人CD46、CD55和CD59与肿瘤免疫逃逸及免疫治疗

膜结合补体调节蛋白CD46、CD55和CD59在肿瘤细胞膜上表达或过表达,保护肿瘤细胞免受免疫系统的攻击,成为肿瘤细胞免疫逃逸的途径之一.如何下调肿瘤细胞表面三者表达或抑制其功能以增强其对补体依赖的细胞毒作用的敏感性备受关注.     

补体调节蛋白CD46、CD55及CD59在肿瘤免疫治疗中的研究进展

以往对肿瘤逃避补体攻击的免疫机制并不是非常清楚,因而对肿瘤的传统免疫治疗,尤其是体液免疫治疗,往往效果并不理想。随着免疫学的发展,经研究发现不同的肿瘤细胞表面往往高表达补体调节蛋白(CD46/MCP,CD55/DAF,CD59/potectin)中的一类或几类分子,由于补体调节蛋白的高表达,从而抑制机体的补体系统对肿瘤细胞的攻击,使肿瘤逃避机体免疫防御。近年来随着对补体调节蛋白在肿瘤中表达及机制的进一步研究,针对补体调节蛋白的单克隆抗体及细胞因子的免疫治疗开始应用于动物实验和临床实验,取得了一定成功。本文阐述补体调节蛋白在肿瘤免疫治疗的研究中所取得的一些认识和进展。     

突变型CD59蛋白对卵巢癌细胞的抑瘤效应

背景与目的：国外报道已在多种实体肿瘤细胞中发现有CD59分子的过表达,并与肿瘤的失控性生长和恶性转化密切相关。本研究探讨突变型CD59在卵巢癌细胞A2780表面的抗补体活性以及与LPS联合抑制A2780细胞增殖的活性。方法：取突变型CD59质粒、野生型CD59质粒分别转染A2780细胞．G418筛选稳定表达细胞克隆,并从基因水平和蛋白水平鉴定CD59突变基因和野生型基因的转染情况。MTT法观察野生型CD59与突变型CD59在A2780细胞表面的抗补体活性,以及突变型CD59在LPS存在时对细胞的抑瘤效应。结果：通过荧光免疫检测、流式细胞术分析、RT—PCR鉴定证明建立了稳定转染野生型和突变型CD59的A2780细胞。MTT结果显示,与野生型CD59相比,突变型CD59失去对补体的抑制功能,与对照组未转染的A2780细胞相比无显著性差异。MTT检测5μg／mLLPS作用30min．对转染野生型CD59、突变型CD59及未转染A2780细胞的增殖抑制率分别为（26．9±2．95）％、（36.3±4．87）％、（29．6±3．16）％,差异有统计学意义（P〈0．05）。结论：CD59的W40位点对其功能具有重要作用,封闭该位点能够提高补体溶细胞作用,并有助于LPS发挥抑制瘤细胞增殖活性．有望应用于肿瘤治疗。     

肝细胞癌患者外周血性激素受体和糖皮质激素受体的含量

动物实验证实协同刺激分子CD80(B7-1)是促进抗肿瘤免疫应答的重要分子.本文应用RT-PCR,FACS技术检测了人肿瘤细胞系及EBV转化的B细胞CD80的表达,结果表明除Raji细胞、EBV转化的B细胞CD80阳性外,3AO、MCF-7、MDA-453、MKN-45、Hela细胞均为阴性.用我室建立的CD80逆转录病毒载体转染包装细胞PA317,筛选高滴度病毒上情感染人肿瘤细胞,得到CD80阳性表达的肿瘤细胞克隆.利用转染前后的人乳腺癌细胞系MDA-453作ICAM-I、HLA class Ⅰ、classⅡ分子表达的分析,结果表明转染有CD80基因的MDA-453细胞ICAM-Ⅰ、HLA classⅠ分子表达上调,class Ⅱ分子的表达未见改变.     

CD40信号和肿瘤免疫

CD40分子是分子量为50 kD的I型跨膜糖蛋白,成熟的CD40分子含有277个氨基酸,属于TNFR超家族成员,表达于APC(antigen presenting cell)、内皮细胞以及某些肿瘤细胞.CD40分子在肿瘤细胞上的表达引起了学者极大的兴趣,研究揭示CD40分子激发不仅可直接作用于肿瘤细胞,其介导的信号还可在多个环节影响肿瘤发生、发展.鉴于此,干预CD40信号可能是肿瘤免疫治疗新的靶点和策略.     

