【作者】 陈礼文；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2011， 博士

【摘要】 人CD83分子是I型免疫球蛋白超家族(IgSF)糖蛋白,由205个氨基酸组成,分子量约45kD,包括胞外段、跨膜段和胞内段三部分,其中胞外段128个氨基酸,含一个V型Ig样功能区并被高度的糖基化;胞内段仅有39个氨基酸,且不含酪氨酸,没有相应免疫受体酪氨酸激活/抑制基序(ITAM/ITIM)。小鼠CD83全长196个氨基酸(胞外段133个),与人有63%同源性。CD83受体尚未鉴定,但可在单核细胞以及树突状细胞(DC)上检测到该潜在的未知受体表达。CD83编码基因位于6号染色体短臂p23带。由5个外显子组成,第1个外显子编码信号肽,第2和3个编码胞外段(其中外显子3主要编码V型Ig样结构域),外显子4和5分别编码跨膜区和胞内段。序列比对表明CD83胞外段含5个半胱氨酸残基,其中第1~4个形成分子内二硫键,与蛋白构象形成有关。靠近跨膜区的第5个半胱氨酸比较保守,可通过分子间二硫键形成CD83同源二聚体。CD83分子伴随MHC II类分子诱导表达在成熟DC表面,因此曾被认为是DC成熟的标志分子之一,但后续研究发现该分子亦可表达在活化的T与B细胞、胸腺基质细胞(TEC)以及一些肿瘤细胞表面。在中枢免疫器官胸腺中,CD83对CD4+ T细胞发育具有关键的调节作用,而在外周免疫系统中,表达在细胞表面的膜型CD83分子(membrane CD83,mCD83)的功能尚待阐明。但是非常有意义的是,大量体外实验证实从细胞表面脱落或体外重组等以可溶性形式存在CD83(solubleCD83,sCD83)对DC介导的T细胞增殖具有明显的免疫抑制作用,继而的动物体内实验也发现重组CD83蛋白对小鼠实验性自身免疫性脑脊髓炎(EAE)以及同种异型皮肤移植急性排斥反应具有很好的预防或治疗作用。因此,sCD83发挥效应的作用模式与分子机制引起高度关注。鉴此,在本研究中,我们首先制备了人CD83-Ig重组蛋白以及鼠抗人CD83单克隆抗体,并利用重组蛋白分析了sCD83分子对T细胞的体外作用。结果表明CD83-Ig以剂量依赖方式有效抑制激发型CD3单抗刺激的外周血单个核细胞(PBMC)增殖以及IL-2与IFN-γ分泌,对纯化外周血T细胞则几乎没有作用,中文摘要人CD83分子单克隆抗体与重组蛋白制备及其抑制T细胞功能的机制研究但CD83-Ig刺激的单核细胞培养上清具有对T细胞的有效抑制效应。通过进一步对上清中的细胞因子分析和特异性抑制剂阻断实验证实,CD83-Ig与单核细胞上的未知受体结合后可介导PGE2上调表达从而发挥对T细胞的抑制效应。因此,本研究揭示了CD83分子通过上调PGE2抑制T细胞效应功能的免疫调控新机制。本论文共分为两个部分。1.人CD83-Ig融合蛋白及鼠抗人CD83单克隆抗体的制备与生物特性鉴定采用重叠PCR(overlap extension PCR)技术构建CD83-Ig融合蛋白,包含人CD83胞外段(1~128位氨基酸)和人IgG1的Fc段。在Fc段铰链区编码序列第326位和335位有2个位点诱变(site-directed mutagenesis),使原本编码的半胱氨酸突变为丝氨酸从而阻止在铰链区形成二硫键,保持重组蛋白为单链状态。重组pIRES2-EGFP-CD83-Ig载体转染CHO细胞株后筛选获得阳性克隆1C4。无血清培养基大量培养1C4后收集上清,经Protein G亲和层析纯化获得重组蛋白。经SDS-PAGE电泳和考马斯亮蓝染色鉴定CD83-Ig分子量为54kD左右,点杂交(Dot-blot)证实重组蛋白可被特异性CD83单抗识别,而且,CD83-Ig与CD83单抗预先孵育可以剂量依赖方式阻断后者与CD83转基因细胞结合,提示该重组蛋白具有良好的生物学特性,可用于后续的功能实验。以高表达膜型CD83分子的转基因细胞株L929/CD83为免疫原,采用经典杂交瘤技术,经反复筛选及克隆化培养,获得一株稳定分泌特异性鼠抗人CD83单抗的杂交瘤细胞株,命名为1E11。快速定性试纸法分析1E11重链为IgG1,轻链为κ链。杂交瘤经体外连续传代培养,液氮冻存,复苏后生长良好,抗体分泌性能稳定。染色体数目分析显示,杂交瘤细胞株的染色体在100条以上。采用本室建立的腹水诱生法制备单抗,腹水形成阳性率约为80%以上,腹水的收获量平均为3 mL/只。经Protein G亲和层析柱分离纯化,腹水中抗体蛋白的得率为1.5~2.0 mg/mL。纯化抗体经Dot-blot鉴定表明可与重组CD83-Ig特异性结合并与商品化CD83单抗HB15e竞争结合同一表位。由此表明,1E11为特异性抗人CD83的单克隆抗体。2. CD83-Ig刺激单核细胞分泌PGE2介导对T细胞的抑制效应(1)体外共培养实验显示,重组CD83-Ig蛋白以剂量依赖方式抑制激发型CD3单抗诱导的PBMC增殖和IL-2与INF-γ分泌:0.5,1,2,5和10μg/mL重组CD83蛋白对PBMC增殖的抑制率分别达到84.7%,72.6%,53.7%,47.8%和43.3%;10μg/mL重组蛋白导致其IL-2分泌减少约4~5倍而IFN-γ分泌减少了60%~70%。与此形成鲜明对比的是,CD83-Ig对激发型CD3和CD28单抗联合刺激的纯化T细胞的增殖并无明显的抑制效应。(2)利用重组蛋白对未知的CD83受体表达分析表明,CD83-Ig主要结合至CD14+单核细胞却几乎不与新鲜乃至经激发型CD3和CD28刺激1到3天的T细胞结合。进一步的实验显示,可溶性CD83-Ig刺激单核细胞上清经离心去除细胞成分后用来培养激发型CD3与CD28单抗联合刺激的T细胞时,可观察到约50%的T细胞增殖抑制以及大约75%和45%的IL-2和IFN-γ分泌抑制。(3)对重组蛋白刺激的单核细胞上清分析表明在目前已知的与单核细胞有关的抑制性因子中,PGE2可被CD83-Ig介导上调表达。western blotting分析表明单核细胞内特异性催化PGE2合成的COX-2亦可被CD83-Ig介导上调表达并与PGE2分泌呈基本一致的时间动力学特点。最后,COX-2选择性抑制剂NS-398几乎完全逆转CD83-Ig介导的对PBMC的抑制效应以及CD83-Ig刺激的单核细胞上清对T细胞的抑制功能。综上所述,本研究成功地构建了重组pIRES2-EGFP-CD83-Ig载体,筛选了稳定表达人CD83-Ig蛋白CHO细胞株并制备了鼠抗人CD83单抗1E11,这为进一步探讨CD83分子表达与功能奠定了厚实的物质基础。继而的研究发现重组CD83-Ig通过刺激单核细胞分泌PGE2发挥对T细胞的免疫抑制效应。在此模式中,sCD83作为负向调控效应T细胞功能分子,在抗原递呈细胞启动的特异性免疫应答中发挥负性调控作用。本研究将为更好地理解CD83分子功能及将可溶性CD83作为生物因子用于免疫干预提供有价值的理论依据。更多还原

