细胞十分重要的工具。椐此制备各种抗原表位不同的H-2Kd/肽-aAPCs用于同种T细胞识别的研究,有力地证明同种T细胞是pMHC特异性的。根据同种T细胞具有pMHC特异性的特点,用H-2Kb-Ig/TRP2180-188-aAPCs从BALB/c鼠体内成功制备出黑色素瘤特异性同种T细胞,同时过继治疗小鼠黑色素瘤,有一定效果。因此本研究为抗原特异性T细胞的研究奠定了基础,为阐明同种抗原的本质和同种识别机制以及利用同种抗原特异性T细胞治疗肿瘤提供了实验依据。更多还原

【Abstract】 Activation of antigen-specific T cell plays a central role in initiating adaptive immune response. Antigen-presenting cell (APC) provides two signals for T-cell activation. The signal one is through T cell receptor (TCR) engaging the peptide/MHC complex on the surface of APC, which provides the basis for antigen specificity. The signal two through the other receptor on T cell (such as CD28, LFA-1 and CD2) provides a costimulatory signal following engagement of the ligand on the surface of APC (such as B7, ICAM-1 and LFA-3). TCR and other receptor on the surface of T cell upon ligand binding can initiate T cell activation by mediating signal transduction pathways and improving the total affinity of T cell-APC interaction. CD28 expressed on resting and activated T cells, the most potent costimulatory receptor, can provide costimulatory signal by binding B7 or anti-CD28 antibody. According to these, the artificial antigen-presenting cells (aAPCs) can be prepared by coating peptide/MHC complex and anti-CD28 antibody to the cell-size beads, which can provide signal one and signal two for T cell activation. Use of the aAPCs enabled stringent control of the antigens presented to T cells. So the aAPCs can be used for the study of alloreactive T cell recognition. Our research results showed alloreactive T cell is pMHC specific. Based on this, tumor specific alloreactive T cells induced by H-2Kb-Ig/TRP2180-188-aAPCs can be prepared which delay the growth of melanoma specifically. The following is a brief introduction to this research:1. The establishment of techniques for preparation recombinant peptide/MHC class I complexesBACKGROUND: Recombinant peptide/MHC class I complexes is a useful tool for the study of T cell recognition. AIM: To build the techniques for preparation of recombinant all sorts of peptide/MHC class I complexes and construct an HLA-A*2402/BRLF1198-206 tetramer. Since HLA-A*2402 is one of the most common human MHC class I allele in East Asia, we choose HLA-A*2402 and its binding peptide (EBV antigen BRLF1 198-206), to form the HLA-A*2402/BRLF1198-206 complexes and construct HLA-A*2402/BRLF1198-206 tetramers.METHODS: the HLA-A*2402-BSP-pET21d recombinant plasmid was cloned, and the HLA-A*2402-BSP fusion protein was induced by IPTG to expressed highly as insoluble aggregates in E.coli. After initial purification, the aggregates were dissolved in 8 M urea. Then the two subunits refolded to form an HLA-A*2402/BRLF1198-206 complex by dilution method in the presence of an antigenic peptide [NH2-TYPVLEEMF-COOH of EB virus BRLF1 198-206]. After concentrate and buffer exchange of refolding products, the BirA enzyme was used to biotinylate the refolded complex. The refolded and biotinylated products were detected by ELISA and Werstern blot. HLA-A*2402/BRLF1198-206 tetramers was prepared by mixing the biotinylated peptide-HLA complex with phycoerythrin (PE) labeled avidin at a molar ratio of 4:1.To the end, the HLA-A*2402/BRLF1198-206 tetramer is applied to detect antigen-specific cytotoxic T lymphocytes (CTLs) induced by aAPCs in vitro.RESULTS: The HLA-A*2402-BSP-pET21d recombinant plasmid was cloned successfully. ELISA and Werstern blot results demenstrate the refolded complex has been refolded and biotinylated successfully. FACS results show HLA-A*2402/BRLF1198-206 tetramers we prepared can bind its targets specifically and efficiently(10.7% vs 0.11%). CONCLUSION: HLA-A*2402/BRLF1198-206 tetramers can