【作者】 陆盛军；

【导师】 吴雄文；

【作者基本信息】 华中科技大学 ， 免疫学， 2009， 博士

【摘要】 中国人群常见HLA-A2亚型间抗原特异性差异的实验研究研究生：陆盛军导师：吴雄文教授MHC分子(鼠为H-2，人为HLA)的功能是将抗原肽提呈给T细胞识别，诱导抗原特异性T细胞发生增殖活化，进而产生免疫效应。T细胞抗原受体(TCR)识别的配体是抗原肽／MHC复合物(pMHC)。不同型别MHC分子之间的抗原特异性差异已经广为人知，例如应用同种异体移植的体外模型—混合淋巴细胞培养，能够观察到不同型别MHC分子诱导淋巴细胞增殖。但是，人们尚未十分明确相同血清型的各亚型之间在抗原特异性方面是否存在差异，阐明这个问题有利于多肽疫苗设计和随机移植供者选择。HLA-A2是HLA-A座位上最常见的型别，在汉族人中HLA-A2阳性者占50％左右，其亚型在中国人群中分布频率较高的有HLA-A~*0201、HLA-A~*0203、HLA-A~*0206、HLA-A~*0207。虽然，这些亚型之间仅在抗原肽结合槽部位存在极少数几个氨基酸的差异，仍有研究提示，不同HLA-A2亚型之间由于抗原肽结合槽部位氨基酸的差异，使得它们在抗原提呈和T细胞识别方面有一定的差异。但是由于HLA具有高度的多态性和单倍型遗传的特点，很难找到仅HLA-A2亚型存在差异的样本，目前尚缺乏HLA-A2亚型间抗原特异性存在差异的实验证据。我们在前期工作中获得了由HLA-A~*0201胞外段和人IgGl的Fc段构成的融合蛋白(HLA-A~*0201／IgG二聚体)，借助该二聚体的Fc段与单核细胞表面Fc段受体(FcR)高亲和力结合，将此HLA-A~*0201二聚体分子结合于HLA-A2阴性(HLA-A2-ve)的单核细胞表面，后者作为刺激细胞与同一个体的淋巴细胞进行共培养，结果显示表面结合HLA-A~*0201／IgG二聚体的HLA-A2-ve单核细胞能够刺激自身淋巴细胞发生增殖，并产生相应的特异性CTLs。很显然，该共培养体系中淋巴细胞的增殖反映了单个表位的HLA-A2与非HLA-A2之间的抗原特异性差异，即上述共培养体系能用于单一pMHC分子的抗原特异性的实验观察。为了对HLA-A2常见亚型间的抗原特异性差异进行实验研究，我们对原有的共培养体系进行改进，改进之处在于选用HLA-A2阳性但非HLA-A~*0201型别的单核细胞和淋巴细胞，刺激细胞和效应细胞之间仅有HLA-A2的亚型不同，而不是HLA-A2与非HLA-A2的差别。将表面结合HLA-A~*0201／IgG二聚体的单核细胞作为刺激细胞，与自身淋巴细胞共培养，通过检测共培养体系中淋巴细胞的增殖强度，反映HLA-A*0201与特定HLA-A2亚型之间抗原特异性的差异。鉴于HLA-A2亚型之间抗原特异性的差异较小，为了控制个体间淋巴细胞增殖活性的差异对实验结果的影响，我们采用“增殖系数”来衡量HLA-A2亚型之间抗原特异性的差异，而非直接采用增殖指数或增殖细胞频率。具体做法是：试验个体(HLA-A2+ve)的单核细胞表面结合HLA-A~*0201／IgG二聚体后与自身淋巴细胞共培养后的增殖细胞频率为试验组增殖细胞频率，该试验个体(HLA-A2+ve)淋巴细胞与HLA-A2-ve的单核细胞混合培养的增殖细胞频率作为阳性参照，而该HLA-A2+ve个体的淋巴细胞与自身单核细胞共培养所测得的增殖细胞频率作为背景参照，采用公式((试验组增殖细胞频率-背景参照的增殖细胞频率)／(阳性参照的增殖细胞频率-背景参照的增殖细胞频率))对原始检测值进行校正，获得的校正值称为增殖系数。我们选择了不同HLA-A2亚型型别组(包括HLA-A~*0201型别组与其它HLA-A2常见亚型型别组)的外周血样本进行了上述实验，将自身肽Tyr368-376(NH2-Y MDGTMSQV-COOH)加载的HLA-A~*0201／IgG二聚体结合到单核细胞表面，与自身淋巴细胞共培养后检测淋巴细胞增殖系数。通过检测不同HLA-A2亚型型别组淋巴细胞对表面结合自身肽／HLA-A~*0201二聚体的自身单核细胞的增殖反应强度(用增殖系数表示)，观察到HLA-A~*0201型别组与HLA-A~*0203型别组的增殖系数之间有统计学差异，其增殖系数分别为0．012±0．109，0．124±0．067(P≤0．05)；HLA-A~*0201型别组与HLA-A~*0206型别组的增殖系数之间可观察到显著统计学差异，其增殖系数分别为0．012±0．109，0．225±0．184(P≤0．01)；HLA-A~*0201型别组与HLA-A~*0207型别组的增殖系数之间未观察到统计学差异，其增殖系数分别为0．012±0．109，0．050±0．109(P＞0．05)。我们的实验结果提示，HLA-A~*0201与HLA-A~*0206之间以及HLA-A~*0201与HLA-A~*0203之间存在抗原特异性差异，而HLA-A~*0201与HLA-A~*0207之间未观察到抗原特异性差异。本研究的意义：1．首次将单核细胞表面结合pMHC分子作为刺激细胞的培养体系用于亚型特异性差异的实验研究。本文研究的是HLA-A2主要亚型分子的抗原特异性差异，问题涉及的HLA分子范围虽局限，但是为研究其它MHC分子亚型间的抗原特异性差异提供了一个新的实验研究策略。2．用实验方法展示出HLA-A2某些亚型分子之间存在的抗原特异性差异。本文首次用实验方法显示出HLA-A~*0201与HLA-A~*0206之间以及HLA-A~*0201与HLA-A~*0203之间存在抗原特异性差异；但HLA-A~*0201与HLA-A~*0207之间未观察到抗原特异性差异。本研究的不足之处：本研究仅使用了HLA-A*0201这一个HLA-A2亚型型别的二聚体进行实验，所得到的实验结果只能显示HLA-A~*0201与其它非HLA-A*0201型别之间的差异，而非HLA-A~*0201型别的HLA-A2亚型相互之间的差异无法显示出来。今后研究设想：为进一步证实HLA-A~*0201分别与HLA-A~*0203和HLA-A~*0206之间的抗原特异性差异，我们认为有希望从以下几个方面改善和提高本论文：1．构建获得HLA-A~*0203、HLA-A~*0206分子与IgG1的CH1-Fc片段的融合蛋白，借助新构建和已构建的分子，利用共培养体系。2．尽量采用高亲和力自身肽，采取多种自身肽的混合表位以提高刺激强度的策略，使个体共培养体系类的增殖更明显。更多还原

