人小细胞肺癌食蟹猴模型的建立

目的:建立各方面更接近人类的小细胞肺癌(SCLC)动物模型,为SCLC的基础和临床研究提供参考.方法:以非人灵长类动物食蟹猴作为实验动物,经切除脾脏后,通过支气管镜直接注入人类SCLC细胞株Nci-h446至食蟹猴的支气管黏膜,诱导建立人SCLC的食蟹猴模型.通过胸部CT扫描和经支气管镜活检送病理,鉴定模型建立的成功率.结果:人小细胞肺癌食蟹猴模型的建立所需时间最少需要9个月,但最早约6个月即能发现细胞水平的变化(核异质变).食蟹猴模型的生活状态、生化水平及影像学改变都与人类肺癌的发病过程相似.6只食蟹猴成功生长出肺癌的有3只,模型建立成功率为50％.按接种部位计算,12个接种细胞浓度为1×107 mL-1的位点,成功生长出肺癌的3个,成功率为16.7％;接种细胞浓度为1×107 mL-1的位点,出现细胞核异质以上改变(包括癌变)的5个,占41.7％;但接种细胞浓度为1×105 mL-1和接种细胞浓度为1×106 mL-1的位点,成功率为0.所有36个接种位点成功率为8.3％,出现细胞核异质以上改变(包括癌变)的百分率为13.9％.结论:通过支气管镜直接接种人类SCLC细胞株Nci-h446至食蟹猴的支气管黏膜,能成功建立人SCLC的食蟹猴模型,但细胞浓度至少达1×107 mL-1.     

抗人CD3人/鼠嵌合抗体基因的构建

为了降低鼠源抗人CD3单抗的免疫原性．增加其在人体内的生物活性及治疗作用，使该抗体能更广泛更有效地长期多次用于人体治疗肿瘤、器官移植排斥反应及自身免疫性疾病。本文采用PCR技术从分泌抗CD3单抗的杂交瘤细胞HIT3a的mRNA中分离克隆了抗体的轻重链可变区基因的cDNA。以此轻重链可变区cDNA为特异探针从HIT3a基因文库中分离带有调控序列的功能性轻重链可变区基因，并将其插入到含有人k轻链及人71重链恒定区基因的哺乳动物表达载体中成功地构建了抗人CD3人／鼠轻重链嵌合抗体基因，为研制人抗CD3入／鼠嵌合抗体完成了关键性的第一步。     

抗人CD3人/鼠嵌合抗体的表达及特性

将从HIT3a基因库分离克隆的功能性抗CD3轻重链可变区基因插入含有人k轻链及γ1重链恒定区基因的哺乳动物表达载体中，构建了抗CD3嵌合抗体基因,用Lipofectin将抗CD3人／鼠嵌合轻重链基因共转染SP2／0细胞表达，获得稳定传代和稳定表达抗CD3嵌合抗体的转染杂交瘤细胞株C-HIT3a，ELISA，免疫荧光和PCR实验证实嵌合抗体与原鼠CD3单抗有相同的特异性和亲和性。体外生物活性的初步分析表明嵌合抗体与鼠CD3单抗对人的PBMC细胞的增殖有相同类型的作用，高剂量抗体抑制人T细胞的增殖，低剂量显示明显的激活增殖作用，当与IL-2联合应用其激活增殖作用增加20倍。细胞毒实验表明嵌合CD3抗体或由该抗体介导的免疫活性细胞(CD3-AK)对多种肿瘤细胞有明显的杀伤活性，较单用IL-2或单用LAK细胞其杀伤活性增加25倍。     

