發(fā)布時間：2018-05-14 13:41

  本文選題：同種識別 + 同種T細胞��； 參考：《華中科技大學》2007年博士論文

【摘要】： 抗原特異性T細胞的活化是機體啟動適應性免疫應答的關(guān)鍵事件。在此過程中,T細胞的活化需要抗原呈遞細胞(antigen-presenting cell,APC)提供的雙信號,第一信號由APC上的抗原肽/MHC復合物(peptide/MHC, pMHC)和T細胞上的T細胞抗原受體(TCR/CD3)結(jié)合提供,該信號決定T細胞識別抗原的特異性;第二信號即共刺激信號,由APC上的共刺激分子(B7、ICAM-1和LFA-3等)與T細胞上的相應受體(CD28、LFA-1、CD2等)結(jié)合后產(chǎn)生,該信號為T細胞活化不可或缺的。TCR以及其他T細胞表面分子和APC表面相應配體結(jié)合,通過介導信號轉(zhuǎn)導事件和提高T細胞-APC相互作用的總體親和力而觸發(fā)T細胞活化。T細胞表面的CD28分子在功能上是作為一個重要的共刺激受體,其結(jié)合B7分子或者抗CD28抗體后,可以提供共刺激信號。據(jù)此,在細胞樣大小(4-5um)的聚苯乙烯乳膠微粒上吸附pMHC和抗CD28抗體分子,使之成為能夠為T細胞活化提供雙信號的人工抗原呈遞細胞(artificial antigen-presenting cell,aAPC)。顯然aAPC表面呈遞給T細胞的pMHC易于控制,克服了傳統(tǒng)抗原呈遞細胞因表面pMHC分子復雜,而不利于制備抗原特異性T細胞的缺點。因此,本研究在構(gòu)建aAPC的基礎上,將其用于同種T細胞識別的研究和腫瘤實驗治療的研究。我們的研究結(jié)果表明同種T細胞是pMHC特異性的,根據(jù)同種T細胞具有pMHC特異性的特點,用aAPC制備單一pMHC特異性的同種T細胞,過繼治療小鼠黑色素瘤,取得一定效果。本研究的主要內(nèi)容及結(jié)果如下: 1.重組的抗原肽/MHC-I復合物分子制備技術(shù)的建立 背景:重組的抗原肽/MHC-I復合物分子對構(gòu)建人工抗原呈遞細胞和研究T細胞識別機制是十分有用的工具。 目的:構(gòu)建一種有功能的HLA-A*2402/BRLF1198-206四聚體,同時建立一套可用于制備各種重組的抗原肽/MHC-I復合物分子的制備技術(shù)。由于HLA-A*2402是亞洲人群中最常見型別之一,我們選擇HLA-A*2402分子與HLA-A*2402限制性的EB病毒抗原肽BRLF1198-206來構(gòu)建HLA-A*2402/BRLF1198-206四聚體。 方法:克隆HLA-A*2402-BSP-pET21d重組質(zhì)粒,經(jīng)IPTG誘導原核表達目的蛋白,提取包涵體并初步純化后溶于8M尿素中。在HLA-A*2402限制性的EB病毒抗原肽BRLF1198-206存在條件下,將得到的HLA-A*2402-BSP融合蛋白和β2m進行稀釋復性折疊,形成折疊產(chǎn)物HLA-A*2402/BRLF1198-206復合物單體。通過一定的緩沖液更換與產(chǎn)物濃縮,在BirA酶的作用下,將折疊產(chǎn)物進行生物素化。用Western blot與ELISA方法來鑒定折疊和生物素化的產(chǎn)物。通過生物素與親合素4:1的特異性結(jié)合關(guān)系,4個生物素化的HLA-A*2402/BRLF1198-206單體與1個熒光標記的親合素結(jié)合而四聚化形成HLA-A*2402/BRLF1198-206四聚體。最后,用HLA-A*2402/BRLF1198-206四聚體來檢測aAPCs體外誘導的特異性細胞毒T淋巴細胞(CTLs)。 結(jié)果:HLA-A*2402-BSP-pET21d重組質(zhì)粒被成功克隆,HLA-A*2402-BSP融合蛋白以包涵體的形式在細菌內(nèi)實現(xiàn)高效表達(12L飽和菌液共得300 mg蛋白,純度為67%) , Western blot與ELISA證實產(chǎn)物的折疊和生物素化成功。