【Abstract】 A novel co-culture system to reveal the heterogeneity ofantigen specificity among major HLA-A2 subtypes inChinese populationPh.D.candidate:Shengjun LuSupervisor:Prof.Xiongwen WuMajor histocompatibility complexes （MHC） is employed in presentingantigenic peptides to T lymphocytes and elicites immune responses by inducingproliferation and activation of antigen specific T lymphocytes.In this process,antigenic peptide/MHC compounds （pMHC） instead of MHC itself alone act as theligands of T cell receptors.Difference in the antigen specificity among serologicalallotypes of MHC （eg.HLA-A2 vs HLA-A24） is well known,which can be shownby lymphocyte proliferation in mixed lymphocyte culture （MLC）,an in vitro modelof allograft transplantation.Nevertheless,it is not addressed yet that whether there isalso difference in antigen specificity among subtypes of a serologically typed MHC（eg.HLA-A*0201 vs HLA-A*0206）.Understanding of this issue will contributemuch to vaccine design strategy and benefit donor selection in clinical unrelatedtransportation.HLA-A2 is one of the most prevalent serological HLA-A types in variousethnic populations.Individuals with HLA-A2 antigen count for some 50% inChinese people.The most frequent allele of HLA-A2 is HLA-A*0201.Otherprominent HLA-A2 subtypes in Chinese population include HLA-A*0203,HLA-A*0206,HLA-A*0207.These HLA-A2 subtypes are nearly identical in theirpeptide sequences,with only 1-3 amino acids substitutions among them. Nevertheless,some data implies that they functionally differ in the properties of theirantigen presentation process and in their specificity of CTL recognition.With theobstacle of the features of MHC （with multiple gene,high polymorphism andhaplotype inherence）,no proper experimental system has been developed yet tostudy the heterogeneity of major HLA-A2 subtypes in their antigen specificity.Experimental evidence is absent about whether they are identical or not in theirantigen specificity.The aim of this study is to explore an experimental system toreveal this issue.A divalent HLA-A*0201/IgG1-Fc molecule （HLA-A*0201 dimer） consisting ofthe extracellular domains of HLA-A*0201 and the Fc-CH1 segment of human IgG1gamma chain,is employed in this study.The