PcDNA3.0/p27~(kip1)真核表达质粒对小鼠哮喘模型Th1/Th2的影响

目的:探讨细胞周期负性调控蛋白PcDNA3.0/p27kip1重组真核表达质粒转染小鼠哮喘模型对Th1/Th2比例失衡的调节作用。方法:小鼠各10只分为A组(正常对照组)、B组(哮喘模型组)、C组(质粒治疗组)三组。用卵蛋白致敏法构建小鼠哮喘模型,以本实验室构建的PcDNA3.0/p27kip1重组真核表达质粒经静脉转染小鼠哮喘模型,36小时后Western blot检测肺组织中p27蛋白表达量;流式细胞仪分析小鼠脾CD4+、CD8+T细胞比例;ELISA检测小鼠血清IL-4、IL-5、IL-13、IFN-γ水平。结果:B组与A组比较,哮喘发病后肺p27蛋白表达量明显降低;脾CD4+T细胞显著增高而CD8+T细胞显著降低;血清IL-4、IL-5、IL-13水平明显升高而IFN-γ明显降低。C组与B组比较,肺p27蛋白表达量明显增加,脾CD4+T细胞比例显著降低而CD8+T细胞显著升高;血清IL-4、IL-5、IL-13水平明显降低而IFN-γ明显升高。结论:PcDNA3.0/p27kip1静脉给药可在哮喘模型肺组织中表达,并通过基因干预调控细胞周期的方式调节Th1/Th2比例失衡,为哮喘防治提出了新思路。     

基因工程鼠Muc1-MBP融合蛋白的制备及其免疫活性测定

目的:制备鼠Muc1-MBP融合蛋白,探讨其免疫活性.方法:将pMAL-Muc1转化大肠杆菌,通过IFTG诱导、SDS-PAGE和Western blot鉴定Mucl的表达,Amylose resin亲和层析纯化Muc1-MBP.将Muc1-MBP融合蛋白免疫小鼠,采用ELISA方法检测小鼠血清Muc1特异性抗体的水平;MTT法测定小鼠抗Muc1特异的CTL活性;ELISPOT法测定脾脏淋巴细胞IFN-γ的分泌.结果:获得稳定表达Muc1的菌株,制备了分子量67 kD较纯的Muc1-MBP融合蛋白.Muc1-MBP融合蛋白免疫小鼠产生高效价Muc1特异抗体,效价在8 000~16 000之间,Muc1-MBP融合蛋白可诱导CTL杀伤活性,对人乳腺癌MCF-7和小鼠Lewis肺癌LLC1靶细胞的杀伤率分别为(74.5±5.9)%和(60.5±10.4)%,与对照组比P<0.05,差异显著;Mucl.MBP融合蛋白免疫可诱导脾脏淋巴细胞分泌IFN-γ,特异性活化Th1细胞.结论:成功制备莺组鼠Muc1-MBP融合蛋白,其不仅能诱导特异体液免疫应答,而且可诱导细胞免疫应答,尤其能诱导对人类MCF-7细胞较小鼠lewis肺癌LLC1细胞更加强烈的CTL杀伤活性,提示异种同源蛋白疫苗具有比同源蛋白疫苗更好的抗肿瘤作用.     