制備的HLA-A*2402/BRLF1198-206四聚體能特異有效地結(jié)合相應T細胞(10.7% vs 0.11%)。 結(jié)論:成功地制備了HLA-A*2402/BRLF1198-206四聚體,所摸索出的技術(shù)路線和實驗方法同樣適用于制備其他型別MHC I類分子及其相應抗原肽形成的復合物分子。從而為下面構(gòu)建人工抗原呈遞細胞和研究同種T細胞識別機制奠定了基礎。 2.人工抗原呈遞細胞制備技術(shù)的建立 背景:依據(jù)T細胞活化的原理,人工抗原呈遞細胞被設計用來刺激抗原特異性T細胞。人工抗原呈遞細胞表面呈遞給T細胞抗原分子可以嚴格被控制,克服了傳統(tǒng)抗原呈遞細胞因表面分子復雜不利于研究抗原特異性的T細胞識別的缺點。因此,構(gòu)建aAPC利于同種T細胞識別的研究。 目的:構(gòu)建人工抗原呈遞細胞HLA-A2/LMP2A426~434-aAPCs同時建立一套可用于制備各種人工抗原呈遞細胞的制備技術(shù)。 方法:將HLA-A2/LMP2A426~434復合物分子和抗CD28抗體分子吸附在細胞大小的聚苯乙烯乳膠微球表面制成HLA-A2/LMP2A426~434-aAPCs。采用流式細胞儀表型分析。然后,通過HLA-A2/LMP2A426~434-aAPCs和HLA-A2陽性個體人外周血淋巴細胞(PBLs)混合培養(yǎng),LMP2A426~434特異性T細胞被誘導和擴增。用HLA-A2/LMP2A426~434四聚體染色法、對特異靶細胞加載抗原肽LMP2A426~434 T2細胞的細胞毒實驗和用抗HLA I類分子構(gòu)象抗體(W6/32)封閉靶細胞的細胞毒實驗來檢測這aAPCs誘導T細胞的特異性。 結(jié)果:流式細胞儀表型分析顯示HLA-A2/LMP2A426~434-aAPCs表面吸附有HLA-A2/LMP2A426~434復合物分子(85.48% vs 12.02%)和抗CD28抗體分子(72.38% vs 10.54% )。四聚體染色法和細胞毒實驗結(jié)果表明構(gòu)建的HLA-A2/LMP2A426~434-aAPCs能有效地誘導LMP2A426~434特異性T細胞的生成。 結(jié)論:成功地構(gòu)建了HLA-A2/LMP2A426~434-aAPCs,所摸索出的技術(shù)路線和實驗方法同樣適用于制備其他pMHC人工抗原呈遞細胞。從而為下面同種T細胞的研究奠定了基礎。 3.用重組的抗原肽/MHC-I復合物分子和aAPCs探索肽在T細胞同種識別機制中作用的研究 背景: MHC型別不同的供受者移植后會發(fā)生強烈的排斥反應(簡稱同種反應),其機制主要是同種反應性T細胞(簡稱同種T細胞)針對表達同種異體組織抗原(簡稱同種抗原)的細胞進行識別和應答。強烈的同種反應是免疫學中尚未完全闡明的現(xiàn)象,對同種抗原的本質(zhì)和同種識別機制一直存在不同的解釋。 目的:用重組的抗原肽/MHC-I復合物分子和人工抗原呈遞細胞探索同種CD8+ T細胞直接識別同種抗原表位的機制。 方法:用H-2Kd限制性的來自BALB/c鼠內(nèi)源性的五個自身肽段制備五種H-2Kd/自身肽四聚體來檢測被BALB/c鼠皮膚致敏的C57BL/6鼠體內(nèi)的CD8+同種T細胞的肽特異性的同種T細胞的頻率。另外, ELISPOT實驗和CFSE染色增殖實驗中,用表面包被有不同pMHC分子H-2Kd/肽復合物的微球作為人工抗原呈遞細胞特異刺激皮膚致敏的同種T細胞來研究CD8+同種T細胞的pMHC特異性。 結(jié)果:FACS表型分析證實人工抗原呈遞細胞H-2Kd/肽-aAPCs表面吸附有一定密度的H-2Kd/肽分子。四聚體染色結(jié)果表明五種H-2Kd/自身肽四聚體可以和五種同種反應肽T細胞特異結(jié)合,他們并不是重疊的,而是具有pMHC特異性的獨立的同種反應T細胞亞群。