超抗原SEA诱导T细胞增殖和无能的体内外研究
摘要
超抗原是一类强大的免疫激活剂,它无需抗原提呈细胞的加工处理,而是以完整的蛋白质形式结合于MHC-Ⅱ类分子沟槽外侧,激活大量的T细胞,同时产生大量细胞因子。超抗原的这种高效免疫活性,使其成为一种新的肿瘤免疫治疗模式。我们的实验室多年来一直致力于肿瘤免疫治疗的研究,通过克隆SEA基因,对SEA进行原核表达、分离纯化,制备了活性较高的SEA蛋白;利用纳米乳剂为载体包裹肿瘤特异性抗原MAGE-1-HSP70融合蛋白与SEA配比制备出一种新型肿瘤纳米疫苗,并研究了其理化和生物学特性以及抗肿瘤免疫效应;同时构建了肝癌靶向性SEA/CD80基因共表达重组腺病毒载体,在动物实验中发现其能有效地激发机体的抗肿瘤免疫反应。
但是在进一步的研究中人们发现,超抗原的多次刺激导致了T细胞的免疫耐受,即一部分抗原特异性的T细胞发生程序性死亡,余下的抗原反应性T细胞对超抗原的再次刺激表现为无反应性,即无能。超抗原的这一特性给人们带来了负面影响,限制了其在肿瘤治疗中的应用,因此超抗原的理论和作用机制的研究又成为热点。但是要想将超抗原SEA免疫治疗应用于临床,只研究理论机制是不够的,超抗原本身的剂量、途径和使用方法等对体内外T细胞免疫效应的正负影响及规律,对于SEA的临床实际应用同样具有重要的指导意义,而这在国内外尚未见系统地研究报道,也是我们研究肿瘤超抗原疫苗的一个空缺。因此本研究的目的就是通过建立小鼠体内和体外无能T细胞模型,从增殖反应、细胞膜表型变化和细胞因子的合成与释放等方面系统全面地探讨超抗原SEA影响T细胞应答反应的量效关系及规律特点,为超抗原在肿瘤临床免疫治疗中的合理而有效地应用提供理论依据。实验内容主要概括如下:
1.超抗原SEA对小鼠脾淋巴细胞活化和增值的体内外影响
目的:分别用SEA单次刺激体内或体外培养的小鼠脾淋巴细胞,观察SEA促进淋巴细胞活化和增殖的规律和特点。方法:①采用MTT法测定SEA刺激后脾淋巴细胞的增殖反应。②通过FACS检测SEA初次刺激后脾淋巴细胞膜表型的变化。③用CTLL-2细胞测定细胞培养上清或血清中IL-2的活性,并用FACS检测细胞内IL-2的水平。结果:在体外,使脾淋巴细胞显著增殖的SEA浓度范围是1-1000ng/ml,选择100ng/ml作为观察SEA体外刺激能力的最佳剂量,其既能刺激T细胞的显著增殖,又不会导致过早的细胞死亡。在体内,根据对T细胞刺激程度和效力维持时间,选择腹腔注射为最佳注射途径;SEA剂量在5-15μg范围内均能促进CD3表达显著上升,最佳剂量为10μg。体内外结果均显示,SEA单次刺激促进了T细胞活化的主要膜表面分子(CD69和CD25)的表达显著升高,使CD4与CD8的表达同步上升,IL-2的合成和释放快速增加。结论:在一定浓度范围内的SEA对体内外的T细胞均具有强烈的活化和增殖作用,其对CD4和CD8亚群具有相同的增殖效果。为下一步研究超抗原诱导T细胞无能的实验提供依据和设计思路。
2.超抗原SEA诱导T细胞无能的体内外研究
目的:建立T细胞无能的小鼠体内和体外模型,观察超抗原SEA反复刺激诱导无能T细胞的规律和特点。方法:①在体外,用MTT法测定间隔48h多次SEA刺激后的脾淋巴细胞的增殖反应;在体内,间隔不同天数反复注射SEA后,通过FACS检测脾淋巴细胞中CD3的表达。②通过FACS分析SEA反复刺激后,体内外淋巴细胞膜表型CD4、CD8、CD69和CD25的变化。③测定细胞培养上清或血清中细胞因子(IL-2、TNF-α、IFN-γ、IL-4和IL-10、)的水平,及细胞内IL-2和IL-10的阳性百分率。④在不同时间点,SEA再刺激体外或体内诱导的无能T细胞,通过MTT法的测定观察其反应性的恢复情况。
结果:①经SEA初次刺激后的体内或体外培养的T细胞,对SEA的再刺激仍显示有继续增殖的能力。在体外,刺激T细胞第二次增殖的SEA最佳浓度为100ng/ml。与SEA初次刺激相比,细胞膜表型CD8表达上升的幅度显著高于CD4,CD69的表达没有明显上升,但仍保持在较高的水平;细胞因子IL-4和IL-10的含量均显著升高。在体内,间隔不同的天数,SEA体内第二次注射均可引起CD3表达升高。间隔3天组中,SEA的再注射主要促进了CD4表达的上升;间隔28天组中CD4和CD8的表达均有上升,且趋势相近。与SEA初次注射相比,血清及细胞内IL-2的水平仍有小幅度的递增,IL-10水平则显著上升。体内外,连续两次SEA刺激均能导致CD25不同程度的表达升高。②在体外,T细胞受间隔48h连续三次SEA刺激后,无明显的增殖反应;CD4和CD8的表达没有继续上升;CD69的表达显著下降;而CD25的表达在末次刺激后48h内保持在较高的水平。在体内,间隔不同天数的SEA连续三次注射后,小鼠脾淋巴细胞中CD3的表达均有不同程度的下降。各组中CD8的表达水平基本相似,CD4和CD25却有明显的不同:间隔3天组中,CD4的表达在末次注射后3天内明显降低;CD25没有上升的趋势,但在末次注射后3天内仍保持一定水平的表达。