【Abstract】 As a type I immunoglobulin superfamily glycoprotein, human CD83 contains 205 amino acids, with a molecular weight of ~45kD. The extracellular compartment of CD83 is composed of 128 amino acids which are highly glycosylated and possess a V-type Ig-like domain. The 39 amino acids of its cytoplasmic tail contains no tyrosine, thus possibly no direct signal is transduced by the short compartment as the lack of immunoreceptor tyrosine-based inhibition or activation motifs (ITIM or ITAM). The murine CD83 is a 196 amino acid protein which shares 63% amino acid sequence homology with that of human. The counter receptor for CD83 has not yet been identified although experiments using recombinant CD83 protein had demonstrated its presentation on monocytes as well as immature and mature DCs.The protein-coding gene for CD83 is located in 6p23 and including five exons. Exon 1 is coding for a signaling peptide, exon 2 and exon 3 are coding for the extracellular domain of CD83 (among which exon 3 is responsible for the largest part of the V-type Ig-like domain). Exon 4 and exon 5 encode the transmembrane region and the intracellular domain of CD83, respectively. Sequence alignment showed that CD83 possess five cysteine residues in its extracellular domain, including a conserved cysteine residue near the transmembrane region that can form dimmer by intermolecular disulfide bond. The other four cysteines form intramolecular disulfide bonds which are involved in protein conformation.CD83 is predominantly expressed on mature DC as well as activated T and B lymphocytes and has been a marker for the former. In thymus, CD83 expressed on thymic stromal cells is essential for the conventional development of double-positive thymocytes to single CD4-positive T cells. However, the function of membrane CD83 (mCD83) in the peripherial immune system remains elusive. In contrast, a great deal of in vitro experiments demonstrated that soluble CD83 (sCD83), either dropped from CD83-positive cells or artificial recombination, exerted significant inhibitory effects on DCs-stimulated T cells. Furthermore, recombinant CD83-Ig or CD83ext prevented the paralysis associated with experimental autoimmune encephalomyelitis (EAE) as well as delayed acute cellular rejection of MHC-mismatched mouse skin allografts. However, the underlying mechanisms for the inhibitory roles of sCD83 remain to be an engima.Here we firstly prepared human CD83-Ig fusion protein as well as mouse monoclonal antibody against human CD83 and employed