【作者】 唐春莲；

【导师】 李雍龙；

【作者基本信息】 华中科技大学 ， 病原生物学， 2010， 博士

【摘要】 寄生虫能逃避宿主的免疫攻击而继续生存的现象称为免疫逃避,其机理为分子模拟与伪装、封闭抗体的产生等多种方式逃避宿主的体液和细胞免疫攻击,因而寄生虫能在宿主体内长期存在并引起慢性感染。新近证实,调节性T细胞与寄生虫逃避宿主免疫攻击有关,即“病原体可能会利用调节性T细胞当做逃生的窗口”,如利什曼原虫感染部位聚集了大量的调节性T细胞而有利于原虫的存活,若用抗体阻断调节性T细胞则有利于机体清除原虫。调节性T细胞最早于1995年由Sakaguchi等描述,主要包括自然性和诱导性调节性T细胞两种类型。诱导性调节性T细胞由外来抗原诱导产生,自然性调节性T细胞即CD4+CD25+调节性T细胞,来源于胸腺并维持于外周,组成性表达CD25、Foxp3、CTLA-4和GITR等分子。CD25分子即IL-2受体的α链,对于维持调节性T细胞增殖及功能具有重要作用,但CD25分子同时也表达于活化的效应细胞表面,因而不具有特异性。Foxp3为核转录因子,是调节性T细胞的特征性标志。CTLA-4(细胞毒性T淋巴细胞相关抗原4)是一种共抑制性受体,CTLA-4与机体的免疫耐受功能相关。CD4+CD25+调节性T细胞能抑制CD4+和CD8+效应T细胞的激活,其抑制功能是由调节性T细胞与效应细胞之间直接接触或者由调节性T细胞分泌抑制性细胞因子IL-10和TGF-β来实现的,单独及联合使用相应的阻断性抗体有利于机体清除寄生虫感染。日本血吸虫疫苗的研究经历了死疫苗、减毒活疫苗、基因工程疫苗和核酸疫苗的漫长过程,仍未取得较好的效果。WHO推荐的六种候选疫苗包括谷胱苷肽S转移酶均不能稳定地获得40%以上的保护力。既然CD4+CD25+调节性T细胞能抑制效应T细胞反应,因此推测日本血吸虫在宿主体内免疫逃避及日本血吸虫疫苗的免疫保护性效果不佳均可能与调节性T细胞有关。本文一方面探讨了日本血吸虫感染与调节性T细胞之间的关系,采用相应抗体阻断调节性T细胞,观察其对机体清除日本血吸虫感染能力的影响及其作用机制。另一方面探讨了日本血吸虫疫苗与调节性T细胞之间的关系,采用相应抗体阻断调节性T细胞观察其对日本血吸虫疫苗免疫保护性效果的影响及其作用机制。本课题分为以下二个部分：一、CD4+CD25+ Tregs在日本血吸虫免疫逃避中的作用及其机制研究目的：通过观察日本血吸虫感染对CD4+CD25+ Tregs的影响及使用anti-CD25 mAb对CD4+CD25+ Tregs的影响,了解CD4+CD25+ Tregs在日本血吸虫免疫逃避中的作用及其机制。方法：动物实验分为三个部分第一部分6～8周龄雌性BALB/c小鼠随机分成二组,即正常对照组和感染组,感染组每只小鼠感染日本血吸虫尾蚴40条,分别在感染后2周、3周、4周和5周剖杀小鼠,流式细胞术检测各组小鼠脾淋巴细胞中CD4+CD25+ Tregs的比例。第二部分6-8周龄雌性BALB／c小鼠随机分成二组,即正常对照组和anti-CD25 mAb组,抗体组腹腔注射300μg anti-CD25 mAb,对照组注射等体积的PBS,使用抗体后2周剖杀小鼠,无菌取脾,制备单个脾淋巴细胞悬液,流式细胞术检测各组小鼠脾淋巴细胞中CD4+CD25+ Tregs百分比。第三部分6-8周龄雌性BALB/c小鼠随机分成二组,即感染对照组和anti-CD25 mAb组。每只小鼠感染日本血吸虫尾蚴40条,感染后2周,抗体使用组每只小鼠腹腔注射300μg anti-CD25 mAb,感染对照组注射等体积的PBS。感染后6周剖杀各组小鼠。计数虫荷和每克肝脏内虫卵数,计算减虫率和每克肝脏组织中减卵率。无菌取各组小鼠脾脏,制备单个脾淋巴细胞悬液,收集脾细胞培养上清,夹心ELISA法检测脾细胞上清中细胞因子含量。结果：1)日本血吸虫感染后2周、3周、4周和5周,小鼠脾淋巴细胞中CD4+CD25+ Tregs百分比由感染前的1.99±0.10%分别上升至2.36±0.37%、2.8±0.05%、2.35±0.09%和2.68±0.05%。提示日本血吸虫感染可诱导机体CD4+CD25+ Tregs升高。2)正常对照组小鼠脾淋巴细胞中CD4+CD25+ Tregs百分比平均值为1.99±0.10%,使用anti-CD25 mAb后2周百分比平均值为0.20±0.05%,抗体组明显低于对照组(P<0.01)。提示anti-CD25 mAb可部分阻断CD4+CD25+ Tregs。3)使用anti-CD25 mAb组小鼠虫荷较未使用抗体组减少18.99%,每克肝脏虫卵数减少15.86%。提示CD4+CD25+ Tregs下降有利于宿主对血吸虫的清除。4)使用anti-CD25 mAb组小鼠脾细胞培养上清中细胞因子IFN-γ,、IL-5明显高于感染对照组。结论：Anti-CD25 mAb能部分阻断CD4+CD25+ Tregs,使用后有利于机体清除日本血吸虫。二、CD4+CD25+ Tregs对日本血吸虫GST疫苗保护性效果的影响及其作用机制目的：通过观察日本吸虫GST疫苗对CD4+CD25+ Tregs的影响及anti-CD25 mAb对CD4+CD25+ Tregs表达的影响,探讨CD4+CD25+ Tregs对GST疫苗免疫保护效果的影响及其作用机制。方法：雌性BALB／c小鼠随机分成五组,正常组、感染对照组、anti-CD25 mAb组、GST组以及anti-CD25 mAb与GST联合组。GST组和联合组小鼠背部皮下多点注射日本血吸虫GST疫苗50μg/鼠,每次间隔2周,共免疫三次,末次免疫后2周,每只小鼠感染日本血吸虫尾蚴40条,感染后2周,anti-CD25 mAb组和联合组小鼠腹腔注射anti-CD25 mAb 300μg/鼠,对照组注射等体积的PBS。分别在感染后2周、3周、4周和5周剖杀四组小鼠。无菌取各组小鼠脾脏,制备单个脾淋巴细胞悬液,流式细胞术检测脾淋巴细胞中CD4+CD25+ Tregs百分比；收集脾细胞培养上清,夹心ELISA法检测脾细胞培养上清中细胞因子含量。感染后5周剖杀的小鼠同时计数虫荷和每克肝脏虫卵数,计算减虫率和每克肝组织中减卵率。小鼠肝组织石蜡切片HE染色,观察各组小鼠虫卵肉芽肿的变化。