【作者】 徐竹蔚；

【导师】 金伯泉； 张赟；

【作者基本信息】 第四军医大学 ， 免疫学， 2009， 博士

【摘要】 免疫球蛋白超家族(immunoglobulin superfamily,IgSF)成员CD226分子是一种广泛表达于多种免疫细胞膜表面的Ⅰ型跨膜糖蛋白,在2000年第七届国际人类白细胞分化抗原协作组大会上获得新的CD命名。此前,该分子被称为“T细胞谱系特异性活化抗原1(T lineage- specific activation antigen 1,TLiSA1)”、“血小板与T细胞活化抗原1(platelet and T cell activation antigen 1,PTA1)”和“DNAX辅助分子1(DNAX accessory molecule-1,DNAM-1)”。人CD226基因于1996年克隆成功,位于染色体18q22.3,其cDNA全长2664bp,开放读框编码336个氨基酸,其基因存在单核苷酸多态性(single nucleotide polymorphism,SNP)。小鼠CD226基因由我室于2002年成功克隆,位于染色体18 E4,除了cDNA全长为2487bp的原型分子以外,还存在三种异型。完整的CD226分子由胞膜外区、跨膜区和胞浆区组成,分子量为65~67 kDa,胞膜外区有2个IgSF V样结构域和潜在的糖基化位点,胞浆区有磷酸化及与信号分子相互作用的位点。不同种属的CD226分子在核苷酸水平和氨基酸水平均高度同源,蛋白结构也十分相似,是一个在进化上是一个相对保守的分子,提示该分子在机体免疫应答过程和其他生物学功能中可能扮演了极为重要的角色。此外,CD226分子与CD96以及其他Nectin和Nectin样分子(Nectin-like molecule,Necl)有较高的同源性。CD226广泛表达于T细胞、NK细胞、NK T细胞、活化的血管内皮细胞、巨核/血小板谱系、单核细胞、胸腺细胞、某些B细胞亚群、部分造血干细胞和肥大细胞等免疫细胞表面,在免疫系统以外则分布于海马和小脑皮质的神经突触部位。2003年,人CD226的两种配体鉴定成功,分别是CD112/Nectin-2和CD155/Necl-5/PVR(脊髓灰质炎病毒受体)。CD226与其配体的相互作用参与CTL和NK细胞的活化、分化和细胞毒作用,调节CD4+ T细胞的增殖和分化,介导内皮细胞与其他细胞的黏附,促进血小板的活化与聚集,参与造血调控,介导肥大细胞的脱颗粒作用,参与NK T细胞和胸腺细胞的抗凋亡作用、树突状细胞的成熟、免疫突触和神经突触的形成,并与肿瘤、自身免疫性疾病、移植排斥及病毒感染等疾病密切相关。人CD226除以膜型(mCD226)的形式表达在细胞膜表面外,还以可溶型(sCD226)的形式存在于血清和细胞培养上清中。前期小规模临床标本检测发现,在肿瘤和多种自身免疫性疾病患者的血清中,sCD226的水平均显著高于正常人,但是目前关于CD226与临床疾病的研究仅限于膜型和/或可溶型CD226的表达及其基因SNP与疾病的关系,而CD226在肿瘤及自身免疫性疾病中的作用和可能的分子机制尚不清楚。本研究首先制备和鉴定了一批抗人IgG Fc段、谷胱甘肽-S-转移酶(glutathione-S-transferase,GST)、麦芽糖结合蛋白(maltose-binding protein,MBP)和硫氧化还原蛋白(thioredoxin,Trx)等常用标签蛋白(tag)的单克隆抗体(monoclonal antibody,mAb),在此基础上建立或改良了定量检测GST、Fc、MBP和Trx或其融合蛋白的双抗体夹心ELISA试剂盒,敏感性分别达到了1.2、15.6、1和0.4 ng/ml。应用Fc ELISA试剂盒检测上清中hCD226-Fc水平的方法,克隆化了高水平分泌该融合蛋白的CHO-hCD226-Fc细胞株,大量制备细胞培养上清,并用抗Fc mAb标记的Sepharose 4B亲和层析柱纯化了hCD226-Fc融合蛋白,用SDS-PAGE、Western blot和流式细胞术(flow cytometry,FCM)鉴定其纯度和生物学活性,以此作为人sCD226 ELISA试剂盒的标准品以及体外杀伤实验中模拟天然sCD226作用的试剂。另外,还对我室前期研制的定量检测人sCD226的双抗体夹心ELISA试剂盒进行了改良。应用条件优化后的ELISA试剂盒检测了259例肿瘤患者(包括消化、呼吸、血液、妇科、骨骼、神经系统等多种来源的肿瘤)及129例正常人血清中sCD226的水平,发现肿瘤患者血清中sCD226的水平(0.2-60 ng/ml,中位数11.5 ng/ml)明显高于正常人(0.1-23 ng/ml,中位数6.3 ng/ml,P<0.001);而在用FCM检测外周血单个核细胞(peripheral blood monouclear cell,PBMC)上mCD226的表达时,发现肿瘤患者PBMC上mCD226的表达(14例,1.1%-56.1%,中位数17.45%)明显低于正常人(12例,43.1%-77.8%,中位数65.3%,P<0.001)。在体外NK细胞杀伤肿瘤细胞的模型中用重组表达的可溶型hCD226-Fc融合蛋白模拟天然sCD226的作用,发现hCD226-Fc能显著抑制NK细胞对其靶细胞K562细胞的杀伤,浓度为1μg/ml时抑制率即可达到34%,而且这种抑制作用呈剂量依赖性。在佛波酯(phorbol 12-myristate 13-acetate,PMA)活化的正常人PBMC和Jurkat细胞上,3种蛋白酶抑制剂(金属蛋白酶抑制剂1-10-phenanathroline、丝氨酸蛋白酶抑制剂AEBSF和半胱氨酸蛋白酶抑制剂NEM)可以在上调mCD226的表达的同时下调培养上清中sCD226的水平,由于多种蛋白酶在许多肿瘤中含量可能有所增加,提示蛋白酶解作用可能是肿瘤患者血清中sCD226的形成机制。用免疫沉淀和Western blot的方法鉴定了血清和细胞培养上清中sCD226的分子量,发现在肿瘤患者和正常人血清以及PMA刺激的PBMC和Jurkat细胞培养上清中,均存在~50 kDa的特异性条带,这与CD226分子的胞膜外区分子量相符。另外,血清样本中还存在一个~45 kDa的条带,这一条带在PMA刺激的细胞培养上清中不存在,提示不同蛋白酶对CD226的酶解作用可能发生在该分子胞膜外区的不同位置。在搞清了肿瘤患者sCD226的表达、作用和来源的基础上,我们提出了如下假说:肿瘤患者中升高的蛋白酶可能通过酶解作用导致了PBMC上mCD226表达下调而血清中sCD226的水平上调,高水平的sCD226与其膜型配体CD112/CD155分子相结合,减弱了杀伤细胞上mCD226分子与肿瘤细胞表面膜型CD112/CD155的结合,从而抑制了杀伤细胞活化信号的产生,并最终导致肿瘤细胞发生免疫逃逸。