抗人卵巢癌-抗人CD3单链双特异性抗体体外介导的细胞毒作用及其机制

目的了解抗人卵巢癌-抗人CD3（BHL—I）单链双特异性抗体（scBsAb）体外介导的外周血淋巴细胞（PBL）对靶细胞SKOV3的细胞毒作用及可能的机制。方法二苯基溴化四氮唑蓝（MTr）法检测BHL-I体外介导PBL对SKOV3细胞的杀伤作用；逆转录-聚合酶链反应（RT—PCR）检测BHL—I介导的细胞毒作用过程中效应细胞PBL对穿孔素（perforin）、颗粒酶（GrB）mRNA表达的影响；酶联免疫吸附（ELISA）法检测细胞培养上清液中人肿瘤坏死因子-α（hTNF—α）和人干扰素-γ（hIFN-γ）含量的变化。结果BHL-I体外介导PBL对SKOV3细胞的细胞毒作用显著高于对MCF-7细胞的作用（P〈0.01），并且在效靶比为12．5：1、作用时间为36h、BHL-I浓度为25μg/ml时，PBL对靶细胞的细胞毒作用最为显著；BHL—I体外介导的细胞毒作用中，PBL表达perforin、GrBmRNA及混合细胞培养上清液中hTNF—α和hIFN-γ的含量均显著升高，并且白介素2（IL-2）的存在有利于这些因子的表达。结论BHL—I介导PBL对靶细胞的细胞毒作用具有一定的特异性，并且IL-2能增强BHL—I介导PBL的细胞毒作用。     

单价抗CD20抗体诱导人B细胞淋巴瘤Raji细胞的凋亡

背景与目的:抗CD20抗体和片段已应用于非霍奇金淋巴瘤的临床治疗,但仍需要开发新的抗CD20抗体和片段(未修饰的或放射性标记的),以治疗对美罗华(利妥昔单抗)无反应的患者。鼠源性抗CD20抗体HI47的嵌合抗体片段Fab和F(ab)'2已被构建。本研究目的是观察HI47(鼠抗-CD20抗体)和其嵌合抗CD20抗体片段抑制肿瘤细胞生长和诱导肿瘤细胞凋亡的作用。方法:用免疫荧光法测定抗CD20抗体与CD20+人B细胞淋巴瘤Raji细胞的结合能力;MTT法测定抗CD20抗体片段对Raji细胞生长的影响;用膜联蛋白Ⅴ染色和DNA琼脂糖凝胶电泳检测和验证抗CD20抗体片段诱导Raji细胞凋亡。结果:HI47和其嵌合的抗CD20抗体片段均可与CD20+Raji细胞结合,结合率可达90%以上;HI47不能与美罗华竞争结合Raji细胞;HI47和其嵌合的抗CD20抗体片段浓度为100μg/ml对Raji细胞的抑制率分别为:(57.0±1.5)%、(65.2±2.5)%、(77.2±3.2)%;单价的抗CD20抗体片段Fab(20μg/ml)能够诱导Raji细胞的凋亡,早期凋亡率为17%。结论:HI47的嵌合抗体片段对Raji细胞有抑制作用,能诱导Raji细胞的凋亡。     

转cry1Ab/Ac基因大米对食蟹猴小肠黏膜形态及上皮内淋巴细胞和杯状细胞数量的影响

目的：研究转cry1Ab/Ac基因大米对食蟹猴小肠黏膜形态及上皮内淋巴细胞和杯状细胞数量的影响。方法：以转cry1Ab/Ac基因大米为受试物,选用24只食蟹猴,雌雄各半,随机分成3组,即普通大米对照组、转cry1Ab/Ac基因大米低、高剂量组。对照组、低剂量和高剂量组给予的猴饲料中转cry1Ab/Ac基因大米含量分别为0、17.5%和70%。每天饲喂2次,每次（100±10）g,持续6个月,恢复期4周。分别进行一般毒性指标的检测：临床症状观察、体质量、体温、心电图、血液学、血清生化、尿生化、脏器质量以及组织病理学检查。解剖取十二指肠、空肠、回肠进行HE染色和PAS染色,观察小肠黏膜形态、上皮内淋巴细胞及杯状细胞数目。结果：饲喂6个月后,与普通大米对照组相比,转cry1Ab/Ac基因大米低剂量组和高剂量组食蟹猴的一般常规检查未见异常。小肠黏膜形态各组均未见病理改变,低、高剂量组的上皮内淋巴细胞和杯状细胞数与对照组相比差异均无统计学意义（P>0.05）。结论：饲喂转cry1Ab/Ac基因大米6个月对食蟹猴小肠黏膜形态、上皮内淋巴细胞及杯状细胞数量未见不良影响。     