结果：1)日本血吸虫感染后2周,感染对照组与GST组脾淋巴细胞中CD4+CD25+ Tregs百分比平均值分别为2.36±0.37%和3.36±0.06%；感染后3周,其百分比平均值分别为2.8±0.05%和2.97±0.08%；感染后4周,其百分比平均值分别为2.35±0.09%和2.47±0.09%；感染后5周,其百分比平均值分别为2.68±0.05%和3.03±±0.13%。提示GST疫苗能诱导机体CD4+CD25+ Tregs升高。2)使用anti-CD25 mAb后1周(即感染后3周),感染对照组与anti-CD25 mAb组小鼠脾淋巴细胞中CD4+CD25+ Tregs百分比平均值分别为2.8±0.05%和0.13±0.04%；使用anti-CD25 mAb后2周(即感染后4周),百分比平均值分别为2.35±0.09%和0.9±0.23%；使用anti-CD25 mAb后3周(即感染后5周),百分比平均值分别为2.68±0.05%和1.93±0.03%。提示使用anti-CD25 mAb后1周能部分封闭CD4+CD25+ Tregs,使用anti-CD25 mAb后2周、3周其百分比逐渐上升,但仍明显低于感染对照组。3)GST组小鼠对日本血吸虫减虫率及减卵率分别为24.98%和32%,而GST与anti-CD25 mAb联合组减虫率及减卵率分别为43.43%和49%。提示anti-CD25 mAb可作为佐剂增强GST疫苗的保护性效果。4)日本血吸虫感染早期以Thl型免疫反应为主,感染后4周雌虫排卵后以Th2型免疫反应为主,而使用anti-CD25 mAb后Th1型细胞因子IFN-y仍维持在较高水平,有利于机体清除日本血吸虫。提示anti-CD25 mAb增强GST疫苗免疫保护性效果的机制为增强机体内Thl型免疫反应。5)病理组织学检查显示各感染组单个虫卵肉芽肿直径及浸润细胞无明显差异。提示anti-CD25 mAb未明显增强小鼠肝脏虫卵肉芽肿的病理变化。结论：日本血吸虫GST疫苗能诱导CD4+CD25+ Tregs比例上升,因而会影响GST疫苗的保护性效果；anti-CD25 mAb能部分阻断CD4+CD25+ Tregs,同时增强Thl型免疫反应,因而能提高GST疫苗的免疫保护性效果,因此anti-CD25 mAb能作为佐剂增强GST疫苗的保护性效果。本研究结论如下：1)寄生虫的免疫逃避与CD4+CD25+ Tregs有关,CD4+CD25+ Tregs升高有利于血吸虫逃避宿主的免疫攻击。2)首次证明日本血吸虫GST疫苗的保护性效果与宿主的CD4+CD25+ Tregs有关,GST疫苗在感染背景下能诱导小鼠脾淋巴细胞中CD4+CD25+ Tregs升高,从而影响GST疫苗的保护性效果。3)首次证明anti-CD25 mAb可增强日本血吸虫GST疫苗的保护性效果,其作用机制是通过阻断CD4+CD25+ Tregs和增强Thl型免疫反应来实现的。4)首次提出anti-CD25 mAb可作为佐剂用来增强日本血吸虫GST疫苗的保护性效果。更多还原

【Abstract】 Pathogens can escape from the host immune assault by antigen mask and blockage antibody, and they can live in the host and result in chronic infection. Most recently, regulatory T cells have been reported to be involved in immune evasion of pathogens. Many parasites including Leishmania major can result in the clustering of regulatory T cells, which favors the pathogen to survive while the antibody blocking regulatory T cells contributes to parasite killing.Regulatory T cells were firstly described by Sakaguchi et al in 1995, which were divided into natural and induced regulatory T cells. Natural regulatory T cells are originated from thymus and maintain in periphery, which constitutively expressed markers of CD25, Foxp3, CTLA-4 and GITR. CD25 molecule, IL-2 receptor a band, is also expressed on the surface of activated effector cells, while Foxp3 is important for the suppressive function and is specific for CD4+CD25+ Tregs. CD4+CD25+ Tregs can suppress the activation of CD4+ and CD8+ effector T cells by direct contact with CD4+CD25+ Tregs and effector T cells or suppressive cytokines of IL-10 and TGF-0. CTLA-4 (cell cytotoxic T lymphocyte associated-antigen), a co-inhibitory receptor, is correlated with the host immune tolerance. Combination treatment of blockage antibody or treatment alone favors for the host clearing parasites.Studies on vaccines against Schistosoma japonicum (S. japonicum), including dead vaccine, attenuated live vaccine, genetically engineering and nucleic acid vaccine, have often yielded disappointing results. Gluthatione-S-transferase (GST) is one of the candidated vaccines against S. japonicum recommended by WHO, but not usually acquires 40% protective efficacy. Since CD4+CD25+ Tregs can suppress effector T cells, the poor protective efficacy of vaccine against S. japonicum may be related to CD4+CD25+ Tregs and blockade antibody can enhance the protective efficacy of vaccine.In this study, one hand, we explore the relationship between CD4+CD25+ Tregs and infection with S. japonicum and the effect and mechanism of blockade antibody on the protective efficacy against S. japonicum. On the other hand, we explore the relationship between GST vaccine against S. japonicum and CD4+CD25+ Tregs and the effect and mechanism of anti-CD25 mAb on the protective efficacy of GST vaccine against S. japonicum.This study was divided into two parts:Part 1:The effect and mechanism of CD4+CD25+ Tregs on the immune evasion of S. japonicum.Objective:To explore the effect and mechanism of CD4+CD25+ Tregs on the immune evasion of S. japonicum and the effect of S. japonicum infection and anti-CD25 mAb on the percentages of CD4+CD25+ Tregs.Methods:Animal experiments include three parts Part 1:BALB/c female mice were randomly divided into 2 groups:normal and infected group. Each mouse in infected group was infected with 40 S. japonicum cercarie. Mice in two groups were sacrificed at 2,3,4 and 5 weeks after infection respectively. Flow cytometry was performed to detect the percentage of CD4+CD25+ Tregs in spleen cells.Part 2:BALB/c female mice were randomly divided into 2 groups:normal, and anti-CD25 mAb group. Each mouse in anti-CD25 mAb group were injected intraperitoneally 300μg of anti-CD25 mAb and that of control group was injected equal volume of PBS. All mice were succumbed at 2 weeks after treatment. Spleens were obtained sterile and prepared single cell suspension. Flow cytometry was performed for detection of the frequency of CD4+CD25+ Tregs.Part 3:BALB/c female mice were randomly divided into 2 groups, infected control and anti-CD25 mAb group. Each mouse were infected 40 S. japonicum cercarie. Mice were injected intraperitoneally 300μg anti-CD25 mAb at 2 weeks after infection, and equal volume PBS as control. All mice were sacrificed at 6 weeks after infection to measure worm burden and eggs number in liver, reduction rate of worm and reduction rate of eggs per gram liver was evaluated. Spleens were obtained sterile and prepared single cell suspension. The levels of cytokines in spleen cell cultural suprernatant were