peptide/HLA-A*0201 dimer was ableto be introduced as an allogeneic epitope onto HLA-A2-ve monocytes via interactionof the Fc and FcR.Autologous PBLs proliferation can be observed after co-culturedwith the dimer-loaded HLA-A2-ve monocyte.This data indicates that the co-culturesystem can be used to show the antigen specificity of a single pMHC epitope.A modified MLC,lymphocytes-autologous monocytes co-culture （LAMC） isdeveloped to show the heterogeneity of major HLA-A2 subtypes in their antigenspecificity.A few improvements are made in LAMC.Firstly,an additionalHLA-A*0201 molecule is attached to the stimulating cell of other HLA-A2 subtypethan HLA-A*0201.Secondly,the effector cells are autologous lymphocytes insteadof allogeneic ones used in conventional MLC.Appearantly,the lymphocyteproliferation in the LAMC indicates the heterogeneity of HLA-A*0201 and thegiven HLA-A2 subtype in their antigen specificity.Counts of proliferating cells in the LAMC is applied but not directely used inshowing the antigen specificity heterogeneity,for a single pMHC epitope ownslimited ability to promote lymphocyte proliferation,which may be submerged by thevariation of lymphocyte proliferation among individual samples.To avoid this problem,we introduce the proliferation efficiency （Pe） to redress the absolute countsof proliferating cells,the Pe is figured out with a formula listed below.In which,‘Tf’means the proliferating PBLs counts in the LAMC with the peptide/HLA-A*0201dimer loaded autologous monocytes （HLA-A2+ve）;‘Bf’is the backgroundproliferating frequency in the LAMC of the native HLA-A2+ve monocyte andautologous PBLs;‘Pf’is the positive proliferating frequency of the HLA-A2+vePBLs in MLC HLA-A2-ve monocytes instead of autologous monocytes.Pe=（Tf-Bf）/（Pf-Bf）With a self-peptide Tyr368-376 （NH₂-YMDGTMSQV-COOH） fromintracellular tyrosinase,the LAMC shows the heterogeneity of major HLA-A2subtypes in their antigen specificity.The Pe is observed significantly differentbetween HLA-A*0201 and HLA-A*0203 （with Pe values of 0.012±0.109 vs 0.124±0.067,P≤0.05）,and between HLA-A*0201 and HLA-A*0206 （with Pe values of0.012±0.109 vs 0.225±0.184,P≤0.01）;no statistical Pe difference betweenHLA-A*0201 and HLA-A*0207 is observed （with Pe values of 0.012±0.109 vs0.050±0.109,P＞0.05）.Our observations suggest antigen specificity ofHLA-A*0201 and HLA-A*0203,and that of HLA-A*0201 and HLA-A*0206 aredifferent,antigen specificity of HLA-A*0201 and HLA-A*0207 is not observeddifferent.更多还原