them as effective tools to observe the role of CD83 on CD3+ T cells and explored the underlying mechanism. We demonstrated that CD83-Ig inhibits proliferation and production of IL-2 and IFN-γby T cells, and the inhibitory effect of CD83 is mediated by monocytes. PGE2, but not IL-10 or TGF-β, was specifically upregulated by CD83-Ig in monocytes. Consistent with higher levels of PGE2, COX-2 expression was also increased upon CD83-Ig treatment. Finally, application of COX-2 selective inhibitor NS-398 fully prevented CD83-Ig-triggered inhibition of T cell responses. Our study establishes a new immune regulatory mechanism by CD83 via stimulation of PGE2 production in monocytes. The dissertation is composed of two parts.1. Preparation and identification of human CD83-Ig fusion protein and mouse monoclonal antibody against human CD83By using overlap extension PCR techniques, we prepared CD83-Ig fusion protein which is composed of extracellular domain of human CD83 cDNA (amino acids 1-128) and the Fcγportion of human IgG1 (hinge, CH2, and CH3). The recombinant protein is a single strand as the Fc part has two site-directed G→C mutations at sites 326 and 335, which mutate cysteine to serine and thus interfere with the interchain dilsulfide bond linkage within hinge region. Using a protein G column, soluble CD83-Ig was purified from newborn calf serum free media conditioned with transfectant CHO cells. Coomassie staining showed the size of fused protein was ~54KD and it can be recognized by specific anti-CD83 mAb in Dot-blot assay. Furthermore, competition analysis showed that preincubation of CD83-Ig with PE-conjugated HB15e abrogated in a dose dependent way the binding of the latter to L929/CD83 transfectants. So, it is likely that fused CD83-Ig protein is considered bioactive and thus relevant functional studies can be performed based on this protein. After pretreatment with mitomycin C, L929/CD83, a transgenic cell line highly expressing CD83 molecule, was used to immunize BALB/c mice. Spleencytes from the immunized mice were fused with mouse myeloma cells SP2/0 by using the classical hybridoma technique and L929/CD83 transfectants were employed to screen anti-CD83 mAb secreting hybridomas. Through repeated sub-cloning and screening, one hybridoma cell line (1E11) against CD83 was eventually obtained. The hybridoma grew well after long-term culturing and storage in liquid nitrogen. Fast-strip analysis showed that subclass of 1E11 is IgG1 and the light chain belongs toκ. After primed with pristine, ascite was induced in BALB/c mice by intraperitoneal injection of well-grown 1E11 hybridoma and the output is average to 3 mL each mouse. Affinity chromatography was used to purify mAb, and the