补体调节蛋白CD 46、CD 55和CD 59在胃肠肿瘤中的研究进展

胃肠肿瘤细胞的细胞膜高表达补体调节蛋白（complement regulatory proteins，CRPs）中的一种或几种分子。这些高表达分子可抑制机体的体液免疫和细胞免疫，可能是导致肿瘤细胞逃避机体免疫监视的机制之一。近年来许多学者在研究CRPs在胃肠肿瘤中的表达及其作用机制，以及将CRPs相关的单克隆抗体（monoclonal antibodies，McAbs）应用于肿瘤免疫治疗，取得了一定疗效。本文将CRPs在胃肠肿瘤研究中所取得的进展作一综述。     

CD147及其与肿瘤关系的研究进展

CD147分子是一种广泛存在于人体各个组织器官的属于免疫球蛋白超家族的糖蛋白,参与机体多种生理过程.CD147已被证实在多种体内外肿瘤细胞中表达增加,其与肿瘤发展的关系是目前肿瘤细胞生物学研究领域的热点之一.目前认为CD147能刺激肿瘤细胞周围的纤维母细胞产生基质金属蛋白酶,从而促进肿瘤的浸润、转移.现综述CD147分子的结构、功能及其与肿瘤发展的关系.     

细胞黏附分子CD15在乳腺癌的表达及与C-erbB-2、nm23、cath-D的关系

目的研究细胞黏附分子CD15在乳腺癌表达的临床病理意义及其与C-erbB-2、nm23、cath-D相互关系.方法应用了微波修复免疫组化技术S-P法检测了84例乳腺癌及20例乳腺良性病变中CD15的表达.结果 CD15主要在肿瘤细胞膜及细胞浆内表达;在乳腺良性病变阳性率低(20%),在乳腺癌中阳性率为57.14%(48/84),其表达与乳腺癌组织学分级高、淋巴结有转移、C-erbB-2表达、cath-D间质表达呈正相关;而与nm23表达负相关.结论细胞黏附分子CD15在乳腺癌细胞增殖、分化及转移过程中发挥重要作用,其表达可作为反映乳腺癌低分化及易转移的有用指标.     

肺癌患者抗原激活系统血液快速固有免疫反应比较实验研究

[目的]在体外采用自然实验体系研究肺癌患者系统血液快速固有免疫反应的变化。[方法]全血细胞自然实验管（0．2ml全血细胞悬液,0．2ml5×10^6／mlS180细胞悬液,0．3ml血浆）和全血自然对照组（0．2ml全血细胞悬液,0．2ml生理盐水,0．3ml血浆）．均37℃温育1h,用流式细胞仪测定红细胞CD59和白细胞的CD25分子的表达。[结果]肿瘤细胞能激活全血细胞,增强正常人和肺癌患者的CD59和CD25分子的表达,但CD59表达量正常人实验组（33．04±2．93）明显高于正常人对照组（29．96±5．40）、肺癌患者实验组（29．60±6．48）和肺癌患者对照组（28．36±4．69）（P〈0．01）;CD25表达量正常人实验组（7．29±3．00）明显高于正常人对照组（5．28±1．78）,肺癌患者实验组CD25表达量（5．99±1．96）和肺癌患者对照组（4．60±1．78）（P〈0．01）。[结论]在体外肿瘤细胞能激活系统血液快速固有免疫反应,但肺癌患者抗肿瘤的系统血液快速固有免疫反应水平明显低于正常人抗肿瘤的系统血液快速固有免疫反应水平。     

肺癌细胞自分泌VEGF对mCRPs的调控及其机制

目的：研究人非小细胞肺癌A549细胞自分泌VEGF对膜结合补体调节蛋白(membranebounal complement regulatory proteins, mCRPs)的调控及其机制。方法: RTPCR法检测CD46、CD55、CD59、VEGF及其受体(KDR和FLT1)和 IL8及其受体(CXCR1 CXCR2) mRNA在人非小细胞肺癌A549细胞的表达。MTT法检测抗VEGF抗体和抗IL8抗体对A549细胞增殖的影响,流式细胞术检测抗VEGF抗体和抗IL8抗体对A549细胞mCRPs表达水平的影响,Western blotting检测抗VEGF抗体对转录因子KLF2和磷酸化NFκB p65蛋白表达的影响。结果：A549细胞表达膜结合型CD46、CD55和CD59 mRNA,亦表达VEGF及其受体（KDR和FLT1）和 IL8及其受体（CXCR1和CXCR2） mRNA。抗VEGF抗体明显抑制A549细胞的增殖（P<0.05）。终质量浓度0.1 μg/ml抗VEGF抗体封闭72 h,CD55和CD59 mRNA表达下降,膜结合的CD55和CD59蛋白分子表达降低(均P<0.05);胞质和核KLF2蛋白相对定量值分别从063和0.88下降至0.42和0.66,胞质和胞核磷酸化NFκB p65蛋白相对定量值分别从0.44和0.28下降至0.37和019。结论：A549细胞可能通过自分泌VEGF增加NFκB p65和KLF2转录因子水平,从而上调CD55和CD59的表达。     

