利用aAPC制备pMHC特异性T细胞及其在同种T细胞识别和抗肿瘤实验研究中的应用

发布时间：2018-05-14 13:41

本文选题：同种识别 + 同种T细胞　；参考：《华中科技大学》2007年博士论文

【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。
[Abstract]:The activation of antigen - specific T cells is a key event in the organism to initiate adaptive immune response . In this process , activation of T cells requires a dual signal provided by antigen - presenting cells ( APC ) , which determine the specificity of T - cell recognition antigens ; the second signal , the co - stimulatory signal , is activated by the antigen peptide / MHC complex ( peptide / MHC , pMHC ) on the APC and the corresponding receptor on the T cell ( CD28 , lfa - 1 , CD - 2 , etc . ) . The CD28 molecule on the surface of the T - cell is functionally active as an important co - stimulatory receptor , which binds to the B7 molecule or anti - CD28 antibody to form an artificial antigen - presenting cell ( aAPC ) capable of providing a dual signal for T cell activation . The results showed that the allogeneic T cells were pMHC - specific , and the allogeneic T cells with single pMHC specificity were prepared by using aAPC .

1 . Establishment of recombinant antigen peptide / MHC - I complex molecule preparation technology

Background : Recombinant antigen peptide / MHC - I complex molecules are useful tools for constructing artificial antigen presenting cells and studying T cell identification mechanisms .

Objective : To construct a functional HLA - A * 2 / BRLF1198 - 206 tetramers , and to establish a set of preparation techniques which can be used to prepare various recombinant antigen peptide / MHC - I complex molecules . Since HLA - A * 2 2 is one of the most common types in Asian population , we choose HLA - A * 402 molecule and HLA - A * 2 - restrictive EB virus antigen peptide BRLF1198 - 206 to construct HLA - A * 402 / BRLF1198 - 206 tetramers .

Methods : The recombinant plasmid was cloned and purified by IPTG . The recombinant plasmid was purified and then dissolved in 8 M urea . The results showed that the specific binding relationship between HLA - A * 2 2 / BRLF1198 - 206 and HLA - A * 2 2 / BRLF1198 - 206 was detected by Western blot and ELISA .

Results : The recombinant plasmid was successfully cloned and HLA - A * 2 - BSP - pET21d recombinant plasmid was successfully cloned , and HLA - A * 2 2 - BSP fusion protein was successfully expressed in bacteria in the form of inclusion body . The purity of the recombinant plasmid was 67 % . Western blot and ELISA confirmed that the product was folded and biotinylated successfully . The HLA - A * 2 - 2 / BRLF1198 - 206 tetramers prepared were able to bind to the corresponding T cells ( 10.7 % vs 0.11 % ) .

Conclusion : We have successfully prepared HLA - A * 2 / BRLF1198 - 206 tetramers , and the technical and experimental methods are also applicable to the preparation of complex molecules formed by other MHC class I molecules and their corresponding antigen peptides . The results provide the basis for the establishment of artificial antigen presenting cells and the research of the same T cell identification mechanism .

2 . Establishment of artificial antigen presenting cell preparation technology

Background : According to the principle of T cell activation , artificial antigen presenting cells are designed to stimulate antigen - specific T cells .

Objective : To construct artificial antigen presenting cells HLA - A2 / LMP2A426 - 434 - aA and to establish a set of preparation techniques which can be used to prepare various artificial antigen presenting cells .

Methods : HLA - A2 / LMP2A426 - 434 complex molecule and anti - CD28 antibody were adsorbed on the surface of polystyrene latex microspheres . HLA - A2 / LMP2A426 - 434 - awere prepared by flow cytometry .

Results : HLA - A2 / LMP2A426 - 434 complex molecules ( 85.48 % vs 12.02 % ) and anti - CD28 antibody molecules ( 72.38 % vs 10.54 % ) were found on the surface of HLA - A2 / LMP2A426 ~ 434 - asurfaces by flow cytometry . The results of tetrameric staining and cytotoxicity indicate that HLA - A2 / LMP2A426 ~ 434 - acan effectively induce the formation of LMP2A426 ~ 434 specific T cells .