detected by sandwich ELIS A after stimulation by ConA.Results:1) At 2,3,4 and 5 weeks after infection, the percentage of CD4+CD25+ Tregs were prompted from 1.99±0.10% to 2.36±0.37%,2.8±0.05%,2.35±0.09% and 2.68±0.05% respectively. It demonstrats that S. japonicum infection can induce the expansion of CD4+CD25+ Tregs.2) The percentage of CD4+CD25+ Tregs in normal group was 1.90±0.10%, which was 0.20±0.05% at 2 weeks post-CD25 mAb administration. It demonstrats that anti-CD25 mAb can partially block CD4+CD25+ Tregs.3) The reduction rate of worm burden at 3 weeks post-anti-CD25 mAb was 18.99% and the reduction rate of eggs of per gram liver was 15.86%. It demonstrates that the reduction of CD4+CD25+ Tregs favors the clearance of S. japonicum in the host body. 4) The levels of IFN-γand IL-5 were increased by anti-CD25 mAb administration.Conclusion:Anti-CD25 mAb favors the host to clear S. japonicum by reducing CD4+CD25+ Tregs.Part 2:The effect and mechanism of CD4+CD25+ Tregs on the protective efficacy of GST vaccine against S. japonicumObjective:To explore the effect and mechanism of CD4+CD25+ Tregs on the protective efficacy of GST vaccine against S. japonicom by the effect of vaccine GST and anti-CD25 mAb on the expression of CD4+CD25+ Tregs.Methods:BALB/c female mice were randomly divided into 5 groups:normal group, infected control group, anti-CD25 mAb group, GST group and co-treated group with anti-CD25 mAb and GST. Each mouse in GST group and co-treated group was injected 50μg of GST each mouse at 2-week interval,3 times immunization totally. At 2 weeks after the last immunization, each mouse was infected with 40 S. japonicum cercarie. Each mouse in anti-CD25 mAb and co-treated group were injected intraperitoneally 300μg of anti-CD25 mAb at 2 weeks after infection and equal volume of PBS was as control. Four groups six each were sacrificed at 2,3,4 and 5 weeks after infection respectively. Flow cytometry was performed to detect the percentage of CD4+CD25+ Tregs. The levels of cytokines in spleen cell cultural supernatant were detected by sandwich ELISA. Worm burden and eggs in per gram liver were valuated at 5 weeks after infection. Liver tissues were stained with HE to detect egg granuloma.Results:1) The percentages of CD4+CD25+ Tregs in infected control and GST group were 2.36±0.37% and 3.36±0.06% at 2 weeks after infection respectively; The percentages of CD4+CD25+ Tregs in infected control and GST group were 2.8±0.05% and 2.97±0.08% at 3 weeks after infection; 2.35±.09% and 2.47±0.09% at 4 weeks after infection; 2.68±0.05% and 3.03±0.13% at 5 weeks after infection respectively. It demonstrates that GST vaccine can induce the expansion