呼吸道合胞病毒G蛋白基因重组质粒诱导小鼠免疫保护性及机制研究

目的 构建编码呼吸道合胞病毒(RSV)G蛋白基因的重组质粒,观察其对RSV感染小鼠的保护性及特异性免疫效应,为研制安全有效的RSV新疫苗提供线索和实验依据.方法 利用基因重组方法构建含RSV G蛋白编码基因的重组质粒,测序和酶切鉴定,Western blot检测目的 基因经体外表达后的免疫原性.重组质粒免疫小鼠后以RSV感染,病毒滴定法检测肺组织标本中RSV滴度,HE染色法检查肺组织病理改变,ELISA检测血清抗体水平,双抗体夹心ELISA测定肺泡灌洗液(BALF)内Th1/Th2细胞因子表达量,流式细胞术检测BALF中T淋巴细胞亚群数量及活化状态.结果 成功构建了编码RSV G蛋白基因的重组质粒pcDNA3.1~G.Western blot证实目的 蛋白在体外具有免疫原性.被免疫小鼠感染RSV后肺组织病毒滴度降低,肺组织中未见明显的炎性细胞浸润.小鼠血清中产生较高滴度的抗RSV-G IgG.小鼠BALF细胞中CD4~+CD25~+T淋巴细胞比例显著增加,并且IFN-γ(Th1)、IL-4(Th2)两类细胞因子表达显示平衡.结论 编码RSV G蛋白基因的重组质粒pcDNA3.1~G能诱导产生CD4~+ CD25~+T细胞亚群,对小鼠具有明显的保护作用.     

铜绿假单胞菌重组Bb-OprF疫苗诱导小鼠细胞免疫应答的研究

目的研究铜绿假单胞菌重组双歧杆菌(rBb)-OprF疫苗免疫小鼠,PAO1株攻击后产生的细胞免疫应答。方法将rBb-OprF疫苗分别采用皮下注射、肌肉注射、鼻腔黏膜和口服灌胃4种途径免疫Balb/c小鼠,免疫后8周用5×106CFU的PAO1株攻击,攻击1周后杀鼠取脾,MTT法检测特异性脾淋巴细胞增殖,流式细胞术检测脾CD4+T和CD8+T细胞比率,用ELISA法检测脾细胞培养上清IFN-γ、IL-12、TNF-α和IL-10的水平。结果脾T淋巴细胞增殖明显;脾细胞CD4+和CD8+T细胞亚群显著增加;脾细胞培养上清IFN-γ、IL-12、TNF-α和IL-10的水平显著升高,其中IFN-γ最为显著。结论铜绿假单胞菌rBb-OprF疫苗可诱导小鼠产生混合型的Th1和Th2免疫应答。     

细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗诱导Balb/c小鼠免疫应答的动态观察

目的动态观察细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗免疫Balb/c小鼠后诱导的免疫应答。方法热絮凝法提取转基因苜蓿的叶蛋白,再用无菌双蒸水将叶蛋白提取液的浓度配制成20μg/μl。88只Balb/c小鼠随机分为2组,分别用100μl灌胃和10μl滴鼻免疫小鼠,每3天1次,连续免疫2个月。在末次免疫后0、2、4、6、8、10、12、14、16、18和20周各组随机剖杀4只小鼠,眼球取血,常规酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测血清中IgG及其亚类和IgE水平;取脾,分离脾细胞,流式细胞仪(flow cytometr,FCM)检测脾CD4+和CD8+T淋巴细胞亚群的百分比,体外经脾细胞悬液或加入Eg粗抗原(EgAg)、伴刀豆球蛋白A(ConA)或脂多糖(LPS)刺激培养,四甲基偶氮唑盐比色法(MTT法)检测免疫鼠脾T淋巴细胞增殖情况,ELISA法检测脾细胞培养上清液中IL-12、IL-10、IFN-γ和TNF-α水平。结果在末次免疫后4～6周,2组免疫小鼠的血清IgG及其亚类和IgE水平升高,脾T淋巴细胞增殖水平升高,CD4+和CD8+T细胞亚群百分比分别升高,脾细胞培养上清液中IL-12、IFN-γ、TNF-α和IL-10水平分别升高。结论细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗在免疫早期(4～6周)可诱导免疫鼠脾T淋巴细胞增殖,产生Th1和Th2混合型免疫应答,CD4+和CD8+T细胞亚群在细粒棘球绦虫转Eg95-EgA31融合基因苜蓿疫苗诱导的保护性免疫机制中起重要作用。     

