MUC1模拟表位肽致敏DC治疗宫颈癌荷瘤小鼠

目的 研究黏蛋白1(MUC1)模拟表位肽致敏树突状细胞(DC)对宫颈癌荷瘤小鼠生长和T淋巴细胞亚群的影响。方法 培养小鼠骨髓细胞，诱导并刺激DC成熟，将MUC1模拟表位肽加入成熟DC，形成负载MUC1模拟表位肽的DC。建立稳定表达MUC1的宫颈癌U14细胞株荷瘤BALB/c小鼠模型。空白对照组注射0.2 mL生理盐水；DC组注射2×10⁵个未加MUC1模拟表位肽的DC;DC+MUC1组注射2×10⁵个经过负载MUC1模拟表位肽的DC。免疫1周后，进行二次免疫。结果 肿瘤体积与治疗前比较，空白对照组、DC组和DC+MUC1组均升高(P<0.05)；治疗前，各组间比较差异无统计学意义(P>0.05)；二次免疫1周后，DC+MUC1组低于空白对照组和DC组(P<0.05)，空白对照组和DC组间差异无统计学意义(P>0.05)；小鼠抑瘤率DC组和DC+MUC1组均高于空白对照组(P<0.05),DC+MUC1组高于DC组(P<0.05)；小鼠外周血CD3⁺、CD4⁺、CD...     

MUC1模拟表位肽致敏DC对宫颈癌荷瘤小鼠肿瘤生长及免疫微环境的影响

目的：研究黏蛋白1(MUC1)模拟表位肽致敏树突状细胞(DC)对宫颈癌荷瘤小鼠生长和免疫微环境的影响。方法：培养小鼠骨髓细胞,诱导并刺激DC成熟,MUC1模拟表位肽处理构建负载MUC1模拟表位肽的DC。构建稳定表达MUC1的宫颈癌U14细胞株的荷瘤BALB/c小鼠模型,随机分为3组：空白对照组(注射0.2 ml生理盐水)、DC组(注射2×10⁵个未加MUC1模拟表位肽的DCs)和DC+MUC1组(注射2×10⁵个负载MUC1模拟表位肽的DCs),免疫1周后进行二次免疫。治疗前和二次免疫1周后测量肿瘤体积;二次免疫1周后取小鼠肿瘤组织称重并计算抑瘤率;取小鼠外周血,流式细胞术检测CD3⁺T、CD4⁺T、CD8⁺T细胞、CD4⁺CD25⁺Foxp3⁺Treg水平;ELISA检测小鼠血清IL-6、IL-10、TGF-β、VEGF浓度。结果：与治疗前比较,空白对照组、DC组和DC+MUC1组肿瘤体积增大(P<0.0...     

CpG-ODN治疗小鼠膀胱肿瘤的实验研究

目的 探讨含CpG序列的寡脱氧核糖核苷酸(CpG Oligodeoxynucleotide, CpG-ODN)对小鼠膀胱肿瘤的治疗作用.方法 建立BTT739荷瘤小鼠动物模型,随机分为CpG-ODN治疗组和PBS对照组,于肿瘤细胞接种后第7、14天给予治疗,每组分两亚组,分别用于测量瘤重、体积及用于观察荷瘤小鼠存活情况.ELISA法检测小鼠血清中IL-12水平.流式细胞仪检测肿瘤组织中浸润性树突状细胞表面共刺激分子CD80、CD86的表达.结果 第2次治疗7d后,平均瘤重CpG-ODN组为(3.30±0.81)g,对照组为(4.50±0.47)g(P<0.01),平均体积CpG-ODN组为(3.57±0.84)cm3,对照组为(4.84±0.58)cm3(P<0.01),CpG-ODN组荷瘤小鼠生存期长于PBS对照组(P<0.05).治疗组及对照组小鼠血清中IL-12浓度分别为(391.5±28.4)pg/mL和(257.2±13.7)pg/mL(P<0.01).肿瘤组织中浸润性树突状细胞(Tumor infiltrating dendritic cell, TIDC)表面CD80、CD86表达水平治疗组分别为(17.75±3.13)%、(18.72±2.79)%,对照组分别为(11.32±1.18)%(P<0.01)、(12.65±1.22)%(P<0.01).结论 CpG-ODN对膀胱肿瘤荷瘤小鼠具有抑瘤效应和生存期延长作用,其机制主要是通过诱导DC成熟、促进Th1型细胞因子分泌、诱导免疫应答向Th1细胞分化.     