间隔28天组中,CD4的表达较稳定,没有显著的升降;CD25的表达水平较低。体内外均发现,经SEA多次刺激后,仅IL-10的水平仍继续上升。③体外培养的无能T细胞在7天内几乎没有增殖能力,21天后增殖能力显著提高,接近正常水平。在体内,间隔3天组和间隔28天组中诱导的无能T细胞分别从末次注射后第21天和第28天开始出现较弱的应答反应。结论:①首次在C57BL/6小鼠体内外成功建立了无能T细胞模型。但体内外诱导的无能T细胞经过一段时间的休息,其应答反应是可以逐渐恢复的。②经SEA初次刺激的T细胞,对SEA的再刺激,仍具有潜在的增殖反应。在体外,SEA的第二次刺激主要促进CD8亚群的增殖;在体内,首次证实间隔较短时间的连续两次SEA注射主要促进了CD4亚群的增殖,而间隔较长时间的两次SEA注射则对CD4和CD8亚群产生相似的增殖效果。③在体内实验中,首次证实了注射间隔时间的长短与无能的触发无关,但影响了CD4亚群的克隆清除,即间隔时间越短,被清除的CD4+T细胞越多;间隔时间越长,CD4+T细胞清除的程度越小。④在体内试验中发现,尽管CD25的高表达与无能的诱导密切相关,但不是绝对的,即在CD25低表达的情况下也可触发T细胞无能,并且CD25可能不参与无能状态的维持。⑤体内外试验均证实IL-10水平的升高与诱导T细胞无能密切相关,还可能与无能T细胞的维持有关。
3.超抗原SEA诱导小鼠体内外T细胞无能过程中CD28/CTLA-4的变化
目的:在超抗原SEA诱导小鼠体内外T细胞无能的过程中,观察CD28和CTLA-4变化规律及相互关系。方法:SEA体内或体外单次或多次刺激小鼠脾淋巴细胞后,通过FACS检测细胞膜表面CD28和CD152(CTLA-4)阳性表达率,及胞内CTLA-4的表达。结果:体外和体内的结果相似:SEA初次刺激后,CD28的表达显著上升;而CTLA-4在胞内和膜表面的表达均低。SEA再次刺激后,CD28的表达继续上升;CTLA-4的表达明显增加,胞内CTLA-4的升高尤为显著。SEA连续三次刺激后,CD28的表达显著降低;CTLA-4在膜表面和胞内的表达均开始下降。结论:CTLA-4的负调节作用限制了CD28的表达,抑制T细胞的继续活化,有利于无能的诱导;但CTLA-4在SEA反复刺激后的表达下调,提示其可能不参与无能T细胞的维持。
上述研究显示,超抗原SEA诱导的机体免疫反应是一个有规律的变化过程,在肿瘤免疫治疗中犹如一把双刃剑,当人们正确掌握了它的合适剂量、给药间隔时间以及给药方式的规律,应该说SEA仍然是当前抗肿瘤的一个有力武器。
Superantigen is a powerful immuno-stimulant, which can stimulate T cells bearing certain TCR vβelements when bound as an unprocessed protein outside the antigenic groove of MHC-Ⅱmolecules. Superantigen-activation of lymphocytes results in cytokines production, proliferation and cytotoxicity, which can be used in the tumor therapy. Tumor vaccines have been studied for several years in our lab. We have constraucted the prokaryotic expression vector of SEA and gained SEA protein with biological activity. We have mixed the MAGE-1-HSP70 fusion protein with superantigen SEA as a new complex protein vaccine (MHS), which was encapsulated in nanoemulsion, and evaluated the vaccine’s immune responses and therapeutic effects on tumor. We have also constructed hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 gene driven by AFP gene transcription- regulatory elements, which could inhibit the growth of Hepa1-6 tumor and increase the survival rates of mouse.