抗CD3单抗和rIL-2共刺激诱生扩增人CD3AK的实验研究Ⅳ抗CD3单抗和rIL-2共刺激诱导的PBL增殖与凋亡共存现象

抗CD3单抗TCR／CD3复合物中的CD3分子相互作用，通过信号传导系统引起T细胞功能的不同变化，如诱导T细胞活化增殖，诱导无反应性(unresponsiveness)和细胞凋亡。但抗CD3单抗这几种性质不同，看似矛盾的作用之间有何内在联系及规律是值得深入探讨的。     

人尿源性干细胞的原代培养

目的：建立一种人尿源性干细胞（hUSCs）的原代分离培养及保存方法,为hUSCs的应用奠定基础。方法：取10例成人健康志愿者的尿液分别进行hUSCs原代培养,台盼蓝染色法比较不同冻存液配方复苏后的细胞存活率,进而确定hUSCs的最佳冻存液配方;采用四甲基噻唑蓝（MTT）法检测hUSCs细胞的增殖活性;流式细胞术测定间充质干细胞表面抗原CD44和CD90的表达情况。结果：10例健康成人志愿者中6例成功提取了hUSCs并体外培养形成集落;台盼蓝染色法确定配比为DMSO∶FBS∶hUSCs培养基=1∶4∶5的冻存液细胞存活率最高,与其他组比较差异具有统计学意义（P < 0.05）;MTT法检测显示hUSCs生长曲线呈S形且具有良好的增殖活性;流式细胞术鉴定干细胞表面抗原CD44和CD90呈阳性表达。结论：成功建立了一种人尿源性干细胞的原代分离培养及保存方法。     

激动性CD40抗体通过调节Th1/Th2平衡增强抗肿瘤作用的机制研究

  目的  探讨激动性CD40抗体中单链抗体（CD40ScFv）和单克隆抗体（CD40mAb）抑制小鼠乳腺癌细胞生长情况及对癌组织中Th1和Th2平衡的改变,分析通过调节Th1/Th2平衡达到抗肿瘤作用的机制。  方法  将含CD40ScFv基因片段的重组质粒转化至Rosetta大肠杆菌中,诱导表达并经纯化后获得有功能的CD40ScFv蛋白,行SDS-PAGE和Western blot检测。体外培养小鼠乳腺癌4T1细胞,在Balb/C小鼠皮下成瘤建立模型,分为CD40ScFv组、CD40mAb组和生理盐水组（NS组）,分别给予CD40ScFv、CD40mAb和生理盐水,观察各组小鼠肿瘤体积的变化。取出瘤体组织,ELISA法检测肿瘤组织上清中IL-12的浓度。提取肿瘤组织浸润淋巴细胞（TILs）,应用流式细胞技术检测TILs中Th1与Th2的比例。  结果  诱导表达的CD40ScFv鉴定表达成功,分子大小约为27 KDa,蛋白纯化后经BCA测定浓度为1.12 mg/mL。CD40ScFv组与CD40mAb组肿瘤大小分别为（3.044±0.239）cm3与（2.749±0.261）cm 3,均小于NS组的（3.933±0.326）cm3,差异具有统计学意义（P <0.05）。CD40ScFv组、CD40mAb组肿瘤组织中IL-12浓度分别为（396.27±48.13）pg/mL、（457.63±58.37）pg/mL,均显著高于NS组的（79.51±14.97）pg/mL,差异具有统计学意义（P <0.05）。CD40ScFv组与CD40mAb组的Th1/Th2细胞比值分别为6.32±0.87与5.54±0.71,均显著高于NS组的1.79±0.38,差异具有统计学意义（P < 0.05）。  结论  激动性CD40抗体在体内通过调节Th1与Th2的比例,发挥抑制肿瘤生长的作用,其中通过促进DC活化后分泌IL-12是影响肿瘤微环境中Th1/Th2平衡的重要机制之一。      