体外观察MUC1模拟表位融合肽在DC细胞内呈递的研究

目的研究MUC1模拟表位融合多肽[人免疫缺陷病毒的转录活化因子(TAT)-MUC1模拟表位多肽-内质网靶向肽]在DC细胞内的呈递。方法取小鼠骨髓及脾细胞诱导培养成熟DC细胞,利用荧光显微镜对MUC1模拟表位融合肽在DC细胞内的呈递进行体外观察。结果 MUC1模拟表位融合肽在37℃5%CO_2浓度的暗孵箱内脉冲成熟DC细胞40 min后,DC MUC1组细胞内可检测出强荧光,DC HBcAg组细胞内荧光显示较弱,DC空白组无荧光显示;2 h后,DC MUC1组细胞荧光显示进一步增强,DC HBcAg组与DC空白组细胞内荧光强度无明显变化。结论该模拟表位融合肽能够高效地进入DC胞质并具有DC细胞高选择性靶向。     

肿瘤微环境中IL-10限制SOCS1基因沉默的树突状细胞疫苗的抗肿瘤作用

目的观察SOCS1基因沉默的DC疫苗对荷黑素瘤小鼠的抗肿瘤作用及肿瘤微环境中IL-10对该DC疫苗抗瘤作用的影响。方法从小鼠股骨分离骨髓细胞,GM-CSF和IL-4联合诱导DCs分化,然后感染携带沉默SOCS1基因的Len-SOCS1-shRNA慢病毒;用TRP2抗原肽负载沉默SOCS1的DC细胞制备DC疫苗,LPS诱导成熟,流式细胞仪分析DC细胞表面MHCⅡ和CD86的表达,real-time PCR分析该DC细胞的SOCS1、IL-10和IL-12p40表达。用B16细胞或降低IL-10基因表达的B16(B16-IL-10-/-)制备荷瘤小鼠模型,瘤内注射DC疫苗(3×106/只),观察肿瘤生长和荷瘤小鼠生存期。采用不连续梯度离心法分离肿瘤浸润淋巴细胞,流式细胞仪观察CD8+T细胞的分布;并采用微量细胞毒的方法分析CTL活性。结果 Len-SOCS1-shRNA慢病毒感染DC后,可使SOCS1表达下调80%;下调SOCS1表达的DC细胞MHCⅡ和CD86表达有增加趋势,但与对照组DC相比无明显差异;下调SOCS1表达可降低DC细胞的IL-10表达,提高IL-12p40的表达。沉默SOCS1的DC疫苗对B16荷瘤小鼠的生存率没有明显影响,但可显著提高B16-IL-10-/-荷瘤小鼠的生存率(P<0.05)。下调SOCS1表达的DC疫苗不仅可提高B16-IL-10-/-荷瘤小鼠肿瘤浸润CD8+T淋巴细胞数,还可促进CD8+T细胞IFN-γ的分泌及CTL活性。结论沉默SOCS1可提高DC疫苗的活性,但肿瘤微环境的IL-10依然是限制该DC疫苗有效发挥抗瘤作用的因素。     