肾怡对狼疮小鼠免疫器官内Th1/Th2细胞比例的调节作用

目的：探讨复方中药肾怡对MRL/lpr小鼠免疫器官内Th1/Th2细胞比例的调节作用。方法：将MRL/lpr狼疮小鼠随机分成对照组和中药治疗组，从12周观察到28周后于实验终点处死小鼠。通过流式细胞术分别检测胸腺和脾脏中CD4^ CD30^-(Th1)、CD4^ CD30^ (Th2)淋巴细胞所占百分比，进一步对脾细胞进行CD^ T淋巴细胞分选，并于体外利用PHA—P进行刺激后，通过RT—PCR方法检测IL-4(Th2细胞因子)和IFN—γ(Th1细胞因子)的表达丰度，比较二者的比值在对照组和中药治疗组之间的差别。结果：胸腺中几乎检测不到CD4^ CD30^ T淋巴细胞；脾脏中虽可检测到CD4^ CD30^ T淋巴细胞，但在未加入PHA-P的条件下，CD4^ CD30^ T淋巴细胞在对照组和中药治疗组中所占的比例没有显著差异；经PHAP刺激后对照组和治疗组脾细胞中CD4^ CD30^ T淋巴细胞所占百分比均明显增加，但治疗组中CD4^ CD30^ T淋巴细胞所占百分比的增加程度明显低于对照组；将脾细胞中CD4^ T淋巴细胞分选出来后进行RT-PCR检测，结果显示中药治疗组CD^ T淋巴细胞所表达IL-4／IFN—γ(Th2／Th1)比值明显低于对照组。结论：复方中药肾怡能够纠正MRL/lpr狼疮小鼠脾脏中CD^ T淋巴细胞在PHA-P刺激的条件下朝向Th2过度分化的倾向性。     

雷公藤内酯醇治疗实验性自身免疫性脑脊髓炎的免疫调节机制研究

目的:探讨雷公藤内酯醇治疗实验性自身免疫性脑脊髓炎(EAE)的免疫调节机制。方法:用MOG35-55肽段免疫C57/BL6小鼠,建立EAE动物模型。免疫后将小鼠随机分为治疗组和对照组,治疗组在开始发病后每天给予雷公藤内酯醇(100μg/kg)治疗,对照组于同一天开始每天给予相同体积生理盐水,每天观察小鼠临床症状。疾病高峰期处死小鼠,HE染色检测脊髓炎性细胞浸润;ELISA检测血清中细胞因子含量,3H掺入法检测淋巴细胞增殖;FACS检测中枢神经系统中Th1/Th17和Treg细胞数量。结果:雷公藤内酯醇治疗能够缓解EAE发病,减轻中枢神经系统炎性细胞浸润;抑制EAE小鼠MOG特异性T细胞增殖;减少血清中炎症细胞因子含量;减少病灶处CD4+T细胞IFNγ-和IL-17的分泌;上调CD4+T细胞Foxp3的表达。结论:雷公藤内酯醇通过减少致病细胞Th1和Th17的浸润,增加Treg细胞的数量来治疗EAE。     

口服免疫Ag85A脂质体DNA诱导T细胞亚群应答效应研究

目的:拟采用构建的可在真核细胞内表达Ag85A基因的质粒,以阳离子脂质体为运载体,制成DNA疫苗,经口途径投予小鼠,以观察Ag85A脂质体DNA疫苗诱导免疫应答效应,为口服DNA疫苗的临床应用提供理论和实验依据.方法:ELISA方法检测Ag85A特异性抗体产生水平及血清Th1型细胞因子IFN-γ及Th2型细胞因子IL-4的分泌水平,ELISPOT技术检测口服DNA疫苗后小鼠可分泌IFN-γ和IL-4脾淋巴细胞数量.流式细胞术观察口服DNA疫苗后小鼠脾淋巴细胞CD4+T细胞及CD8+T细胞亚群的变化,从而判断口服DNA疫苗的免疫效果及脂质体是否有免疫增强作用.结果:口服自制Ag85ADNA疫苗可见血清中抗Ag85A特异性抗体的产生;下调了脾CD4+T细胞和CD8+T细胞亚群的数量;分泌IFN-γ的Th1型细胞比率和血清中的IFN-γ水平下降,而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高.口服DNA疫苗组诱导Ag85A特异性抗体产生,脂质体具有免疫佐剂作用.结论:应用阳离子脂质体为运载体,重组Ag85A DNA疫苗口服免疫C57BL/6小鼠,诱导了CD4+及CD8+T细胞亚群的下调及IFN-γ的表达减少,而玎IL-4的分泌呈增高趋势;疫苗可诱导Ag85A特异性抗体的分泌,产生了明显的体液应答.     