CRISPR/Cas9 介导的PD-1基因敲除对食蟹猴T细胞增殖、表型及IFN-γ和IL-2 分泌的影响

目的：探讨CRISPR/Cas9 基因编辑技术敲除食蟹猴T细胞PD-1 基因对T细胞增殖、表型及IFN-γ、IL-2 分泌的影响。方法: 设计靶向食蟹猴PD-1 基因的gRNA,构建并提取质粒,分离食蟹猴外周血单个核细胞（peripheral blood mononuclear cell,PBMC）,加入质粒DNA,用Lonza 4D电转仪进行细胞转染,转染48 h 后用流式细胞术和荧光显微镜技术检测转染效率。提取细胞基因组DNA进行PCR扩增及T7E1 酶切鉴定。在人源性CD3 抗体和IL-2 刺激下诱导食蟹猴T细胞增殖并绘制细胞生长曲线,PI 染色流式细胞术检测T细胞的细胞周期及CD4、CD8 表达水平,ELISA检测IFN-γ、IL-2 的分泌水平。结果: 转染48 h 后,荧光显微镜下见实验组出现绿色荧光蛋白表达的细胞,其转染效率为（21.6±3.2）%;实验组细胞基因组DNA PCR产物经T7E1 酶切显示3 条带,出现目的分裂条带（243、197 bp）。与未转染组比较,（1）实验组T细胞增殖缓慢、集落形成时间延迟、细胞体积较小、折光性较弱;（2）实验组处于G0/G1 期的T 细胞数显著增多（P<0.05）、G2/M 期细胞数显著减少（P<0.05）;（3）实验组T 细胞IFN-γ、IL-2 的分泌水平显著升高（均P<0.05）,但CD4、CD8 表达水平比较差异无统计学意义（均P>0.05）。结论: PD-1 基因敲除可使食蟹猴T细胞阻滞于G0/G1 期从而抑制其增殖,同时上调了IFN-γ、IL-2 的分泌水平。     

人外周血TIM3 基因敲除T淋巴细胞的制备及其抗肿瘤作用

[摘要] 目的: 通过CRISPR-Cas9 基因编辑技术及质粒电转染技术制备T 细胞免疫球蛋白黏液素3（TIM3）基因敲除的人外周血T淋巴细胞,探讨敲除TIM3 基因后能否增强T细胞免疫功能及其抗肿瘤作用。方法: 通过电转染法将hTIM3 sgRNA/Cas9 双质粒共转染EBV阳性胃癌患者外周血T淋巴细胞,电转24 h 后流式细胞术检测转染效率并利用荧光显微镜观察;体外培养过程中观察基因敲除后T细胞增殖活性,流式细胞术验证TIM3 基因敲除效率以及细胞表型的变化。用肿瘤抗原肽活化T细胞检测TIM3 基因敲除后T 细胞分泌细胞因子水平及体外杀伤胃癌AGS-EBV 细胞的能力。结果:电转染法能够有效地将hTIM3 sgRNA/Cas9 双质粒敲除体系转入人外周血T淋巴细胞,其转染效率平均达（41.5±3.6）%,基因敲除效率波动在40.0%～50.0%（均P<0.01）;经抗原活化后的基因敲除组T细胞的增殖活性和免疫表型未见明显变化,表面活化分子中仅见HLA-DR较对照组明显提高（P<0.05）;TIM3 基因敲除T细胞分泌TNF-α、IFN-γ 的水平显著增高（P<0.01 或P<0.05）,体外杀伤胃癌AGS-EBV 细胞的能力也明显增强（均P<0.05）。结论: 用CRISPR-Cas9 基因编辑及质粒电转染技术制备人外周血TIM3 基因敲除T淋巴细胞的方法简易可行,在体外的扩增活化中保持TIM3 基因水平下调并具有更高的免疫应答水平以及更强的抗肿瘤作用。这一新技术的研发为基因工程化细胞免疫治疗提供了新的思路。     

胃腺癌患者外周血中CD4+T淋巴细胞表面抑制性分子PD-1的表达

目的:检测胃腺癌患者外周血中程序性死亡分子1（programmed death-1,PD-1）在CD4+T淋巴细胞上的表达水平,探讨其与胃腺癌的关系。方法:选取未经放、化疗或外科手术治疗的胃癌患者57例、疾病对照22例与健康对照20例,取新鲜抗凝血经流式细胞术（flowcytometry,FCM）检测抑制性分子PD-1的百分比例;同时对胃腺癌患者进行TNM分期比较。结果:胃腺癌患者外周血中PD-1在CD4+T淋巴细胞上的表达水平（10.91±4.36）%比疾病对照（8.14±3.65）%和健康对照（7.37±3.06）%显著升高,Ⅲ-Ⅳ期胃腺癌患者的CD4+PD-1+T细胞的百分比例（11.9±3.85）%显著高于Ⅰ-Ⅱ期（9.08±3.13）%。结论:抑制性分子PD-1在胃腺癌外周血中CD4+T淋巴细胞表面表达增加,并随胃腺癌进展而升高,提示CD4+PD-1+T淋巴细胞与胃腺癌的发生发展相关。     