本研究还用ELISA和胞内细胞因子染色的方法观察了抗人CD226的mAb LeoA1对混合淋巴细胞培养(mixed lymphocyte culture,MLC)和PBMC中细胞因子分泌格局及细胞亚群百分比的调控,发现被上调的有IL-10、GM-CSF和G-CSF等,被下调的有IL-2、IFN-γ、TNF-α、IL-12 p40、IL-15、IL-23和TGF-α等,相关的细胞亚群包括Th1、Th2、CTL、NK以及单核/巨噬和DC。采用信号分子阻断剂的手段,分析了CD226调控MLC细胞因子分泌格局的信号通路中可能涉及的信号分子主要有p38、JNK和PI3K。最后,成功建立了小鼠实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)模型,证实了抗CD226抗体对EAE的发生有明显的防治作用,其分子机制涉及中枢神经系统局部炎细胞浸润的减轻,脾脏CD25阳性的活化细胞数量的减少,以及脾脏IL-10+细胞和调节性T细胞百分率的增加。简言之,本研究的实验结果为阐明CD226分子在肿瘤和自身免疫性疾病中的作用提供了实验依据,并为深入研究CD226参与机体多种生理功能和病理过程的分子机制奠定了基础。更多还原

【Abstract】 CD226 molecule, a member of the immunoglobulin superfamily (IgSF), is a typeⅠtransmembrane glycoprotein which expressed widely by various immunocytes. Although it had several names before including“T lineage- specific activation antigen 1 (TLiSA1)”,“platelet and T cell activation antigen 1 (PTA1)”, and“DNAX accessory molecule-1 (DNAM-1)”, it was designated as CD226 in the 7th Workshop and Conference on Human Leukocyte Differentiation Antigen in 2000.Human CD226 gene, located on chromatosome 18q22.3, was successfully cloned in 1996. The full length of human CD226 cDNA is 2664bp, with an open reading frame encoding 336 amino acids. Single nucleotide polymorphisms (SNP) have been reported in human CD226. Murine CD226 gene, located on chromatosome 18 E4, was cloned in our lab in 2002. Three isoforms of murine CD226 were found in addition to its prototype, the full length of which is 2487bp. The intact CD226 is a 65~67 kDa molecule, consisting of extracellular region, transmembrane region and cytoplasmic region. It contains 2 IgSF V like domains and several potential glycosylation sites in its extracellular region and some phosphorylation sites in its cytoplasmic region. CD226 molecule is highly conserved between different species, with similar nucleotide sequences and protein structures, indicating its important roles in immune responses and other biological functions. In addition, CD226 shares high homology with CD96 and other molecules belonging to Nectin or Nectin-like molecule (Necl) family.CD226 is expressed on the surface of a variety of cells, including T cells, NK cells, NK T cells, activated vascular endothelial cells, megakaryocyte/platel- et lineage, monocytes, thymocytes, a subset of B cells, some haemopoietic stem cells and mast cells. It is also localized in murine hippocampus and cerebellum during adulthood and postnatal development. The ligands for human CD226 were identified to be CD112/Nectin-2 and CD155/Necl-5/poliovirus receptor in 2003. The recognition and interaction between CD226 and CD112/ CD155 is involved in a lot of important biological functions or pathological processes, such as: activation, differentiation, and cytotoxicity of CTL and NK cells; proliferation and polarization of CD4+ T cells; endothelial transmigration of leukocytes; activation and aggregation of platelets; regulation of haematogenesis; degranulation of mast cells; antagonism of apoptosis of NK T cells as well as thymocytes; maturation of dendritic cells; formation of immune synapse and neuro-synapse; and