【作者】 朱参胜；

【导师】 金伯泉；

【作者基本信息】 第四军医大学 ， 免疫学， 2007， 博士

【摘要】 1992年,Wang及其同事发现并且克隆了一种新的细胞膜型分子,当时将其命名为Tactile(T cell activation increased late expression)。随后,在人类白细胞分化抗原协作组会议上将该分子命名为CD96。CD96分子表达于正常T细胞、T细胞克隆以及某些转化的T细胞系。外周血T细胞表达低水平的CD96分子,活化后其表达明显上调,并在刺激后第6~9天表达达到高峰值。在同种异体抗原刺激的条件下,NK细胞上CD96的表达也发生上调。CD96属于免疫球蛋白超家族,胞膜外区包含3个免疫球蛋白样结构域,共有15个N-连接糖基化位点,还有1个高度O-连接糖基化富含丝/苏/脯氨酸残基的茎状结构域,跨膜区有24个氨基酸残基,胞质区含44个氨基酸残基,并有一个富含碱性/脯氨酸的区域。自克隆CD96分子后的十多年间,由于不清楚其配体,该分子的研究无明显进展。直到2004年,Fuchs及其同事发现NK细胞可通过CD96识别PVR(CD155),促进NK细胞对表达CD155靶细胞的黏附,介导NK细胞对靶细胞表面上CD155的内化作用。由于PVR(CD155)高表达于某些肿瘤细胞,这一受体系统可能在NK细胞对肿瘤的识别和杀伤中起重要作用。然而,迄今为止对CD96表达和功能的认识还十分有限。在本课题中,我们通过对CD96分子的生物信息学分析,发现CD96的同源分子有CD166、CD113、CD226和CD86，预测CD96分子的配体结合位点为Y177、T186、T245、T246和V249，可结合配体上GLU-LYS-VAL保守序列。也发现CD96分子的胞质区有1个高度保守的ITIM模体，跨膜区有1个能结合跨膜型接头分子带正电的精氨酸残基。这些结果为进一步探讨CD96分子的功能及其信号转导途径提供了重要的启示。采用PHA刺激上调人T淋巴细胞系HSB-2细胞CD96分子的表达，用RT-PCR成功从该细胞中克隆出CD96分子胞膜外区第一免疫球蛋白V样结构域的cDNA基因，并将该cDNA插入pCDM18-T载体中，经过测序证实该序列正确。从pCD96-D1-T载体中通过双酶切得到CD96-D1 cDNA片段，将该cDNA插入到载体pCDM7中构建出pCDM7-CD96-D1真核表达载体。用脂质体将该载体的质粒转染CHO细胞，表达出CD96-D1-Fc融合蛋白。用抗-Fc mAb亲和层析柱对CD96-D1-Fc融合蛋白进行纯化。以纯化后的CD96-D1-Fc融合蛋白为免疫原制备了17株抗人CD96-D1 mAbs。用ELISA、FCM、Western Blot等方法对这些抗体进行了鉴定。按常规方法制备腹水，用饱和硫酸铵和阴离子交换层析法对抗CD96-D1 mAbs进行纯化。用过碘酸钠氧化法制备辣根过氧化物酶（HRP）标记的抗CD96-D1 mAbs，以建立测定可溶型CD96的夹心ELISA试剂盒。从抗CD96-D1 mAbs和HRP标记的CD96-D1 mAbs中筛选出最佳抗体配对，即包被抗体采用抗CD6-D1-No.5 mAb，检测抗体采用HPR标记的抗CD96-D1-No.13 mAb。对包被抗体和HPR酶标检测抗体的工作浓度进行优化，建立了测定可溶型CD96的夹心ELISA试剂盒。该试剂盒的灵敏度、精密度、准确度和稳定性均令人满意，为可溶型CD96的研究奠定了重要基础。我们对CD96在多种造血、非造血细胞系以及正常人PBmAb，检测抗体采用HPR标记的抗CD96-D1-No.13 mAb。对包被抗体和HPR酶标检测抗体的工作浓度进行优化，建立了测定可溶型CD96的夹心ELISA试剂盒。该试剂盒的灵敏度、精密度、准确度和稳定性均令人满意，为可溶型CD96的研究奠定了重要基础。我们对CD96在多种造血、非造血细胞系以及正常人PBMC中的表达情况进行了系统研究,发现CD96在造血和非造血细胞系中表达十分广泛。CD96也表达于正常人CD4+T细胞、CD8+T细胞、单核细胞和NK细胞。在PHA刺激后，CD4+T细胞、CD8+T细胞和NK细胞上CD96表达明显上调，证实CD96是一种活化表达增加的分子。但PHA对单核细胞CD96的表达上调作用不十分明显，提示不同细胞上CD96分子的表达调节具有不同特点。对CD96分子对细胞因子分泌的影响也进行了研究，发现用CD96-DI mAb交联正常人PBMC细胞上CD96受体，能促进PBMC细胞TNF和IL-10的分泌。单独CD96-D1 mAb的刺激并不能始动IL-2的分泌，但CD96-D1 mAb能促进同种异体抗原诱导的IL-2分泌。另外，CD96-D1 mAb交联CD96对IFN-γ和IL-4的分泌未发生明显影响。这些结果提示CD96分子在免疫调控、炎症反应、T细胞增殖和分化中可能发挥着重要作用。对CD96介导杀伤作用也进行了研究。在CD96-D1 mAb介导的重导向杀伤实验系统中，观察到CD96-D1 mAb能介导MLC中反应细胞对P815细胞的重导向杀伤。也发现CD96-D1 mAb能抑制MLC中反应细胞对K562细胞的杀伤，证实CD96为NK细胞的活化性受体，说明CD96胞膜外N-端第一免疫球蛋白V样结构域为参与CD96/CD155结合的主要结构域。CD96分子具有十分重要的生理功能，尤其是该分子参与NK细胞对肿瘤细胞的杀伤和黏附。本文研究了CD96结构、表达和功能，对弄清其信号转导通路和生理功能具有重要意义。更多还原