Conclusion : HLA - A2 / LMP2A426 ~ 434 - aare successfully constructed . The technical and experimental methods of HLA - A2 / LMP2A426 ~ 434 - aare also suitable for the preparation of other pMHC artificial antigen presenting cells .

3 . Study on the Role of the Recombinant Antigen Peptide / MHC - I Complex Molecule and the Peptide in the Homologous Recognition of T Cell

BACKGROUND : There is a strong rejection response ( the same reaction ) after transplantation of MHC - type donor , and its mechanism is mainly the identification and response of alloreactive T cells ( allogeneic T cells ) for the expression of alloantigens ( the same antigen ) . Strong homologous reactions are not fully elucidated in immunology , and different explanations have been given to the nature of the same antigen and the same identification mechanism .

Objective : To explore the mechanism of direct recognition of allogeneic CD8 + T cells by recombinant antigen peptide / MHC - I complex molecule and artificial antigen presenting cells .

Methods : Five kinds of H - 2Kd / autopeptide tetramers were prepared from five self - peptide fragments of BALB / c mice by H - 2Kd , and the frequency of CD8 + allogeneic T cells in C57BL / 6 mice sensitized by BALB / c mouse skin was detected . In addition , the pMHC specificity of CD8 + allogeneic T cells was investigated by using microballoons coated with different pMHC molecules H - 2Kd / peptide complexes as artificial antigen presenting cells to stimulate skin - sensitized allogeneic T cells .

Results : The results showed that the H - 2Kd / peptide molecule adsorbed on the surface of human antigen - presenting cells H - 2Kd / peptide - aB was adsorbed by FACS analysis . The results showed that the five H - 2Kd / autopeptide tetramers could be specifically combined with five alloreactive peptide T cells . They were not overlapping , but they had pMHC - specific independent alloreactive T - cell subsets . The results of the experiment showed that the frequency of CD8 + allogeneic T cells increased with the number of H - 2Kd / peptide epitopes adsorbed on the surface of the surface .

Conclusion : The alloreactive T cells specifically recognize the specific epitopes formed by non - self MHC molecules and peptides bound thereto . Strong homologous reactions are supported by the hypothesis that a large number of antigenic peptides / non - self MHC complexes specifically stimulate the cumulative effect of a large number of alloreactive T cell clones in the body T cell bank .

4 . Background of the study of tumor specific allogeneic T lymphocytes induced by artificial antigen presenting cells : The response of autologous T cells to tumor antigen peptides presented by their own MHC molecules is usually weak and ineffective . The presence of the same restriction T cell bank is likely to produce T cells for the self peptide binding to non - MHC molecules , since self - tolerance is not a response to the self - peptide presented for its own MHC molecules . Thus , allogeneic T cells provide a promising source of cells for adoptive tumor - specific T cell therapy tumors .

Objective : To induce melanoma - specific allogeneic T cells from BALB / c mice ( H - 2Kd ) by artificial antigen presenting cells H - 2Kb - Ig / TRP2180 - 188 - aB , and adoptive treatment of melanoma in C57BL / 6 mice .

Methods : H - 2Kb - Ig / TRP2180 - 188 complex molecule and anti - CD28 antibody molecule artificial antigen - presenting cell H - 2Kb - Ig / TRP2180 - 188 - awere prepared from BALB / c mice ( H - 2Kd ) .

Results : Dimer staining showed that the same T lymphocyte could specifically bind to the dimer .

The results showed that the induction of allogeneic T lymphocytes could specifically inhibit the growth of melanoma in C57BL / 6 mice .

Conclusion : The specific H - 2Kb - restricted allogeneic T lymphocytes can be induced by H - 2Kb - Ig / TRP2180 - 188 - aon BALB / c mice .

In this study , HLA - A2 / LMP2A426 - 434 cells were constructed by T lymphocyte activation theory . HLA - A2 / LMP2A426 ~ 434 - specific T cells were prepared successfully .

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

【参考文献】