调节性T细胞对日本血吸虫GST疫苗保护性效果的影响及机制研究

发布时间：2018-07-07 19:54

本文选题：日本血吸虫 + GST疫苗　；参考：《华中科技大学》2010年博士论文

【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(P0.01)。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。
[Abstract]:Parasites can escape the host's immune attacks and continue to survive the phenomenon known as immune escape, its mechanism is a variety of ways to escape the host's humoral and cellular immune attacks, such as molecular simulation and camouflage, the production of closed antibodies and so on. Thus parasites can exist in the host for a long time and cause chronic infection. Recently, regulatory T cells are newly confirmed to be sent to the host. The parasite escapes from the host immune attack, that is, "pathogens may use regulatory T cells as escape windows", such as Leishmania infecting sites that accumulate a large number of regulatory T cells to help protozoa survival. If the antibodies are used to block regulatory T cells, it is beneficial to remove the protozoa.
As early as in 1995, regulatory T cells were described by Sakaguchi, including two types of natural and inducible regulatory T cells. Inducible regulatory T cells were induced by external antigens, and natural regulatory T cells, CD4+CD25+ regulated T cells, derived from the thymus and maintained in the peripheral, expressed CD25, Foxp3, CTLA-4 and GITR, and so on. The molecular.CD25 molecule, the alpha chain of the IL-2 receptor, plays an important role in maintaining the proliferation and function of regulatory T cells, but the CD25 molecule is also expressed on the activated cell surface, so it does not have specific.Foxp3 as a nuclear transcription factor, and is a characteristic marker of the regulatory T cell.CTLA-4 (cytotoxic T lymphocyte associated antigen 4). Co suppressor receptor, CTLA-4 and the immune tolerance function of the body,.CD4+CD25+ regulatory T cells can inhibit the activation of CD4+ and CD8+ effect T cells. The inhibitory function is achieved by direct contact between regulatory T cells and effector cells or by the secretion of inhibitory cytokine IL-10 and TGF- beta by regulatory T cells, alone and in combination. The use of corresponding blocking antibodies is beneficial to the elimination of parasitic infections.
The study of Schistosoma japonicum vaccine has experienced a long process of dead vaccine, live attenuated vaccine, genetic engineering vaccine and nucleic acid vaccine. The six candidate vaccines recommended by.WHO, including glutathione S transferase, are not stable to obtain more than 40% protection. Both CD4+CD25+ regulatory T cells can inhibit the effect of T cells Therefore, it is speculated that the immune escape of Schistosoma japonicum in vivo and the poor protective effect of Schistosoma japonicum vaccine may be related to regulatory T cells.
On the one hand, the relationship between Schistosoma japonicum infection and regulatory T cells was discussed, and the corresponding antibodies were used to block regulatory T cells to observe the effect and mechanism of its effect on the organism's ability to scavenging Schistosoma japonicum infection. On the other hand, the relationship between Schistosoma japonicum vaccine and regulatory T cells was discussed, and the corresponding antibody resistance was used. The effects of T cells on the protective effect of Schistosoma japonicum vaccine were observed.
This topic is divided into the following two parts:
The role and mechanism of CD4+CD25+ Tregs in immune escape of Schistosoma japonicum
Objective: to understand the role and mechanism of CD4+CD25+ Tregs in immune evasion of Schistosoma japonicum by observing the effect of Schistosoma japonicum infection on CD4+CD25+ Tregs and the effect of anti-CD25 mAb on CD4+CD25+ Tregs.
Methods: the animal experiment was divided into three parts
The first part of the 6~8 week old female BALB/c mice was randomly divided into two groups, that is, the normal control group and the infection group. The infected group infected 40 Schistosoma japonicum cercariae in each mouse. The mice were killed at 2 weeks, 3 weeks, 4 and 5 weeks after infection, and the proportion of CD4+CD25+ Tregs in the splenocytes of each group was detected by flow cytometry.