CpG增强DC疫苗对黑色素瘤B16-HLA-MAGE-3的免疫效果

目的:观察CpG对抗原肽负载DC疫苗免疫效果的增强作用.方法:分别用50 μg/ml的MAGE-3271-279抗原肽、4 μg/ml的CpG和MAGE-3271-279抗原肽(50 μg/ml)+CpG(4 μg/ml)刺激培养到第6天的小鼠骨髓DC细胞,2天后用LPS再刺激24 小时诱导其成熟;给HLA-A·0201/Kb转基因鼠移植B16-HLA-MAGE-3黑色素瘤,并于小鼠荷瘤的第-4、5和8天分别于颈部皮下注射同系小鼠DC疫苗(2×105/鼠),观察荷瘤鼠肿瘤生长情况;在荷瘤的第23 天处死小鼠,检测脾脏NK细胞活性.给正常HLA-A2转基因小鼠皮下注射DC疫苗,8周后接种肿瘤细胞,观察免疫记忆形成情况.结果:抗原肽负载的DC疫苗,在小鼠荷瘤的第14天之前,小鼠肿瘤生长受到抑制,在第15天后抑瘤作用不明显;CpG与MAGE-3271-279+CpG负载的DC疫苗可显著抑制荷瘤鼠肿瘤的生长,且可诱导较高的NK细胞活性.MAGE-3271-279+CpG负载的DC疫苗在免疫8周后依然可抑制肿瘤细胞的生长,说明可以诱导免疫记忆形成.结论:CpG对抗原肽负载的DC疫苗的免疫效果具有潜在的增强作用.     

利用小鼠皮下人乳腺癌移植瘤模型观察MUC1-MBP抗乳腺癌作用

目的建立小鼠皮下人乳腺癌移植瘤模型,探讨MUC1-MBP蛋白疫苗的抗人乳腺癌作用。方法首先通过筛选最佳条件建立小鼠皮下人乳腺癌移植瘤动物模型,HE染色和免疫组化鉴定。利用已经建立的乳腺癌动物模型,通过肿瘤预防实验和治疗实验,观察MUC1-MBP抗人乳腺癌作用。结果通过5 Gy X射线照射,皮下注射MCF-7人乳腺癌细胞3×10~6个/只,FTY720灌胃4～6 d,成功建立小鼠皮下人乳腺癌移植瘤动物模型。在预防乳腺癌生长实验中,PBS对照组、MBP组和MUC1-MBP组小鼠成瘤率分别为100%、83%和67%,肿瘤平均体积分别为(91.9±56.3)mm~3、(22.3±21.5)mm~3和(17.2±18.0)mm~3。与对照组相比,MUC1-MBP组显著减小(P<0.001),MBP组明显减小(P<0.05),提示MUC1-MBP能明显预防乳腺癌生长,MBP对乳腺癌生长也有一定的预防作用。在治疗乳腺癌生长实验中,PBS对照组、MBP组和MUC1-MBP组小鼠肿瘤生长抑制率分别为0、17%和17%,肿瘤平均体积分别为:(101.7±73.6)mm~3、(56.9±56.7)mm~3和(32.6±32.3)m...     

激发型4-1BB单抗联合凋亡肿瘤细胞负载的树突状细胞在恶性肿瘤免疫治疗中的作用

目的：研究激发型4-1BB单抗（2A）联合凋亡肿瘤细胞负载的DC（AP-DC）疫苗在小鼠B细胞淋巴瘤免疫治疗中的作用.方法：凋亡小鼠B细胞淋巴瘤细胞A20负载的DC用来制备AP-DC.A20荷瘤小鼠分别被注射AP-DC、2A单抗或二者联合.观察肿瘤生长情况及小鼠生存期.流式细胞术检测免疫治疗后荷瘤鼠脾脏T细胞的表型和胞浆内细胞因子;3H-TdR掺入试验检测T细胞体外增殖能力;ELISA法检测荷瘤鼠血清和脾脏T细胞培养上清中IL-2、IFN-γ和IL-10含量.结果：应用激发型4-1BB单抗2A或AP-DC疫苗对荷瘤小鼠开展免疫治疗,可抑制肿瘤生长,延长荷瘤鼠生存期,获得12.5%或25%的肿瘤完全缓解率.但二者联合应用,治疗效果更好,可获得62.5%的肿瘤完全缓解率,并且荷瘤鼠可长期生存.联合治疗后荷瘤鼠脾脏T细胞体外增殖更为显著,CD4^＋IFN-γ^＋细胞的比例也明显增加.IL-2和IFN-γ的分泌水平在联合治疗荷瘤鼠的血清和脾脏T细胞的培养上清中升高更明显,而IL-10的分泌则降低.结论：激发型4-1BB单抗和AP-DC在肿瘤免疫治疗中有协同作用.     