Although exposure of mature T cells to superantigen generally induces a marked proliferative response in vivo, this often followed by unresponsiveness of T cells, also called anergy. Anergy is one of mechanisms of tolerance. Such property of superantigen strongly limits its antitumor therapy effect. The mechanisms of superantigen in tolerance have been highly studied. However, the tumor vaccine could not be used at clinic only by the studies of mechanisms, it is also important to know the changes and regulations of the responses of T cells in vitro and in vivo affected by the doses and operation method of superantigen, which have not been reported comprehensively. In this study, we designed the models of anergic T cells in vitro and in vivo, and evaluated the responses and regulations of the induction of T cells induced by SEA stimulations by detecting comprehensively the proliferation and surface molecule expressions and the levels of cytokines.
1. Activation and proliferation of T cells stimulated by SEA in vitro and in vivo
Objective: to evaluate the regulations and characteristics of proliferation of activated T cells after SEA stimulation in vitro and in vivo. Methods:①Proliferation of spleen cells was detected by MTT assay after SEA stimulation.②surface molecule expressions were analyzed by FACS.③The IL-2 levels were measured with CTLL-2 cells and intracellular IL-2 was analyzed after permeabilized using 0.5% saponin before stained. Results: In vitro, the results showed a strong proliferation after a single SEA stimulation. A dose-dependent relationship was clearly observed and 100ng/ml was the optimal dose of SEA, which could accelerate the proliferation of T cells and could not lead to the early death of the cells. In vivo, we selected i.p. as the optimal method of injection and the optimal dose of SEA was 10μg in our study. The data showed both in vitro and in vivo that the expressions of CD69 and CD25 were markedly upregulated and expressions of CD4 and CD8 increased similarly. At the same time, large amounts of IL-2 were released. Conclusion: a significant activation and proliferation of T cells could be stimulated by SEA both in vitro and in vivo, which was similar to CD4+T cells and CD8+T cells.
2. Anergy of T cells induced by SEA in vitro and in vivo.
Objective: to construct the model of anergy of T cells in vitro and in vivo, and evaluate the characteristics of anergic T cells induced by repeated SEA stimulations. Methods:①In vitro, Proliferation of spleen cells was detected by MTT assay after repeated SEA stimulations at 48h interval. In vivo, the expressions of CD3 were analyzed by FACS after repeated SEA injections at different time-intervals.②The expressions of CD4、CD8、CD69、CD25 were analyzed by FACS.③IL-2 and TNF-αlevels were measured with CTLL-2 cells and L929 cells, and IL-4、IL-10 and IFN-γproductions were measured with sandwich ELISA kits. Intracellular IL-2 and IL-10 were analyzed after permeabilized using 0.5% saponin before stained.④The responsiveness of anergic T cells induced in vitro and in vivo was measured by MTT. Results:①There was a maintained expansion in response to SEA twice stimulations. In vitro, 100ng/ml was the optimal dose of SEA. The twice stimulations induced a different increase between the two T subsets and the up-regulation of CD8+ T cells appeared a dominant position. The expressions of CD25 were going up, while CD69 expressions didn’t appear upregulation. Productions of IL-4 and IL-10 increased markedly. In vivo, the effects on expression of CD4+ and CD8+ T cells were different at different time-intervals between SEA injections. The 3-days intervals led to an obvious increase of CD4, not to the increase of CD8; while the increase of CD4 and CD8 was both seen markedly at 28 days intervals. The expressions of CD25 were seen markedly regulated in both time-intervals. The maintained level of IL-2 was seen and productions of IL-10 increased markedly.