be prepared successfully.The techniques and methods can be used for preparation of recombinant complexes of other types of MHC class I molecules with the corresponding peptides, and lay the foundation for the construction of artificial antigen-presenting cells and the following study of alloreactive T cell recognition.2. The establishment of techniques for preparation aAPCsBACKGROUND: According to the principle of T cell activation, the artificial antigen-presenting cells (aAPCs) are designed to stimulate antigen-specific T cells. Use of the aAPCs enabled stringent control of the antigens presented to T cells. So the aAPCs can be used for the study of alloreactive T cell recognition.AIM: To construct HLA-A2/LMP2A426~434-aAPCs and establish the technology for preparation of aAPCs.METHODS: HLA-A2/LMP2A426~434-aAPCs were generated by coating HLA-A2/LMP2A426~434 complexes and anti-human CD28-specific antibodies to cell-sized latex beads. The immunophenotype of HLA-A2/LMP2A426~434-aAPCs was analyzed by FACS. Then by co-culture of the HLA-A2/LMP2A426~434-aAPCs and peripheral blood lymphocytes from HLA-A2 positive healthy donors, LMP2A426~434 antigen-specific CTLs were induced and expanded in vitro. The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2/LMP2A426~434 tetramer staining, cytotoxicity against HLA-A2/LMP2A426~434 bearing T2 cell and the cytotoxicity inhibited by the anti-HLA class I antibody (W6/32).RESULTS: The analysis of FACS shows that HLA-A2/LMP2A426~434 complexes (85.48% vs 12.02%) and anti-CD28 antibody (72.38% vs 10.54%) are immobilized on microbeads. Tetramer staining and cytotoxicity assay results showed that LMP2A426~434 antigen-specific CTLs could be induced and expanded in vitro by the HLA-A2/LMP2A426~434-aAPCs.CONCLUSION: HLA-A2/LMP2A426~434-aAPCs can be constructed successfully. The techniques and methods can be used for construction of other types of aAPCs and lay the foundation for the study of antigen specific T cell.3. Study of Peptide Involvement in T Cell Allorecognition using recombinant peptide/MHC class I and aAPCsBACKGROUND: Allogeneic transplantation causes acute rejection when MHC molecules of donor and receptor were mismatched (allo-reaction). The major mechanism is that alloreactive T cells recognize the graft cell-expressed allogeneic tissue antigens (alloantigens), and respond to them. The phenomenon of T-cell alloreactivity is a paradox. Alloreactive T cells respond to MHC molecules with exceptional vigor. Typically, 1–10% of T cells respond to allogeneic MHC molecules. This is orders of magnitude above the frequency of T cells specific to antigen presented by self-MHC. The molecular basis for exceptionally high frequency and diversity of T cells responding to allogeneic antigens remains elusive.AIM: To investigate CD8+ T cell allorecognition using recombinant peptide/MHC class I complexes and artificial Ag-presenting cells (aAPCs).METHODS: The frequency of CD8+ alloreactive T cell primed with skin graft was explored using tetramer staining tests by a panel of H-2Kd tetramers representing five self-peptides derived from ubiquitously expressed proteins known to be bound endogenously by this allotype. In addition, in the ELISPOT assay and CFSE proliferation assay, CD8+ alloreactive T cell with pMHC specific was explored using a panel of aAPCs that represent functional densities of H-2Kd molecules displaying antigen peptides.RESULTS: The analysis of FACS showed that peptide/H-2Kd complexes are immobilized on these different H-2Kd/peptide-aAPCs. Tetramer staining results showed the alloreactive populations generated using skin graft contain peptide specific subsets of T cells that respectively bind the