some pathological processes including tumor, autoimmune diseases, transplantation rejection, and virus infection diseases.Human CD226 (hCD226) molecule exists in two forms, one is membrane binding form (membrane CD226, mCD226) on cell membrane, and the other is soluble form (soluble CD226, sCD226) in serum or cell culture supernatant. Previous study on a small scale of samples demonstrated that sCD226 was significantly higher in sera from patients of tumor and autoimmune diseases than from healthy donors. However, the only available studies between CD226 and clinical diseases are focused on the expression of mCD226 and/or sCD226 or individual SNP in certain disease whereas its role and possible mechanism involved in tumor and autoimmune disease remains unclear.In this study, monoclonal antibodies (mAbs) against four frequently used tags including human IgG Fc fragment, glutathione-S-transferase (GST), maltose-binding protein (MBP) and thioredoxin (Trx) were prepared and characterized. Base on that, Sandwich ELISA systems for quantitative detection of Fc, GST, MBP, Trx or corresponding tag fusion proteins were established or improved, with the detection limits being 1.2, 15.6, 1, and 0.4 ng/ml, respectively. Using the improved Fc ELISA, hCD226-Fc level in cell culture supernatant was detected and CHO-hCD226-Fc cell line highly secreting hCD226-Fc fusion protein was re-cloned. Purified hCD226-Fc fusion protein was prepared by collection of culture supernatant from this cell line, and purified with anti-Fc mAb labeld Sepharose 4B affinity column. The purity and biological activity of the obtained hCD226-Fc fusion protein was identified by SDS-PAGE, Western blot, and flow cytometry (FCM), respectively. After characterization, the fusion protein was used to be standard in sCD226 ELISA kit or to mimic natural sCD226 in cytotoxicity assay in vitro. In addition, Sandwich ELISA for quantitative detection of sCD226 was also improved.The improved sCD226 ELISA kit was used to detect sera sCD226 levels in 259 cancer patients (including tumors from various origin such as digestive system, respiratory system, hematological system, gynecological system, skeletal system, and nervous system) and 129 normal subjects. It was found that the concentration of sCD226 was significantly higher in serum from cancer patients (0.2-60 ng/ml, median 11.5 ng/ml) than in serum from normal subjects (0.1-23 ng/ml, median 6.3 ng/ml, P<0.001). Meanwhile, FCM analysis demonstrated that mCD226 expression on peripheral blood mononuclear cells (PBMC) from cancer patients (14 cases, 1.1%-56.1%, median 17.45%) was significantly lower than that from healthy controls (12 cases, 43.1%-77.8%, median 65.3%, P<0.001). In vitro, hCD226-Fc fusion protein could significantly inhibit the cytotoxicity of NK cells against its target cells K562 in a dose-dependent manner and the inhibitory rate was 34% when using 1μg/ml of hCD226-Fc in this assay. Three protease inhibitors (1-10-phenanathroline: a metalloprotease inhibitor; AEBSF: a serine protease inhibitor; NEM: a cysteine protease inhibitor) could significantly decrease sCD226 level while increase mCD226 expression of the cultured PBMC or Jurkat cells treated with PMA (phorbol 12-myristate 13-acetate), indicating that the increased serum sCD226 in cancer patients might be shed from the cell surface by certain protease(s), which are often up-regulated when tumor occurs. Furthermore, molecular weight of natural sCD226 from sera samples of tumor patients or healthy controls as well as cell culture supernatants of PMA stimulated PBMC or Jurkat cells was identified by immunoprecipitation and Western blot. A band with molecular weight of ~50 kDa, coincident with the extracellular domain of CD226 molecule, was found in all the four kinds of samples. Interestingly, another weaker band, with molecular massed of ~45 kDa, was observed in sera samples, but not in supernatant samples, indicating that the variety of proteases in vivo could cause the cleavage of sCD226 from different sites of intact CD226.After investigation of the expression, function and origin of sCD226 in tumor, we speculate the following hypothesis: the up-regulated proteases in tumor could increase sCD226 level in serum and decrease mCD226 expression on PBMC by proteolysis; the increased sCD226 shed from cell surface and released into the circulation could bind membrane CD112/CD155 on tumor cells but could not transduce activating signal, which could reduce the binding between mCD226 and its membrane ligands and inhibit the cytotoxicity against tumor cells. We think this might be one of the immune escape strategies used by tumor cells.Besides, ELISA and intracellular cytokine staining was used to study the effect of anti-hCD226 mAb LeoA1 on cytokine secretion profile and cell populations/subpopulations during mixed lymphocyte culture (MLC). LeoA1 could up-regulate IL-10, GM-CSF, and G-CSF level in MLC supernatant while down-regulate IL-2, IFN-γ, TNF-α, IL-12 p40, IL-15, IL-23, and TGF-αsecretion, and the involved cell populations included Th1, Th2, CTL, NK, and monocytes/macrophages/DCs. Using inhibitors of signaling molecules, we found the regulation of LeoA1 in MLC cytokine secretion and cell population might involve p38, JNK and PI3K.Finally, experimental autoimmune encephalomyelitis (EAE) model was successfully established and we confirmed that anti-CD226 antiody treatment could delay the onset and reduce the severity of disease. The mechanism for this effect of CD226 blockade on EAE could be the reduction of inflammatory cell infiltration in central nervous system, decreased CD25+ activated cells and increased IL-10+ cells and Treg cells in spleen.In conclusion, these results provide experimental data for fully understanding the role of CD226 in tumor and autoimmune diseases, and lay foundation for further exploration of its molecule mechanism involved in multiple biological functions as well as pathological processes.更多还原