CD4+ T lymphocytes: a critical component of antitumor immunity

Both prophylactic and therapeutic vaccines targeting a wide variety of cancers are being developed. Because of the potency of cell-mediated immunity, many vaccine strategies are focused on activating tumor-specific cytotoxic CD8⁺ T lymphocytes. CD4⁺ T lymphocytes are a key element in optimal activation of CD8⁺ T cells and in the maintenance of immune memory, and therefore their activation is critical for cancer vaccine efficacy. This article reviews the mechanisms by which CD4⁺ T cells facilitate tumor immunity and the vaccine strategies that enhance CD4⁺ T cell activity.     

纳米Fe3O4／CD133Ab复合物对胶质瘤U251细胞放疗增敏作用的实验研究

目的 探讨纳米Fe3O4／CD133Ab复合物对脑胶质瘤U251细胞的放射增敏作用。方法 细胞免疫组化法检测脑胶质瘤U251细胞CD133的表达情况;流式细胞分选获得U251-CD133+细胞;MTT法观察纳米Fe3O4／CD133Ab复合物和CD133Ab分别对脑胶质瘤细胞U251及U251-CD133+的细胞毒作用;克隆形成抑制实验检测纳米Fe3O4／CD133Ab复合物和CD133Ab对脑胶质瘤U251细胞放射增敏的影响。结果 脑胶质瘤U251细胞中有CD133分子阳性表达并可以分选出CD133阳性表达细胞;在0.0488～25μg／ml范围内,纳米Fe3O4／CD133Ab复合物作用于脑胶质瘤U251及U251-CD133+细胞24h的细胞毒性呈明显剂量依赖性,与阴性对照组比较,差异有统计学意义（P＞0.05）。纳米Fe3O4／CD133Ab复合物对脑胶质瘤U251和U251-CD133+细胞的增殖抑制率最高值分别为59.46％和56.66％,IC50分别为11.84μg／ml和12.06μg／ml。纳米Fe3O4／CD133Ab复合物+照射组与CD133Ab+照射组比较,细胞存活分数差异有统计学意义（P＜0.05）,在照射4Gy以上时,随X射线剂量增加而放射敏感性增强。结论 纳米Fe3O4／CD133Ab复合物对脑胶质瘤细胞U251和U251-CD133+有细胞毒作用,并且对U251细胞有放射增敏作用。     

Numbers and cytotoxicities of CD3+CD56+ T lymphocytes in peripheral blood of patients with acute myeloid leukemia and acute lymphocytic leukemia

Recent reports have highlighted the role of cellular immunity in anti-tumor defenses. T lymphocytes are known to play important part in anti-cancer immunity. The number and function of T lymphocytes are altered in chronic leukemia patients. CD3⁺CD56⁺ T lymphocytes have also been found to be abnormal in cancer patients. We therefore investigated changes in the number and cytotoxicity of CD3⁺CD56⁺ T lymphocytes in the peripheral blood of acute leukemia (AL) patients (excluding acute promyelocytic leukemia), to improve our understanding of the role of this T lymphocyte subset. We analyzed CD3⁺CD56⁺ T lymphocyte numbers and cytotoxicities in healthy controls, AL patients, and AL patients with complete remission. Lymphocyte counts were performed in peripheral blood and flow cytometry was used to determine cell numbers and cytotoxicities. The absolute number of CD3⁺CD56⁺ T lymphocytes was increased in AL patients (including acute myeloid [AML] and acute lymphocytic leukemia [ALL]) compared with healthy controls (P < 0.05), but their functioning was significantly reduced (P < 0.05). The number of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients who achieved remission following chemotherapy was close to healthy controls (P > 0.05), but their functioning was still significantly reduced (P < 0.05). In addition, the number of CD3⁺CD56⁺ T lymphocytes increased significantly in AML patients with increased peripheral blood white blood cell (WBC) counts, and in ALL patients without increased WBCs. These results suggest that cellular immunity may respond to AML and ALL, but that lymphocyte cytotoxicity remains impaired. Dysfunction of CD3⁺CD56⁺ T lymphocytes in AML and ALL patients may contribute to the failure of the host immune response against leukemic blasts.     