八肽胆囊收缩素对经钥孔戚血蓝蛋白免疫小鼠T淋巴细胞亚群的影响

目的：探讨八肽胆囊收缩素（CCK-8）对经钥孔戚血蓝蛋白（KLH）免疫小鼠T淋巴细胞亚群的影响。方法：雌性BALB/c小鼠KLH免疫同时分别给予不同剂量CCK-8。流式细胞法检测小鼠外周血及脾细胞中CD4+、CD8+T细胞阳性百分率；RT-PCR法检测脾细胞中Th1型细胞因子IFN-γ、Th2型细胞因子IL-4 mRNA表达；ELISA法检测其培养上清中IFN-γ、IL-4水平；HE染色观察小鼠肺组织病理变化。结果：CCK-8下调KLH免疫小鼠外周血及脾细胞中上升的CD4+、CD8+T细胞阳性百分率，降低CD4+/CD8+比值；进一步提高其IFN-γ mRNA表达和培养上清中IFN-γ分泌量，同时下调上升的IL-4 mRNA表达和培养上清中IL-4分泌量；减轻KLH免疫所致小鼠肺部炎症。结论：CCK-8可调节适应性免疫应答，抑制T细胞尤其是CD4+T细胞活性；抑制Th2功能，提高Th1功能，因此可能在变态反应性疾病的发病和防治中具有一定作用。     

减毒鼠伤寒沙门氏菌运送CD8+T细胞表位的细胞免疫应答

目的： 探索减毒沙门氏菌运送CD8＋ T 细胞表位诱导机体产生特异性细胞免疫应答的规律性.方法： 通过构建融合表达OVA 257～264aa和LCMV NP 118～132aa CD8＋ T 细胞表位的原核表达质粒ptG2F, 以电穿孔法转化减毒鼠伤寒沙门氏菌SL7207, 筛选重组菌SL7207（ptG2F）.采用静脉注射免疫C57BL/6和BALB/c小鼠, 间隔2 wk, 分别于第2次和第3次免疫后, 取免疫小鼠脾细胞, 用ELISPOT法检测特异性IFN-γ分泌细胞和IL- 4分泌细胞.结果： 携带CD8＋ T细胞表位的重组菌SL7207（ptG2F）能诱导产生细胞免疫应答.在提呈OVA CD8＋ T细胞表位时, 2次免疫后, 诱导产生的细胞免疫应答趋向于Th1; 而在3次免疫后, 呈现Th1/Th2的平衡转换.在提呈LCMV NP CD8＋ T细胞表位过程中, Th2免疫应答水平高于Th1, 且有增强趋势.结论： 减毒沙门氏菌可以有效运送CD8＋ T细胞表位并诱导产生特异细胞免疫应答, 这为减毒细菌作为运送载体的研究提供了参考依据.     