concentration of protein is 1.5~2.0 mg/mL. Indirect immunofluorescence assay suggested the engagement of purified CD83 mAb to cells was 0.2-1μg/1×106 cells. Dot-blot showed that 1E11 spcifically bind to recombinant CD83-Ig the same way as commercially available anti-CD83 mAb HB15e. Competition experiment indicated that 1E11 recognized the same antigen epitope with that of commercially available HB15e. Thus 1E11 is a specific monoclonal antibody against human CD83 molecule.2. CD83-Ig stimulated monocytes suppress T cell function via production of PGE2CD83-Ig inhibited anti-CD3 mAb triggered proliferation as well as IL-2 and INF-γproduction of PBMCs in a dose dependent manner. The inhibition of proliferation at 0.5, 1, 2, 5, and 10μg/mL recombinant protein was 84.7%, 72.6%, 53.7%, 47.8%, and 43.3% respectively. Accordingly, PBMCs stimulated with 10μg/mL CD83-Ig produced 4~5 fold lower IL-2 and about 60%~70% reduction of IFN-γsecretion as compared with anti-CD3 stimulation alone. However, CD83-Ig had any significant effect on the proliferation of purified T cells stimulated with agonistic anti-CD3 and anti-CD28 mAb. Being as a binding tool, CD83-Ig was found to bind to ~65% of fresh CD14+ monocytes whereas almost no binding of fusion protein was observed in freshly isolated CD3+ cells. Upon being stimulated with anti-CD3 and anti-CD28 mAb for 3 days, the expression of counter receptor on T cells increased slightly to about 2%. Next, cell-free supernatants collected from CD83-Ig-stimulated 24h monocytes cultures resulted in 51% and 48% proliferation suppression of T cells stimulated with anti-CD3 and anti-CD28 mAbs. Likewise, secretion of IL-2 and IFN-γwas inhibited by ~78% and ~45%, respectively. Further analysis of the supernatants showed that PGE2, but not IL-10 or TGF-β, was specifically upregulated by CD83-Ig in monocytes. Consistent with higher levels of PGE2, COX-2 expression was also increased upon CD83-Ig treatment. Finally, application of COX-2 selective inhibitor NS-398 fully prevented CD83-Ig-triggered inhibition of PBMCs as well as of T cells cultured with monocyte supernatants.Taken together, the successful cloning of CD83-Ig-coding gene, construction of recombinant vector, preparation of cell lines stablly express fusion protein as well as production of anti-CD83 mAb have laid a good foundation for further study CD83 expression and functions. Furthermore, we firstly demonstrated that PGE2, produced by monocytes upon CD83 stimulation, was a key mediator of T cell suppression. Our studies reveal a novel regulatory mechanism by which sCD83 suppresses T cell functions and thus provides a new insight on the regulatory function for CD83 in modulating magnitude of immune responses.更多还原