The second part of the 6-8 week old female BALB / c mice were randomly divided into two groups, namely the normal control group and the anti-CD25 mAb group. The antibody group was intraperitoneally injected with 300 mu g anti-CD25 mAb, the control group was injected with equal volume PBS, the mice were killed for 2 weeks after using the antibody, the spleen was taken asepsis, the single splenic lymphocyte suspension was prepared, and the splenic lymphocytes of each group were detected by flow cytometry. Percentage of medium CD4+CD25+ Tregs.
Third parts of the 6-8 week old female BALB/c mice were randomly divided into two groups, namely, infection control group and anti-CD25 mAb group. Each mouse infected with 40 Schistosoma japonicum cercariae and 2 weeks after infection. The antibody group was injected with 300 g anti-CD25 mAb in each mouse, infected with the same volume of PBS. infection in the control group for 6 weeks after 6 weeks. The number of eggs in each gram of liver, the rate of worm reduction and the rate of egg reduction in every gram of liver were calculated. The spleen of mice in each group was taken asepsis, the single splenic lymphocyte suspension was prepared, the splenocyte culture supernatant was collected, and the content of cytokines in the splenocyte supernatant was detected by the sandwich ELISA method.
Result:
1) 2 weeks, 3 weeks, 4 weeks and 5 weeks after Schistosoma japonicum infection, the percentage of CD4+CD25+ Tregs in spleen lymphocytes of mice increased from 1.99 + 0.10% before infection to 2.36 + 0.37%, 2.8 + 0.05%, 2.35 + 0.09% and 2.68 + 0.05%., suggesting that the infection of Schistosoma japonicum could induce the increase of CD4+CD25+ Tregs in the body.
2) the average value of CD4+CD25+ Tregs in spleen lymphocyte in normal control group was 1.99 + 0.10%, and the average value of 2 weeks after using anti-CD25 mAb was 0.20 + 0.05%, and the antibody group was significantly lower than that of the control group (P0.01). It suggested that anti-CD25 mAb could partially block CD4+CD25+ Tregs..
3) the decrease of the number of mice in the anti-CD25 mAb group was 18.99% less than that in the unused antibody group. The decrease of the number of eggs per gram of liver 15.86%. suggests that the decrease of CD4+CD25+ Tregs is beneficial to the host's clearance of Schistosoma.
4) using anti-CD25 mAb group, the cytokine IFN- gamma in spleen cell culture supernatant of mice was significantly higher than that of infection control group (IL-5).
Conclusion: Anti-CD25 mAb can partially block CD4+CD25+ Tregs, which is beneficial to the elimination of Schistosoma japonicum.
Two, the protective effect of CD4+CD25+ Tregs on Schistosoma japonicum GST vaccine and its mechanism.
Objective: To investigate the effect of anti-CD25 mAb on the expression of CD4+CD25+ Tregs by observing the effect of GST vaccine on CD4+CD25+ Tregs and the effect of anti-CD25 mAb on the expression of CD4+CD25+ Tregs, and to explore the effect of CD4+CD25+ Tregs on the protective effect of GST vaccine and its mechanism.
Methods: female BALB / c mice were randomly divided into five groups, the normal group, the infection control group, the anti-CD25 mAb group, the GST group and the anti-CD25 mAb and GST combined group.GST group and the combined group of mice with the back subcutaneous injection of the 50 mu g/ rat of Schistosoma japonicum GST vaccine for 2 weeks each time, three times and 2 weeks after the last immunization, each mouse infected with the Japanese blood sucking. 40 strains of cercariae, 2 weeks after infection, the anti-CD25 mAb group and the combined group of mice were intraperitoneally injected with anti-CD25 mAb 300 mu g/ rats, and the PBS. in the control group was killed in 2 weeks, 3 weeks, 4 weeks and 5 weeks after infection. The spleen of the mice was taken asepsis to prepare the single splenocytes suspension, and the flow cytometry was used to detect the CD4+CD25+ in the spleen lymphocyte. The percentage of Tregs, splenocyte culture supernatant was collected and sandwich ELISA method was used to detect the cytokine content in the supernatant of splenocyte culture. In the 5 weeks after infection, the mice were counted and the number of eggs per gram of liver were counted. The rate of worm reduction and the rate of egg reduction in each gram of liver were calculated. The mouse liver tissue was stained with paraffin slice HE, and the granuloma of eggs was observed in mice in each group. Change.