检测外周血MUC1蛋白双抗体夹心ELISA方法的建立及初步应用

目的为提高外周血MUC1蛋白检测的敏感度,建立了抗MUC1多克隆抗体的夹心ELISA试剂盒,并对其进行了初步测试。方法首先成功构建-表达并纯化了MUC1-GST和MUC1-MBP融和蛋白;通过免疫家兔和大鼠,获得抗MUC1血清,经饱和硫酸铵沉淀、Protein A/G纯化及抗GST和MBP抗体吸收的进一步纯化获得纯的家兔抗人及大鼠抗人MUC1多克隆抗体;通过凝血酶溶解MUC1-GST获得MUC1标准品。经不同的筛选确立了以家兔抗人MUC1抗体作为包被抗体、大鼠抗MUC1抗体作为检测抗体的双抗体夹心试剂盒,敏感度可达到0.2 ng/ml。结果对32例乳腺癌患者和20例健康受试者外周血血清中MUC1蛋白水平的检测结果显示,乳腺癌患者的阳性检出率达到53.1%(17例/32例),而健康对照组检出率为0。结论本研究建立的抗MUC1多克隆抗体双夹心试剂盒有望应用于临床诊断。     

Cytokine production from murine CD4 and CD8 cells after mannan-MUC1 immunization.

Immunotherapy with oxidized mannan-MUC1 fusion protein (M-FP) leads to a T1 immune response characterized by the generation of cytotoxic T lymphocytes (CTL), few antibodies, secretion of interleukin-2 (IL-2), IL-12, and interferon-gamma and tumor protection. Immunotherapy with reduced M-FP or fusion protein (FP) alone leads to a T2 immune response characterized by the generation of MUC1 antibodies, few CTL, IL-4 secretion, and no tumor protection. In these studies, cytokine production from T cells was measured from cultures containing whole spleens. We now report the cytokine secretion patterns from spleen cells separated into CD4+ and CD8+ T cells obtained from mice immunized with either oxidized M-FP, reduced M-FP or FP, or the simultaneous administration of oxidized M-FP and FP. Immunization with oxidized M-FP led to the secretion of T1 cytokines from CD8+ T cells (IL-2, IFN-gamma, and tumor necrosis factor-alpha [TNF-alpha]) and from CD4+ T cells (IL-2 and IFN-gamma). IL-12 production, presumably from activated macrophages, was observed in CD8+ but not CD4+ cultures. Immunization with either reduced M-FP or FP led to the secretion of predominantly T2 cytokines from CD4+ T cells (IL-4 and IL-10) and IL-2 production in both CD4+ and CD8+ T cell cultures. The simultaneous immunization of both oxidized M-FP and FP led to the production of both T1 and T2 cytokines from CD8+ T cells (IL-2, IFN-gamma, and TNF-alpha) and CD4+ cells (IL-2, IFN-gamma, IL-4, and IL-10) and IL-12 production in CD8+ cultures that is, both types of immune responses could occur together. The results demonstrate that the cellular immune response observed in oxidized M-FP-immunized mice is indeed dependent on the T1 cytokine profile secreted by CD8+ T cells, and the simultaneous production of both T1 and T2 cytokines is not cross-inhibitory.     

日本血吸虫重组28GST T细胞表位谱的预测及鉴定

目的 :用软件预测日本血吸虫重组 2 8GST抗原分子T细胞表位谱 ,并鉴定其Th1型细胞表位。方法 :用软件预测重组2 8GSTT细胞表位谱 ,并筛选几种较好的T细胞表位 ,人工合成表位肽或用基因工程制备重组表位肽融合蛋白。体外刺激经照射的尾蚴感染并用 2 8GST加强免疫的C5 7BL/ 6小鼠 (H 2 b)脾细胞 ,通过淋巴细胞增殖试验、ELISA及流式细胞术等 ,分析各种表位的免疫刺激作用 ,鉴定Th1型细胞表位。结果 :在 9个候选的表位中 ,P6 (73～ 86aa)是刺激作用最强的Th1型细胞表位。结论 :重组 2 8GST含有功能性Th1型细胞表位     