CD59 expressed on a tumor cell surface modulates decay-accelerating factor expression and enhances tumor growth in a rat model of human neuroblastoma

It has been hypothesized that complement inhibitors expressed on the surface of tumor cells prevent effective immune-mediated clearance. Whereas there are in vitro data to support this hypothesis, the species-selective activity of complement inhibitors has been a hindrance to investigating the role of membrane-bound complement inhibitors in rodent models of human cancer. The CD59-positive LAN-1 human neuroblastoma cell line was significantly more sensitive to lysis by rat complement than by human complement, illustrating the species selectivity of endogenously expressed complement inhibitors. Transfection of LAN-1 cells with rat CD59, an inhibitor of the terminal cytolytic membrane attack complex, effectively protected the cells from lysis by rat complement in vitro. When LAN-1 cells stably expressing rat CD59 were inoculated into immune-deficient rats, the onset of tumor growth and the rate of tumor growth were significantly enhanced compared with those of control-transfected LAN-1 cells. These data show directly that the expression of a complement inhibitor on a tumor cell promotes tumor growth. Flow cytometric analysis revealed that the endogenous expression of decay-accelerating factor (DAF), an inhibitor of complement activation, was up-regulated on the surface of cells after in vivo growth. Of further interest, higher levels of DAF were present on CD59-transfected cells than on control-transfected cells derived from tumors. Increased DAF expression correlated with decreased complement deposition on the tumor cell surface. These results show that expression of complement inhibitors on a tumor cell has functional consequences with regard to complement deposition in vivo and indicate that CD59 can indirectly effect complement activation and C3 deposition in vivo via a link between CD59 and DAF expression.     

K562 erythroleukemic cells are equipped with multiple mechanisms of resistance to lysis by complement

Resistance of tumor cells to lysis by complement is generally attributed to several protective mechanisms. The relative impact of these mechanisms in the same tumor cell, however, has not been assessed yet. We have analyzed the interaction of the human erythroleukemia tumor cell line K562 with human complement. K562 cells express the membrane complement regulatory proteins CD59, CD55 and CD46. As shown here for the first time, K562 also spontaneously release the soluble regulators C1 inhibitor, factor H, and soluble CD59. Complement resistance of K562 cells is augmented upon treatment with PMA, TNF or even with sublytic complement. Unlike TNF and sublytic complement, PMA enhanced the expression of membrane-bound CD55 and CD59 and led to increased secretion of soluble CD59. In addition, we show that complement-resistant K562 cells express a membrane-associated proteolytic activity, higher than the complement-sensitive K562/S cells. Treatment of complement-resistant K562 cells with serine protease inhibitors enhance their sensitivity to complement-mediated lysis. Inhibitors of protein kinase C (PKC) also sensitize K562 cells to complement lysis, implicating PKC-mediated signaling in cell resistance to complement. Neutralization of the CD55 and CD59 but not of CD46 regulatory activity with specific antibodies significantly increases complement-mediated K562 cell lysis. Treatment of K562 cells with a mixture of inhibitory reagents results in a significant additive enhancing effect on complement-mediated lysis of K562. In conclusion, K562 cells resist a complement attack by concomitantly using multiple molecular evasion strategies. Future attempts in antibody-based tumor therapy should include strategies to interfere with those resistance mechanisms.     

Modulation of CD59 expression by restrictive silencer factor-derived peptides in cancer immunotherapy for neuroblastoma

Tumor cells escape clearance by complement by abundantly expressing CD59 and other membrane complement regulators. Existing strategies for blocking/knocking down these regulators can contribute to tumor immunoclearance in vitro; however, there are numerous difficulties restricting their use in vivo. Here, we report a new strategy for suppression of CD59 expression in neuroblastoma using peptides that target regulators of CD59 expression. We identified the neural-restrictive silencer factor (REST) as a target for modulation of CD59 expression in neuroblastoma. We next designed plasmids that encoded peptides comprising different DNA-binding domains of REST and transfected them into neuroblastoma cell lines. These peptides suppressed CD59 expression, sensitizing neuroblastoma to complement-mediated killing triggered by anti-GD2 therapeutic monoclonal antibody. These CD59-modulating peptides might be effective therapeutic adjuvants to therapeutic monoclonal antibodies used for treatment of neuroblastoma and other cancer types sharing the same mechanism for regulation of CD59 expression.     