MUC1基因疫苗诱导小鼠特异性CTL和体液免疫应答

目的 :观察MUC1基因疫苗诱导小鼠特异性杀伤性T细胞及体液免疫应答的作用。方法 :采用股四头肌肌肉注射 ,将构建的MUC1基因疫苗pcDNA3.1 MUC1免疫雌性BALB/c小鼠 ,每次间隔 3wk ,共 3次。最后 1次免疫后第 3周 ,接种表达MUC1的EMT6乳腺癌细胞进行免疫保护实验。用 4h51Cr释放法检测小鼠脾细胞特异性CTL杀伤活性 ;免疫组化染色法检测小鼠血清特异性抗体的水平。结果 :在效靶比为 10 0∶1、5 0∶1、2 5∶1、12 .5∶1时 ,MUC1基因疫苗免疫组特异性CTL对EMT6靶细胞杀伤活性分别为 5 4 .1%、39.8%、2 6 .4 %和2 0 .1% ,对照组分别为 13.2 %、10 .0 %、8.2 %、7.2 %和 11.7%、9.8%、7.7%、7.0 % ,前者与后二者差异显著 (P <0 .0 1)。免疫组化染色检测显示 ,人乳腺癌组织MUC1呈染色阳性 ;MUC1基因疫苗免疫组仅见 4 0 % (4/ 10 )的小鼠有肿瘤形成 ,而 pcDNA3.1对照组和生理盐水阴性对照组 10 0 %可见肿瘤形成、生长 ,表明MUC1基因疫苗免疫组小鼠具有一定的免疫保护作用。结论 :MUC1基因疫苗可诱导小鼠产生特异性CTL及体液免疫应答 ,对小鼠体内荷瘤可能具有一定的预防作用     

小剂量环磷酰胺治愈荷膀胱癌小鼠过程中外周血血小板计数的动态变化及意义

目的:建立环磷酰胺(CTX)治愈荷膀胱癌T739小鼠的动物模型,观察外周血象特别是血小板计数在化疗治愈肿瘤中的动态变化及其意义.方法:给正常T739小鼠皮下接种小鼠可移植性膀胱移行细胞癌组织,建立荷膀胱癌的小鼠模型.接种后第7天,荷瘤小鼠腹腔内分别注入15、40、100 mg/kg的CTX,等量生理盐水对照,确定CTX治愈肿瘤的量效关系.然后再取12只荷瘤小鼠,随机分成2组,15 mg/ks CTX单次腹腔内注射,等量生理盐水对照,分别在用药后6 h、用药后第2、4、9、14天内眦静脉取血,进行血常规检测,分析荷瘤小鼠外周血象的动态变化.结果:15～100 mg/kg CTX治疗后2周内,荷瘤小鼠肿瘤生长受抑制或肿瘤结节缩小的速率与所用CTX的剂量呈正相关;CTX治疗后2月,荷瘤小鼠的存活率与所用CTX的剂量呈负相关.15 mg/kg CTX单次腹腔内注射在治愈多数荷瘤小鼠的同时对外周血象无明显抑制作用.用药后6 h治疗组荷瘤鼠外周血血小板计数为(1483.4±184.4)×109/L,明显高于对照组小鼠(1086.6±81.0)×109/L,两组数据比较具有统计学意义(P<0.01);用药后第2、4、9、14天治疗组荷瘤鼠外周血血小板计数与对照组小鼠比较差异无统计学意义(P>0.05).结论:15 mg/kg CTX可治愈多数荷膀胱癌T739小鼠.单剂量CTX治疗后6 h荷瘤小鼠外周血血小板计数的一过性增高可能与其抗肿瘤作用有关.     

Vaccination of mice with MUC1 cDNA suppresses the development of lung metastases

C57BL/6 mice were immunized intradermally with various doses of purified pCEP4 plasmid DNA containing full-length MUC1 cDNA (22 tandem repeats). Mice immunized with MUC1 DNA three times at weekly intervals had serum antibodies to a synthetic peptide corresponding to the tandem repeats of MUC1. The antibody titer correlated with the plasmid DNA dose. After the third immunization mice were injected intravenously with 5×10⁵ B16-F10 melanoma cells that had been stably transfected with MUC1 cDNA (F10-MUC1-C8 clone cells). The number of lung metastatic nodules three weeks after inoculation of F10-MUC1-C8 cells was significantly lower in mice immunized with MUC1 plasmid DNA than in mice immunized with the vector DNA alone. Thus, the suppression of lung metastasis was antigen-specific. In vivo depletion of lymphocyte subpopulations by specific antibodies revealed that natural killer cells are the major effector cells responsible for the suppression of lung metastasis. CD4⁺ cells and CD8⁺ cells apparently played some roles too. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of organ site and tumor cell type on MUC1-specific tumor immunity