CD103 is a hallmark of tumor-infiltrating regulatory T cells

Regulatory T cells (Treg) mediate tolerance towards self-antigens by suppression of innate and adaptive immunity. In cancer patients, tumor-infiltrating FoxP3+ Treg suppress local anti-tumor immune responses and are often associated with poor prognosis. Markers that are selectively expressed on tumor-infiltrating Treg may serve as targets for immunotherapy of cancer. Here we show that CD103, an integrin mediating lymphocyte retention in epithelial tissues, is expressed at high levels on tumor-infiltrating FoxP3+ Treg in several types of murine cancer. In the CT26 model of colon cancer up to 90% of the intratumoral FoxP3+ cells expressed CD103 compared to less than 20% in lymphoid organs. CD103+ Treg suppressed T effector cell activation more strongly than CD103(neg) Treg. Expression of CD103 on Treg closely correlated with intratumoral levels of transforming growth factor β (TGF-β) and could be induced in a TGF-β-dependent manner by tumor cell lines. In vivo, gene silencing of TGF-β reduced the frequency of CD103+ Treg, demonstrating that CD103 expression on tumor-infiltrating Treg is driven by intratumoral TGF-β. Functional blockade of CD103 using a monoclonal antibody did however not reduce the number of intratumoral Treg, indicating that CD103 is not involved in homing or retention of FoxP3+ cells in the tumor tissue. In conclusion, expression of CD103 is a hallmark of Treg that infiltrate TGF-β-secreting tumors. CD103 thus represents an interesting target for selective depletion of tumor-infiltrating Treg, a strategy that may help to improve anti-cancer therapy.     

The intratumoural subsite and relation of CD8+ and FOXP3+ T lymphocytes in colorectal cancer provide important prognostic clues

Background:

To find improved tools for prognostic evaluation in patients with colorectal cancer (CRC), we have analysed how infiltration of cytotoxic T lymphocytes (CD8⁺) and regulatory T lymphocytes (FoxP3⁺) correlates to prognosis, not only according to quantity and relation, but also to subsite within tumours of different molecular characteristics (microsatellite instability and CpG island methylator phenotype status).

Methods:

CD8 and FOXP3 expression was evaluated by immunohistochemistry in 426 archival tumour tissue samples from patients surgically resected for CRC. The average infiltration of CD8⁺ and FOXP3⁺ cells was assessed along the tumour invasive front, in the tumour centre and within the tumour epithelium (intraepithelial).

Results:

We found that infiltration of CD8⁺ T lymphocytes within the tumour epithelium provided the strongest prognostic information (P<0.001). At the tumour invasive front and tumour centre, FOXP3 expression withheld the strongest association to prognosis (P<0.001), suggesting FOXP3⁺ T-lymphocyte infiltration to be a better prognostic tool than CD8⁺ T lymphocytes at these intratumoural subsites. We further analysed the possible prognostic impact of the relation between these T-cell subsets, finding that a high intraepithelial CD8 expression was associated with a better patient outcome, independent of FOXP3 infiltration. In groups of low intraepithelial CD8 expression, however, a high infiltration rate of FOXP3⁺ cells at the tumour invasive front, significantly improved prognosis.

Conclusions:

Analyses of intraepithelial infiltration of CD8⁺ T lymphocytes, infiltration of FOXP3⁺ T lymphocytes at the tumour front or centre, and the relation between these subsets, may be a valuable tool for predicting prognosis in colon cancer.     