of CD4+CD25+ Tregs.2) The percentages of CD4+CD25+ Tregs were decreased at 1 week post-anti-CD25 mAb (3 weeks after infection) from 2.8±0.05% to 0.13±0.04%. The percentages of CD4+CD25+ Tregs in infected control and anti-CD25 mAb group were 2.35±0.09% and 0.9±0.23% at 2 weeks post-anti-CD25 mAb administration (4 weeks after infection); 2.68±0.05% and 1.93±0.03% at 3 weeks post-anti-CD25 mAb administration (5 weeks after infection) respectively. It demonstrates that CD4+CD25+ Tregs can be blocked at 1 week after anti-CD25 mAb administration, which was partially restored but still lower than those in infected control group at 2 and 3 weeks post-anti-CD25 mAb adminstration.3) The reduction rate of worm burden and eggs per gram liver in GST group was 24.98% and 32%, which were prompted to 43.43% and 49% in co-treated group respectively. It demonstrates that anti-CD25 mAb can enhance the protective efficacy of GST vaccine as an adjuvant.4) Thl type immune response is predominant in the early stage of S. japonicum infection, which shifts to Th2 immune response at 4 weeks after infection. Thl type cytokine IFN-y was still sustain at higher level by the administration of anti-CD25 mAb and favored the host to clear S. japonicum. It demonstrates that anti-CD25 mAb enhances the protective efficacy of GST vaccine by increasing Thl type immune response.5) Pathogenical analysis denoted that there were no significant differences in the size and infiltrating cells of egg granuloma in each infected group. It demonstrates that anti-CD25 mAb can not enhance the pathogenical injury.Conclusion:GST vaccine against S. japonicum can induce the frequency of CD4+CD25+ Tregs which influence the protective efficacy of GST; anti-CD25 mAb can partially block CD4+CD25+ Tregs and enhance Thl type immune response which can enhance the protective efficacy of GST. So anti-CD25 mAb can act as an adjuvant to enhance the protective efficacy of GST vaccine against S.japoncium.In conclusion, this study demonstrated:1) CD4+CD25+ Tregs are involved in parasites immune evasion, and the expansion of CD4+CD25+ Tregs favors parasites escaping from the host immune assault.2) It is the first time that GST vaccine against S. japonicum can induce CD4+CD25+ Tregs on the infected background and CD4+CD25+ Tregs are involved in the poor protective efficacy of GST vaccine.3) It is the first time that anti-CD25 mAb can enhance the protective efficacy of vaccine GST against S. japonicum by blocking CD4+CD25+ Tregs and enhancement Thl type immune response.4) It is the first time that anti-CD25 mAb can as an adjuvant to enhance the protective efficacy of GST vaccine against S. japoncium.更多还原