We investigated the influence of organ-specific parameters on tolerance and immunity to human MUC1. C57Bl/6 mice (wild-type) and C57Bl/6 transgenic for MUC1 (MUC1.Tg) were challenged in the pancreas with Panc02-MUC1, a C57Bl/6-syngeneic pancreatic cancer cell line expressing human MUC1. Wild-type mice produced immune responses to MUC1 when presented on tumor cells growing in the pancreas; however, the responses to tumors in the pancreas were less effective than responses produced by tumor challenge at the s.c. site. Tumor immunity specific for MUC1 was produced in wild-type mice by two different procedures: (i) s.c. immunization of wild-type mice with a low dose of Panc02-MUC1 or (ii) adoptive transfer of spleen and lymph node cells harvested from wild-type mice previously immunized s.c. with Panc02-MUC1. This demonstrates that immune responses to MUC1 presented at the s.c. site can be detected and adoptively transferred. MUC1.Tg mice were immunologically tolerant to MUC1; however, some immunological protection against orthotopic challenge with Panc02-MUC1 was conferred by adoptive transfer of CD4+ and CD8+ T cells from wild-type mice. These results show that it is more difficult to produce immune responses to tumors growing at the pancreatic site than the s.c. site. Panc02-MUC1 cells growing in the pancreas were accessible to the immune system, and immune responses evoked by s.c. presentation of this molecule in wild-type mice were effective in rejecting tumor cells in the pancreas of both wild-type and MUC1.Tg mice. No effective anti-tumor immune responses against MUC1 were produced in MUC1.Tg mice.     

Potential for novel MUC1 glycopeptide-specific antibody in passive cancer immunotherapy

Abstract

MUC1 is an important target for antibodies in passive cancer immunotherapy. Antibodies against mucin glycans or mucin peptide backbone alone may give rise to cross reactivity with normal tissues. Therefore, attempts to identify antibodies against cancer-specific MUC1 glycopeptide epitopes havebeen made. We recently demonstrated that a monoclonal antibody against the immunodominant Tn-MUC1 (GalNAc-α-MUC1) antigen induced ADCC in breast cancer cell lines, suggesting the feasibility of targeting combined glycopeptide epitopes in future passive cancer immunotherapy.     

针对MUC1黏蛋白DC疫苗的研究进展

树突状细胞(DC)作为抗原递呈细胞在激活肿瘤特异性免疫中发挥重要作用,DC疫苗为肿瘤免疫治疗提供了一种有效手段.MUC1是一种高分子量糖蛋白,属于粘蛋白家族成员,在多种上皮性肿瘤中异常表达,是肿瘤免疫治疗的理想靶抗原.本文综述了MUC1的生物学特征、DC对MUC1的递呈和以MUC1为靶点DC疫苗的抗肿瘤效果.     

Dendritic cell vaccine immunotherapy of cancer targeting MUC1 mucin

The use of dendritic cells (DCs) for cancer vaccination is effective in suppressing cancer progression. This is because the DCs play a crucial role in priming tumor-specific immunity efficiently as antigen-presenting cells. In this study, we analyzed the ability of DCs to elicit tumor-specific immunity and clinical effects of DC vaccine immunotherapy targeting MUC1 tumor antigens. DCs from 14 patients with advanced or metastatic breast or lung cancer (9 positive for MUC1 and 5 negative for MUC1) were loaded with MUC1 antigens or tumor lysate and used for therapeutic vaccination. After vaccination, all the MUC1-positive patients acquired antigen-specific immunity whereas only 1 case with MUC1-negative cancer showed the specific immunity. Clinically, marked effects such as reduction in tumor sizes or tumor marker levels or disappearance of malignant pleural effusion were observed in 7 of the 9 MUC1-positive cases. However, MUC1-negative patients did not respond to DC vaccines, with the exception of 1 case with MAGE3-positive lung cancer. Survival of MUC1-positive patients was significantly prolonged in comparison with MUC1-negative patients (mean survival: 16.75 versus 3.80 months, p=0.0101). These data suggest that MUC1 is sufficiently immunogenic to elicit strong anti-tumor immunity as a tumor antigen and that DC vaccines targeting MUC1 are useful for immunotherapy of cancer.     