【Abstract】 In 1992, Wang and his colleagues identified and cloned cDNA of a novel cell-surface protein named T cell activation increased late expression (Tactile). Subsequently, this molecule was designated as CD96 at Human Leukocyte Differentiation Antigen (HLDA) workshop. CD96 is expressed on normal T cell lines and clones, and some transformed T cells. It is expressed at low levels on peripheral T cells and is strongly up-regulated after activation, peaking 6 to 9 days after stimulation. It is also up-regulated on NK cells activated in mixed lymphocyte culture. CD96 is a member of the immunoglobulin superfamily (IgSF) with three Ig domains highly N-glycosylated and a long serine/ threonine/proline-rich motif in extracellular region, a 24 residue transmembrane region, and a 44 residue cytoplasmic region. The CD96 molecule had not been paid much attention for over a decade due to the lacking knowledge of its ligand. Until 2004, Fuchs and his colleagues showed that CD96 on NK cells recognize poliovirus receptor (PVR/CD155) which promotes NK cell adhesion to target cells expressing PVR (CD155), stimulates cytotoxicity of activated NK cells, and mediates acquisition of PVR from target cells. As PVR is highly expressed in certain tumors, this receptor system may be critical for NK cell recognition of tumors. However, the knowledge about CD96 expressions and functions is still limited at the moment.In present research, we analyzed the sequences of CD96 molecule through bioinformatics methods. The results show that its homologous molecules include CD166, CD113, CD226, and CD86. The predicted binding sites for its ligand are Y177, T186, T245, T246, and V249, which could interact with consensus GLU-LYS-VAL region on its ligand of CD155. Moreover, we identified the putative ITIM motif on its cytoplasmic tail and a positive charged arginine residue on its transmembrane region, which may associate with transmembrane signal adaptors. These results can enlighten our further exploration for CD96 functions and its signal pathways.We employed PHA to stimulate CD96 expressions on HSB2 which is a human T cell line. The first extracellular Ig V-like domain (CD96-D1) cDNA was cloned successfully through RT-PCR from PHA stimulated HSB-2 cells and inserted into pCDM18-T vector. The sequence of CD96-D1 cDNA was ascertained by DNA sequencing. The confirmed cDNA of CD96-D1 was digested from pCD96-D1-T vectors by two restriction endonucleases and inserted into pCDM7 vectors to construct the eukaryotic expression vector pCDM7-CD96-D1. This vector was transfected into CHO cells through lipofection method, and fusion protein of CD96-D1-Fc was expressed and purified by an anti-Fc mAb conjugated affinity column. The fusion protein of CD96-D1-Fc was employed as immunogen to prepare mAbs specific for CD96-D1. 17 clones of hybridomas secreting monoclonal antibodies against CD96-D1 were prepared and the mAbs were characterized by ELISA, FCM, and Western Blot. Ascitic fluids were prepared according to our lab’s routine protocol and primarily purified by ammonium sulfate precipitation and further by anion exchange chromatography. The purified mAbs were labeled with HRP by periodinate sodium. Optimal paired mAbs were selected, i.e., CD96-D1-No.5 mAb was employed as coated mAb, and CD96-D1-No.13 mAb as detecting mAb after HRP labeling. Following optimization of the working concentrations of coated and detecting mAbs, we successfully established ELISA kit for detecting soluble CD96. The sensitivity, precision, and accuracy of the kit were determined and satisfied. The kit’s establishment makes a solid foundation for soluble CD96 research.The expression of CD96 was extensively investigated using fluorescent staining and flow cytometry analysis. The results show that CD96 molecule is expressed broadly in both haematopoietic and non-haematopoietic tissues. The expressions of CD96 on CD4+T cell, CD8+T cell and NK cell were up-regulated after PHA stimulation, confirming that CD96 is an activation increased molecule. Furthermore, we investigated the effects of CD96 on cytokine secretions in PBMC or mixed lymphocyte culture, we found that TNF and IL-10 secretions by PBMC from health doners were increased after CD96 receptors were cross-linked by CD96-D1 mAb, but not IL-2, IFN-γ, and IL-4 secretions. These results suggest that CD96 receptors on PBMC might play important roles in immune regulations, inflammatory responses, T cell expansions and differentiations, to which much attention should be paid. Finally, we determined the effects of CD96 on cytotoxicity. In the experiment of antibody-dependent redirected cytotoxicity, we found that CD96-D1 mAb could mediate cytotoxicity against target P815 cells by effecter cell generated from MLC. Moreover, CD96-D1 mAb could block the cytotoxicity of MLC-generated effecter cells against target K562 cells suggesting that domain 1 at its N-terminal region is involved in CD96/CD155 engagement. These results confirmed that CD96 is an activating receptor.CD96 plays important functions, especially NK cytotoxicity and adhesion with tumors. The structure, expressions, and functions of CD96 were explored through bioinformatics and experimental approaches in our research, which is of great significances for cell signaling study and the investigation of its physiological and pathological significances in vitro and vivo.更多还原