Upregulated expression of complement inhibitory proteins on bladder cancer cells and anti-MUC1 antibody immune selection

Membrane complement inhibitors (CD46, CD55 and CD59) are upregulated in some human cancers indicating that they play a role in immune evasion. We investigated complement inhibitor expression in bladder cancer and examined the hypothesis that selective pressure of an antibody response (anti-MUC1) results in the upregulated expression of complement inhibitors on tumor cells. Paired samples of tumor and normal tissue from 22 bladder cancer patients were analyzed for expression of MUC1, CD46, CD55 and CD59, and matched serum samples analyzed for anti-MUC1 IgM and IgG levels. Relationships between anti-MUC1 antibody levels and complement inhibitor expression were investigated. MUC1 mRNA was upregulated in 86% of tumor samples. CD46 was upregulated in 77%, CD55 in 55% and CD59 in 59% of tumors. Low titer anti-MUC1 IgM was detected in normal human sera, but was elevated in 41% of the bladder cancer patients. Anti-MUC1 IgG was virtually absent from normal sera, but present in 32% of the cancer patients. There was a direct relationship between anti-MUC1 antibody titer and expression level of complement inhibitors. Analysis of the correlation of each antibody with the expression of each complement inhibitor by Spearman's rank test revealed a strong correlation between both anti-MUC1 IgM and IgG levels and increased expression of CD46 and CD55, and combined anti-MUC1 IgM/IgG levels correlated with increased expression of all 3 complement inhibitors. In conclusion, the data demonstrate upregulated complement inhibitor expression and the presence of an anti-MUC1 antibody response in bladder cancer patients and support the hypothesis of antibody-mediated immune selection.     

p53 regulates cellular resistance to complement lysis through enhanced expression of CD59

It has been recently hypothesized that the CD59 gene has two putative p53-responsive elements that may be involved in defense of host cells from damage by the complement system in inflammation. Here we have examined the roles of these putative p53-binding sequences within the CD59 gene in regulation of CD59 expression. We have shown that both of these potential responsive elements bind p53 in vitro. Knocking down expression of p53 using small interfering RNA led to a 6-fold decrease in CD59 protein expression in HeLa cells. We have previously observed a decrease of CD59 in camptothecin-induced apoptotic IMR32 cells, whereas expression was increased in the surviving fraction compared with untreated cells. Here, we have shown that these changes are associated with altered expression levels and acetylation status of p53. We have also shown that acetylation status of p53 regulates CD59 expression on cells exposed to inflammatory cytokines to model inflammation. Our data suggest that p53 and in vivo positive/negative regulators of p53 could be used to modulate susceptibility of tumor cells to complement lysis in chemotherapy.     

A tumor-expressed inhibitor of the early but not late complement lytic pathway enhances tumor growth in a rat model of human breast cancer

Membrane-bound complement inhibitors protect host cells from inadvertent complement attack, and complement inhibitors are often up-regulated on tumors, possibly representing a selective adaptation by tumors to escape elimination by a host antitumor immune response. Relevant in vivo studies using rodent models of human cancer have been hampered by the fact that human complement inhibitors are not effective against rodent complement. Using nude rats and MCF7 cells expressing different rat complement inhibitors, a model of human breast cancer was established to investigate the role of complement and complement inhibitors in tumor progression. Expression of rat CD59, an inhibitor of the terminal cytolytic membrane attack complex of complement, had no effect on the incidence or growth rate of MCF7 tumors. In contrast, expression of rat Crry, an inhibitor of complement activation, dramatically enhanced the tumorigenicity of MCF7 cells. The expression of rat Crry on MCF7 inhibited the in vivo deposition of complement C3 fragments that serve as opsonins for receptors on phagocytes and natural killer cells. These data provide direct in vivo evidence that an inhibitor of complement activation can facilitate tumor growth by modulating C3 deposition. These data indicate an important role for complement opsonization in promoting cell-mediated antitumor immune function, a conclusion further supported by the demonstration that expression of rat Crry, but not rat CD59, on MCF7 cells inhibits rat cell-mediated cytotoxicity in vitro. Rat complement activation on MCF7 tumors was mediated by tumor-reactive antibodies present in the serum of na?ve nude rats, but there was also an IgM response to MCF7 tumors, a situation with similarities to some human cancers. These data support a hypothesis that blocking complement inhibitor function on tumor cells will not only enhance monoclonal antibody-mediated immunotherapy but may also be effective at enhancing a normally ineffective humoral immune response in the absence of administered antitumor antibody.