H-2Kd/KYI, H-2Kd/SYF, H-2Kd/SFV, H-2Kd/YYV and H-2/Kd/NYA tetramers. They do not overlap. ELISPOT assay and CFSE proliferation assay results demonstrated skin grafting enriches for peptide-specific alloreactive CTL CONCLUSION: Peptide-specific recognition by alloreactive T cells predominates, supporting the proposal that the strength of the alloreactive response is primarily due to the large amount of T cells stimulated by the diverse array of peptides on the nonself MHC molecules.4. Exploitation of tumor-specific alloreactive T cells for Tumor Immunotherapy using aAPCsBACKGROUND: Autologous T cell responses to tumor Ags presented by self-MHC are usually weak and ineffective. The existence of the allorestricted repertoire raises the possibility of generating T cells reactive against synthetic self-peptides bound to nonself-MHC molecules, because tolerance to self-antigens is self-MHC restricted. Therefore, allorestricted T cells represent a potent source of tumor-specific T cells for adoptive immunotherapy.AIM: To induce melanoma-specific H-2Kb-restricted alloreactive T cells from BALB/c mice (H-2Kd) using H-2Kb-Ig/TRP2180-188-aAPC in vivo for melanoma immunotherapy.METHODS: H-2Kb-Ig/TRP2180-188-aAPCs were constructed by coating H-2Kb-Ig/TRP2180-188 complexes and anti-mouse CD28-specific antibodies to cell-sized latex beads. CD8+ alloreactive T cells specific for a melanoma-specific, H-2Kb-restricted CTL epitope (TRP2180-188) derived from mouse tyrosinase-related protein 2 (TRP2) from BALB/c mice (H-2Kd) were prepared using H-2Kb-Ig/TRP2180-188-aAPC in vivo. The specificity of the aAPC-induced alloreactive T cells was demonstrated by H-2Kb-Ig/TRP2180-188 dimer staining, ELISPOT assay and cytotoxicity assay. Specific antimelanoma effects of the alloreactive T cells induced by H-2Kb-Ig/TRP2180-188-aAPCs was identified by intratumoral injection with alloreactive T cells.RESULTS: Dimer staining result showed the alloreactive T cells induced by H-2Kb-Ig/TRP2180-188-aAPCs can specifically bind H-2Kb-Ig/pTRP2 dimer instead of control dimer. ELISPOT assay result showed the induced-T cells can generate higher IFN-γspots when they were stimulated by H-2Kb-Ig/TRP2180-188-aAPCs. Cytotoxicity assay results determined the induced-T cells can specifically kill its target. The induced-T cells can delay the growth of melanoma specifically.CONCLUSION: Melanoma-specific H-2Kb-restricted alloreactive CTLs can be obtained from allo-restricted T cells repertoire of BALB/c mice by H-2Kb-Ig/TRP2180-188-aAPCs and can delay the growth of melanoma specifically. In this study, according to the principle of T cell activation, the artificial antigen-presenting cells HLA-A2/LMP2A426~434-aAPCs were constructed and T cells specific for HLA-A2/LMP2A426~434 were successfully prepared using HLA-A2/LMP2A426~434-aAPCs. The techniques and methods can be used for construction of other types of aAPCs. Use of the aAPCs enabled stringent control of the antigens presented to T cells. So the aAPCs was a very important tool for the study of antigen-specific T cell. Based on these, the study of alloreactive T cell recognition using a panel of H-2Kd /peptide-aAPCs determined the alloreactive T cell is pMHC specific. Because the alloreactive T cell is pMHC specific, Melanoma-specific H-2Kb-restricted alloreactive CTLs can be obtained from allo-restricted T cells repertoire of BALB/c mice by H-2Kb-Ig/TRP2180-188-aAPCs in vivo and can delay the growth of melanoma specifically. Therefore, our research lays the foundation for further study of antigen-specific T cell and offers experimental basis for the mechanism of allorecognition and tumor immunotherapy using tumor-specific allreactive T cells.更多还原