双抗体间接夹心ELISA法检测乳腺癌患者外周血MUC1蛋白水平的评价

目的优化双抗体间接夹心ELISA试剂盒,并探讨其在乳腺癌患者中检测MUC1黏蛋白水平的应用价值。方法用基因重组MUC1-GST和MUC1-MBP融和蛋白免疫家兔和大鼠,获得抗MUC1血清,并对其纯化,获得纯化的家兔抗人及大鼠抗人MUC1多克隆抗体;经不同的筛选确立了以家兔抗人MUC1抗体作为包被抗体、大鼠抗人MUC1抗体作为检测抗体的双抗体间接夹心试剂盒,敏感度可达到0.2 ng/ml。结果应用建立的试剂盒对40例乳腺癌,18例乳腺良性疾病和120健康对照者血清中MUC1蛋白水平的进行检测,检测结果绘制ROC曲线,分析得出以2.75 ng/ml为乳腺癌患者与乳腺良性疾病患者的临界值,以1.86 ng/ml为乳腺疾病与正常人为临界值,检测结果表明本研究对乳腺癌诊断的阳性率高达97.5%,乳腺良性疾病的阳性率为66.7%,正常人特异性为96.7%。对于乳腺癌同一病例样本用酶联免疫法CA15-3诊断试剂盒进行对比检测,其检出率为3.33%,特异度为100%。绘制ROC曲线对比显示,本研究所建立的双抗体夹心ELISA方法对乳腺癌诊断的准确度明显高于CA15-3试剂盒。结论本研究成功建立了特异性强,灵敏度良好的双抗体间接夹心ELISA试剂盒,有望开发为临床辅助诊断的常规试剂盒,尤其有望应用于乳腺癌的大规模筛查及早期诊断。     

Deviation of the allergic IgE to an IgG response by gene immunotherapy

The Th1/Th2 type immune response to E. coli beta-galactosidase (beta-gal) was compared to that to gene vaccination with plasmid (p) DNA encoding beta-gal. BALB/c mice were immunized with beta-gal in alum or a pDNA construct consisting of a CMV-based promoter and the beta-gal gene (pCMV-LacZ). Beta-gal in alum induced IgG1 and IgE antibodies and the CD4+ T cells from these mice secreted interleukin 4 (IL-4) and IL-5 but no interferon-gamma (IFN-gamma) after in vitro antigen stimulation. In contrast, mice immunized with pCMV-LacZ formed predominantly IgG2a antibodies and their CD4+ T cells secreted IFN-gamma but no IL-4 and IL-5. These data indicate that beta-gal induced a Th2 and the pCMV-LacZ a Th1 response to beta-gal. The pDNA induced Th1 response dominated over the Th2 response. Mice primed with pCMV-LacZ failed to produce IgE antibodies after a booster injection of beta-gal in alum. Boosting of mice primed with beta-gal in alum with pCMV-LacZ resulted in a 75% decrease in the IgE antibody titer within 6 weeks and IgG2a antibody formation and CD4+ T cells that secreted IFN-gamma in amounts similar to T cells from pDNA primed mice. As shown by adoptive cell transfer, both CD4+ and CD8+ T cells from pDNA immunized mice inhibited an IgE response to beta-gal in alum in the recipient mice. pDNA immunization also inhibited the eosinophilic infiltration of the lung of ovalbumin (OVA) immunized mice after OVA inhalation challenge in an animal model of the late phase reaction. The mechanism of the pDNA induced Th1 immune response was shown to be the result of stimulation by distinct non-coding immunostimulatory DNA sequences (ISS) in the backbone of the pDNA. The ISS induced antigen presenting cells to secrete cytokines that cause naive T cells to differentiate into Th1 cells (e.g. IFN-alpha, IL-12). The data indicate that gene vaccination induces a Th1 immune response that is capable of down-regulating a preexisting Th2 response and IgE antibody formation. Thus, immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.     