O-glycosylated versus non-glycosylated MUC1-derived peptides as potential targets for cytotoxic immunotherapy of carcinoma

Due to the fact that many cellular proteins are extensively glycosylated, processing and presentation mechanisms are expected to produce a pool of major histocompatibility complex (MHC) class I-bound protein-derived peptides, part of which retain sugar moieties. The immunogenic properties of the presented glycosylated peptides in comparison to their non-glycosylated counterparts have not been determined clearly. We assessed the cellular immunogenicity of MUC1 (mucin)-derived peptides O-glycosylated with a Tn epitope (GalNAc) using HLA-A*0201 single chain (HHD)-transfected cell lines and transgenic mice. For part of the compounds Tn moiety did not interfere with the HLA-A*0201 binding. Moreover, part of the glycopeptides elicited effective cytotoxic responses, indicating recognition of the glycopeptide-HLA-A*0201 complex by the T cell receptor (TCR) and subsequent cytotoxic T lymphocyte (CTL) activation. The CTLs exhibited a substantial degree of cross-reactivity against target cells loaded with glycosylated and non-glycosylated forms of the same peptide. The studied (glyco)peptides showed cellular immunogenicity in both MUC1-HHD and HHD mice and induced effective lysis of (glyco)peptide-loaded target cells in CTL assays. However, the elicited CTLs did not induce selective lysis of human MUC1-expressing murine cell lines. Moreover, immunization with (glyco)peptide-loaded dendritic cells (DCs) did not induce significant immunotherapeutic effects. We conclude that Tn glycosylated MUC1-derived peptides can be presented by MHC class I molecules, and may be recognized by specific TCR molecules resulting in cytotoxic immune responses. However, the studied glycopeptides did not offer significant benefit as targets for cytotoxic immune response due apparently to (a) cross-reactivity of the elicited CTLs against the glycosylated and non-glycosylated forms of the same peptide and (b) low abundance of glycopeptides on tumour target cells.     

Heat shock fusion protein induces both specific and nonspecific anti-tumor immunity

Mucin 1 (MUC1) is a tumor antigen, and the most important epitopes that can induce cytotoxic T lymphocytes (CTL) reside in the variable-number tandem repeats (VNTR). Heat shock protein (HSP) complexes isolated from tumors have been shown to induce specific anti-tumor immunity. HSP alone can also induce nonspecific immunity. To explore the possibility to utilize the specific anti-tumor immunity induced by MUC1 VNTR and the nonspecific immunity induced by HSP, we constructed a recombinant protein (HSP65-MUC1) by fusing Bacillus Calmette-Guérin-derived HSP65 with the MUC1 VNTR peptide and tested its ability to induce anti-tumor activities in a tumor challenge model. The growth of MUC1-expressing tumors was significantly inhibited in mice immunized with HSP65-MUC1, both before and after tumor challenge. A much larger percentage of immunized mice survived the tumor challenge than non-immunized mice. Correlating with the anti-tumor activity, HSP65-MUC1 was shown to induce MUC1-specific CTL as well as nonspecific anti-tumor immunity. In the human system, HSP65-MUC1-loaded human DC induced the generation of autologous MUC1-specific CTL in vitro. These results suggest that exogenously applied HSP65-MUC1 may be used to treat MUC1 tumors by inducing the epitope-specific CTL as well as nonspecific anti-tumor responses mediated by the HSP part of the fusion protein.     