Result:
1) for 2 weeks after Schistosoma japonicum infection, the average percentage of CD4+CD25+ Tregs in the spleen lymphocytes of the infected control group and the GST group was 2.36 + 0.37% and 3.36 + 0.06% respectively. The average percentage of the percentage was 2.8 + 0.05% and 2.97 +, respectively 3 weeks after the infection, and the average percentage of the infection after infection was 2.35 + 0.09% and 0.06%. The average percentages were 2.68 + 0.05% and 3.03 + 0.13%. respectively, suggesting that GST vaccine can induce CD4+CD25+ Tregs elevation.
2) the average value of CD4+CD25+ Tregs percentage in the spleen lymphocytes of the infected control group and the anti-CD25 mAb group was 2.8 + 0.05% and 0.13 + 0.04%, respectively after the use of anti-CD25 mAb after 1 weeks of infection, and the average value of the percentage was 2.35 + 0.09% and 0.9 + 0.23% in the 2 weeks after the use of anti-CD25 mAb (i.e., 4 weeks after infection). (5 weeks after infection), the percentage averages were 2.68 + 0.05% and 1.93 + 0.03%., respectively, indicating that CD4+CD25+ Tregs could be partially closed for 1 weeks after anti-CD25 mAb. The percentage of anti-CD25 mAb increased gradually in the 2 weeks and 3 weeks after the use of mAb, but it was still significantly lower than that in the infection control group.
3) the rate of worm reduction and egg reduction of Schistosoma japonicum in GST group were 24.98% and 32% respectively, while the rate of worm reduction and the rate of egg reduction in the combination group of GST and anti-CD25 mAb were 43.43% and 49%., respectively, suggesting that anti-CD25 mAb could be used as an adjuvant to enhance the protective effect of GST vaccine.
4) the early infection of Schistosoma japonicum was dominated by Thl type immune response. After 4 weeks of infection, the female parasite was dominated by Th2 type immune response. After anti-CD25 mAb, the Th1 cytokine IFN-y remained at a high level, which was beneficial to the organism to remove the Schistosoma japonicum. The mechanism of anti-CD25 mAb to strengthen the immune protective effect of GST vaccine was the enhancement mechanism Type Thl immune response in the body.
5) histopathological examination showed that there was no significant difference between the diameter of single egg granuloma and infiltrating cells in each infection group. It was suggested that anti-CD25 mAb did not significantly enhance the pathological changes of egg granuloma in the liver of mice.
Conclusion: the GST vaccine of Schistosoma japonicum can induce the increase in the proportion of CD4+CD25+ Tregs and thus affect the protective effect of the GST vaccine. Anti-CD25 mAb can partially block the CD4+CD25+ Tregs and enhance the Thl type immune response, so it can improve the protective effect of the GST vaccine. Therefore, anti-CD25 mAb can be used as an adjuvant to enhance the protection of the vaccine. Sexual effect.
The conclusions of this study are as follows:
1) the immune evasion of parasites is related to CD4+CD25+ Tregs, and the increase of CD4+CD25+ Tregs is beneficial for schistosomiasis to evade the host's immune attack.
2) the protective effect of the GST vaccine of Schistosoma japonicum is related to the host's CD4+CD25+ Tregs for the first time. The GST vaccine can induce the increase of CD4+CD25+ Tregs in the spleen lymphocyte of mice in the infected background, thus affecting the protective effect of the GST vaccine.
3) it is the first time that anti-CD25 mAb can enhance the protective effect of GST vaccine of Schistosoma japonicum, and its mechanism is achieved by blocking the CD4+CD25+ Tregs and enhancing the Thl immune response.
4) for the first time, anti-CD25 mAb can be used as an adjuvant to enhance the protective effect of GST vaccine against Schistosoma japonicum.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【相似文献】