MUC1/Y胞外段在大肠杆菌中的表达和鉴定

目的：获得MUC1／Y胞外段重组蛋白，研究其生物学功能，为肿瘤治疗提供实验依据。方法：利用RT—PCR从MCF7细胞中获得MUC1／Y胞外段编码基因，将其克隆到原核表达载体pET-32a中，并在BL21（DE3）大肠杆菌中进行表达；以亲和层析法对MUC1／Y重组蛋白进行纯化；利用纯化的MUC1／Y蛋白免疫家兔制备MUC1／Y多克隆抗体，然后对乳腺癌组织进行组化染色。结果：在大肠杆菌BL21（DE3）中成功表达了分子量为30000的Trx—MUC1／Y融合蛋白，经镍亲和层析一步纯化所获得的蛋白质纯度〉90％，用Trx-MUC1／Y融合蛋白免疫家兔获得的抗血清对乳腺癌组织的初步组化检测证明MUC1／Y融合蛋白具有很好的生物学活性。结论：成功表达并纯化了具有生物学活性的MUC1／Y胞外段重组蛋白。     

Delivery of antigen in allogeneic cells preferentially generates CD(4+) Th1 cells

We have examined the possibility of evoking antigen-specific T cell immune response by using allogeneic cells as a source of adjuvant and also as a vehicle to deliver antigen. The mice were immunized with different preparations of antigen-pulsed allogeneic and syngeneic splenocytes. It was observed during the study that the animals immunized with antigen-pulsed mitomycin C treated allogeneic cells elicited antigen specific CD(4+) Th1 cell response. Predominant release of IL-2, interferon (IFN)-gamma and IgG2a-isotype also occurred. In contrast, mice immunized with antigen-pulsed syngeneic cells chiefly enhanced the production of interleukin (IL)-4 and IgG1-isotype. Further, allogeneic macrophages induced better T cell response than B cells or splenocytes and prominently induced the expression of B7-1 and B7-2. Immunization with antigen-pulsed macrophages provided better recall responses compared to B cells. This was manifested by the high LFA-1alpha and low CD45RB expression on T cells. Because it is already known that mitomycin C-treated cells undergo apoptosis and dendritic cells engulf apoptotic cells, we therefore propose that generation of T cell response using antigen-pulsed allogeneic cells may be due to the engulfment of these cells by dendritic cells, which may then process and present antigen entrapped in allogeneic cells to activate naive CD(4+) T cells and differentiate them to Th1 cells. This study therefore provides a rational basis for manipulating antigen-specific responses by immunizing with antigen-pulsed allogeneic cells.     

T cells expressing the Vgamma1 T-cell receptor enhance virus-neutralizing antibody response during coxsackievirus B3 infection of BALB/c mice: differences in male and female mice

Coxsackievirus B3 infection causes severe cardiac inflammation in male but not female mice. CD3+ T cells and T cells expressing the Vgamma4 T cell receptor (TCR) predominate in the cardiac inflammatory cell infiltrate in infected male BALB/c mice. Infected females have mostly CD19+ (B lymphocyte) and Vgamma1+ cells. No significant differences in CD11b+ (monocyte) cells were observed between the sexes. Infected males showed a predominant CD4+Th1 (IFNgamma+) response, whereas females showed a predominant CD4+Th2 response. The importance of IFNgamma for myocarditis susceptibility and IL-4 for protection was confirmed using IFN-gamma-/- and IL-4-/- mice. Antibody depletion of Vgamma1+ cells augmented myocarditis susceptibility, whereas antibody depletion of Vgamma4+ cells was protective. Cardiac virus titers inversely correlated with virus neutralizing antibodies and showed that Vgamma1+ cells are important for virus neutralizing antibody response. IFNgamma affected the Vgamma4+ cell response in the heart, as IFNgamma-/- mice had few Vgamma4+ cells; but exogenous administration of recombinant IFNgamma to IFNgamma-/- mice restored myocarditis susceptibility, Th1 bias, and Vgamma4+ cell infiltration of the myocardium. These results demonstrate that two gammadelta+ T cell populations, Vgamma1+ and Vgamma4+, have different functions during myocarditis, in that Vgamma1+ cells promote humoral immunity and protection whereas Vgamma4+ cells are pathogenic.     

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.