Expression of Fas/FasL in CD8+ T and CD3+ Foxp3+ Treg Cells - Relationship with Apoptosis of Circulating CD8+ T Cells in Hepatocellular Carcinoma Patients

Aims: Dysfunction of the host immune system in cancer patients can be due to a number of factors, includinglymphocyte apoptosis. Several studies showed that Foxp3+T cells take part in inducing this process by expressingFasL in tumor patients. However, the relationship between apoptosis, CD8+T cells and Foxp3+T cells in HCCpatients is still unclear. The present study was designed to investigate the correlation between apoptosis levelsand Fas/FasL expression in CD8+T lymphocytes and Foxp3+T cells in patients with HCC. Methods: CD8+T cellsand CD3+Foxp3+T cells were tested from peripheral blood of HCC patients and normal controls and subjectedto multicolor flow cytometry. The expression of an apoptosis marker (annexin V) and the death receptor Fas inCD8+T cells and FasL in CD3+Foxp3+T cells were evaluated. Serum TGF-β1 levels in patients with HCC weremeasured by enzyme-linked immunosorbent assay. The relationship between apoptosis and Fas expression, aswell as FasL expression in CD3+Foxp3+T cells was then evaluated. Results: The frequency of CD8+T cells bindingannexin V and Fas expression in CD8+T cells, were all higher in HCC patients than normal controls and theproportion of apoptotic CD8+T cells correlated with their Fas expression. Serum TGF-β1 levels correlated inverselywith CD3+Foxp3+T cells. Conclusions: Fas/FasL interactions might lead to excessive turnover of CD8+T cellsand reduce anti-tumor immune responses in patients with HCC. Further investigations of apoptosis inductionin Fas+CD8+T cells in vitro are required.     

肝癌细胞通过Fas/FasL途径触发T淋巴细胞凋亡的双重阻断研究

目的通过阻断Fas/FasL介导的T细胞凋亡,建立快速制备大量激活的肿瘤特异性细胞毒性T淋巴细胞(CTL)的方法。方法选择FasL表达阳性的肝癌标本,分离肝癌细胞和肿瘤浸润淋巴细胞(TIL),然后在体外将两者混合,并在CD28单抗共刺激下,激活制备特异性CTL。应用可溶性Fas受体阻断肝癌细胞通过Fas/FasL途径触发激活T细胞凋亡,与对照组比较用流式细胞仪和DNA ladder Test同时检测阻断凋亡作用,通过3H掺入法和51Cr释放法了解T细胞增殖力及杀伤活性。结果流式细胞术仪检测未阻断组较阻断组和未阻断对照组凋亡率明显升高,未阻断组凋亡率达47．82％±0．13％,对照组静息性T淋巴细胞组为3．76%±0．25％,阻断组为8．22％±0．26％(P<0．01),DNA ladder显示未阻断组T淋巴细胞出现明显梯状条带,阻断组为阴性。51Cr释放法表明阻断后T淋巴细胞杀伤活性增加,较未阻断组及未阻断对照组差异有显著性(P<0．01)。3H掺入法检测证实可溶性Fas受体阻断激活T细胞凋亡后,细胞增殖明显升高,较未阻断组差异有显著性(P<0．01)。结论体外实验可获得大量激活T细胞,并可杀伤肿瘤细胞。     

Serial killing of tumor cells by cytotoxic T cells redirected with a CD19-/CD3-bispecific single-chain antibody construct

Certain bispecific antibodies exhibit an extraordinary potency and efficacy for target cell lysis by eliciting a polyclonal T-cell response. One example is a CD19-/CD3-bispecific single-chain antibody construct (bscCD19xCD3), which at femtomolar concentrations can redirect cytotoxic T cells to eliminate human B lymphocytes, B lymphoma cell lines and patient-derived malignant B cells. Here we have further explored the basis for this high potency. Using video-assisted microscopy, bscCD19xCD3 was found to alter the motility and activity of T cells from a scanning to a killing mode. Individual T cells could eliminate multiple target cells within a 9 hr time period, resulting in nuclear fragmentation and membrane blebbing of target cells. Complete target cell elimination was observed within 24 hr at effector-to-target cell ratios as low as 1:5. Under optimal conditions, cell killing started within minutes after addition of bscCD19xCD3, suggesting that the rate of serial killing was mostly determined by T-cell movement and target cell scanning and lysis. At all times, T cells remained highly motile, and no clusters of T and target cells were induced by the bispecific antibody. Bystanding target-negative cells were not detectably affected. Repeated target cell lysis by bscCD19xCD3-activated T cells increased the proportion of CD19/CD3 double-positive T cells, which was most likely a consequence of transfer of CD19 from B to T cells during cytolytic synapse formation. To our knowledge, this is the first study showing that a bispecific antibody can sustain multiple rounds of target cell lysis by T cells.