The Tat‐conjugated N‐terminal region of mucin antigen 1 (MUC1) induces protective immunity against MUC1‐expressing tumours

Mucin antigen 1 (MUC1) is overexpressed on various human adenocarcinomas and haematological malignancies and has long been used as a target antigen for cancer immunotherapy. Most of the preclinical and clinical studies using MUC1 have used the tandem repeat region of MUC1, which could be presented by only a limited set of major histocompatibility complex haplotypes. Here, we evaluated N‐terminal region (2–147 amino acids) of MUC1 (MUC1‐N) for dendritic cell (DC)‐based cancer immunotherapy. We used Esherichia coli‐derived MUC1‐N that was fused to the protein transduction domain of human immunodeficiency virus Tat protein for three reasons. First, mature DCs do not phagocytose soluble protein antigens. Secondly, tumour cells express underglycosylated MUC1, which can generate epitopes repertoire that differs from normal cells, which express hyperglycosylated MUC1. Finally, aberrantly glycosylated MUC1 has been known to impair DC function. In our study, Tat‐MUC1‐N‐loaded DCs induced type 1 T cell responses as well as cytotoxic T lymphocytes efficiently. Furthermore, they could break tolerance in the transgenic breast tumour mouse model, where MUC1‐positive breast cancers grow spontaneously. Compared with DCs pulsed with unconjugated MUC1‐N, DCs loaded with Tat‐conjugated MUC1‐N could delay tumour growth more effectively in the transgenic tumour model as well as in the tumour injection model. These results suggest that the recombinant N‐terminal part of MUC1, which may provide a diverse epitope repertoire, could be utilized as an effective tumour antigen for DC‐based cancer immunotherapy.     

MUC1 mRNA体外负载树突状细胞诱导细胞毒性T细胞对非小细胞肺癌的杀伤作用

 目的：将体外扩增的黏蛋白1 （MUC1） mRNA转染入成熟的树突状细胞（DCs）,观察其体外诱导的特异性抗肿瘤效应。方法：将分离提纯的单核细胞培养诱导为DC并用流式细胞术鉴定。构建pcDNA3.1（+）-MUC1质粒,体外转录为mRNA,电穿孔法转染DCs。定量 PCR检测转染的DCs中MUC1的表达;MTT法检测T细胞增殖情况;流式细胞术检测CD8+ 在T细胞的表达;LDH释放法测定细胞毒性,ELISA检测IFN-γ分泌水平。结果：流式细胞术结果表明成熟DCs标志表型的表达明显高于对照组。定量PCR结果说明转染后的DCs MUC1 mRNA相对表达量增高。转染组DCs与T细胞按1∶10共培养时,刺激增殖能力明显高于未转染组,且CD8+  T细胞表达率高于未转染组,诱导产生特异性的细胞毒性T细胞杀伤表达MUC1蛋白的靶细胞,而未转染组的杀伤作用较弱。转染组DCs与T细胞共培养的上清中IFN-γ的分泌水平高于未转染组。结论：电穿孔法可以将MUC1 mRNA成功转染至DCs,产生特异性杀伤效应,为以MUC1为靶点的非小细胞肺癌的免疫治疗提供实验和理论依据。     

Identification of an HLA-A*0201-restrictive CTL epitope from MUC4 for applicable vaccine therapy

Recent research has indicated that MUC4 plays an important role in the development of many tumors and may prove useful as a novel cancer immunotherapy target. We aimed to identify HLA-A*0201-restrictive cytotoxic T lymphocyte (CTL) epitopes of the cancer-associated antigen MUC4. The MUC4 sequence was scanned for immunogenic peptides using HLA-binding prediction software. Dendritic cells (DCs) from peripheral blood mononuclear cells (PBMCs) were induced by cytokines. Five possible CTL epitopes were selected by software analysis, synthesized, and used to pulse mature DCs. The CD8⁺ T cells from PBMCs from an HLA-A*0201 healthy donor were stimulated with autologous MUC4-peptide-loaded DCs and expanded in vitro. T cell activation was assessed by ELISPOT, and cytotoxicity was determined by ⁵¹chromium (⁵¹Cr)-release assays. Our results show that CTLs induced by peptide P01204 could lyse T2 cells pulsed with peptide P01204 and HCT-116 cells (MUC4⁺, HLA-A2⁺). Compared with a control peptide, P01204 increased the number of IFN-γ producing T cells. Overall, these results suggest that P01204 is a novel HLA-A*0201-restrictive CTL epitope of the cancer-associated antigen MUC4. This will provide a foundation for the development of tumor-specific peptide vaccines.