ELISPOT實驗和CFSE染色增殖實驗結(jié)果顯示CD8+同種T細胞反應頻率隨著aAPCs表面吸附的H-2Kd/肽表位種類的數(shù)量增高而增高。 結(jié)論:同種反應性T細胞特異識別由非自身MHC分子和其結(jié)合的肽所形成的特異表位。支持強烈的同種反應是由于眾多抗原肽/非自身MHC復合物特異刺激機體T細胞庫中數(shù)量巨大的同種反應性T細胞克隆的累積效應引起的假說。 4.利用由人工抗原呈遞細胞誘導腫瘤特異性同種T淋巴細胞治療腫瘤的研究背景:自身T細胞對自身MHC分子呈遞的腫瘤抗原肽的反應通常是微弱和無效應的。同種限制性T細胞庫的存在有可能產(chǎn)生針對非自身MHC分子結(jié)合的自身肽的T細胞,因為自身耐受是針對自身MHC分子呈遞的自身肽無反應。因此,同種T細胞為過繼腫瘤特異性T細胞治療腫瘤提供了很有發(fā)展?jié)摿Φ募毎麃碓础?目的:用人工抗原呈遞細胞H-2Kb-Ig/TRP2180-188-aAPCs從BALB/c鼠(H-2Kd)體內(nèi)誘導黑色素瘤特異性同種T細胞,并過繼治療C57BL/6鼠的黑色素瘤。 方法:制備表面包被有H-2Kb-Ig/TRP2180-188復合物分子和抗CD28抗體分子人工抗原呈遞細胞H-2Kb-Ig/TRP2180-188-aAPCs。用此aAPCs從BALB/c鼠(H-2Kd)體內(nèi)同種T細胞庫中誘導針對H-2Kb限制性來自酪氨酸酶相關(guān)蛋白2(TRP2)黑色素瘤特異性抗原表位TRP2180-188的同種T淋巴細胞。體外用二聚體染色實驗、ELISPOT實驗、細胞毒實驗來檢測誘導的同種T淋巴細胞的特異性和殺傷活性。同時體內(nèi)過繼誘導的同種T淋巴細胞治療C57BL/6鼠黑色素瘤。 結(jié)果:二聚體染色結(jié)果顯示誘導的同種T淋巴細胞能特異結(jié)合二聚體 H-2Kb-Ig/pTRP2而不結(jié)合對照肽二聚體。ELISPOT實驗結(jié)果表明誘導的同種T淋巴細胞對H-2Kb-Ig/pTRP2-aAPCs的抗原特異刺激分泌高的IFN-γ斑點數(shù)。細胞毒實驗結(jié)果說明誘導的同種T淋巴細胞能特異殺傷相應靶細胞。體內(nèi)過繼治療結(jié)果證實誘導的同種T淋巴細能有效地特異抑制黑色素瘤在C57BL/6鼠體內(nèi)的生長。 結(jié)論:黑色素瘤特異性H-2Kb限制性的同種T淋巴細胞能夠從BALB/c鼠體內(nèi)用H-2Kb-Ig/TRP2180-188-aAPCs誘導獲得,過繼治療黑色素瘤有一定療效。 本研究依據(jù)T淋巴細胞活化理論體外構(gòu)建人工抗原呈遞細胞HLA-A2/LMP2A426~434-aAPCs,用其成功制備出HLA-A2/LMP2A426~434特異性T細胞。所摸索出的技術(shù)路線和實驗方法,同樣適用于制備其他類別人工抗原呈遞細胞。構(gòu)建的人工抗原呈遞細胞具有表位易于控制的特點,是研究抗原特異性T細胞十分重要的工具。椐此制備各種抗原表位不同的H-2Kd/肽-aAPCs用于同種T細胞識別的研究,有力地證明同種T細胞是pMHC特異性的。根據(jù)同種T細胞具有pMHC特異性的特點,用H-2Kb-Ig/TRP2180-188-aAPCs從BALB/c鼠體內(nèi)成功制備出黑色素瘤特異性同種T細胞,同時過繼治療小鼠黑色素瘤,有一定效果。因此本研究為抗原特異性T細胞的研究奠定了基礎,為闡明同種抗原的本質(zhì)和同種識別機制以及利用同種抗原特異性T細胞治療腫瘤提供了實驗依據(jù)。
[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 .

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392

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本文編號：1888074