②the unresponsiveness could be induced after three SEA stimulations in vitro and in vivo. In vitro, there was no proliferation of T cells after third SEA stimulation. No up-regulations of CD4 and CD8 expressions were seen and the expression of CD69 decreased significantly. The CD25 expression only kept high levels during 48h, and then gradually decreased. Only IL-10 productions still increased. In vivo, It was CD4+T cells that were reduced markedly after three SEA injections at 3 days interval. The expression of CD25 was not increased, which kept high levels during 3 days after last injections. At 28 days interval, the expression of CD4 was not seen markedly up or down, and the CD25 expression was low. Only IL-10 production increased markedly after injections.③The responsiveness of anergic T cells induced in vitro could be seen after 21 days in culture. In vivo, faint responsiveness of anergic T cells induced by SEA injections at 3 days interval and at 28 days interval could be seen respectively after 21 days and 28 days. Conclusion:①the model of T cells anergy were constructed successfully in vitro and in vivo. The responsiveness of anergic T cells could be restored gradually after some times.②there were still expansion of SEA-reactive T cells after SEA re-stimulation. In vitro, the second stimulation mainly resulted in the proliferation of CD8+T cells. In vivo, it was firstly proved that only the proliferation of CD4+T cells was induced by twice SEA injections at a short interval, while the proliferation of both subsets was induced by twice SEA injections at a long interval.③there was no relationship between the induction of anergy and the time-interval. However, the time of interval affected the clonal deletion. the time of interval was shorter, the more amount of CD4+T cells were deleted..④Although the high expression of CD25 was related with the induction of anergy, the unresponsivnession of T cells could be induced in the state of low expression of CD25.⑤IL-10 was related to the induction and maintenance of anergy.
3. CD28/CTLA-4 expressions during induction of T-cell anergy in vitro and in vivo.
Objective: to evaluate characteristics of CD28 and CTLA-4 expressions in induction of T cells anergy in vitro and in vivo. Methods: the spleen cells were stimulated with SEA single or repeatedly in vitro and in vivo. Cells were collected and analyzed for the expressions of CD28 and CTLA-4. Results: Expression of CD28 was significantly upregulated after single SEA treatment, with low expression of CTLA-4. Twice SEA stimulations led to increases of CTLA-4, while upregulation of CD28 was also seen. The expression of CTLA-4 and CD28 were gradually down after three SEA stimulations. Conclusion: The downregulation of CD28 and upregulation of CTLA-4 accelerated the induction of anergy, and CTLA-4 might have no relation with the maintenance of anergy.
但是在进一步的研究中人们发现,超抗原的多次刺激导致了T细胞的免疫耐受,即一部分抗原特异性的T细胞发生程序性死亡,余下的抗原反应性T细胞对超抗原的再次刺激表现为无反应性,即无能。超抗原的这一特性给人们带来了负面影响,限制了其在肿瘤治疗中的应用,因此超抗原的理论和作用机制的研究又成为热点。但是要想将超抗原SEA免疫治疗应用于临床,只研究理论机制是不够的,超抗原本身的剂量、途径和使用方法等对体内外T细胞免疫效应的正负影响及规律,对于SEA的临床实际应用同样具有重要的指导意义,而这在国内外尚未见系统地研究报道,也是我们研究肿瘤超抗原疫苗的一个空缺。因此本研究的目的就是通过建立小鼠体内和体外无能T细胞模型,从增殖反应、细胞膜表型变化和细胞因子的合成与释放等方面系统全面地探讨超抗原SEA影响T细胞应答反应的量效关系及规律特点,为超抗原在肿瘤临床免疫治疗中的合理而有效地应用提供理论依据。实验内容主要概括如下:
1.超抗原SEA对小鼠脾淋巴细胞活化和增值的体内外影响
目的:分别用SEA单次刺激体内或体外培养的小鼠脾淋巴细胞,观察SEA促进淋巴细胞活化和增殖的规律和特点。方法:①采用MTT法测定SEA刺激后脾淋巴细胞的增殖反应。②通过FACS检测SEA初次刺激后脾淋巴细胞膜表型的变化。③用CTLL-2细胞测定细胞培养上清或血清中IL-2的活性,并用FACS检测细胞内IL-2的水平。结果:在体外,使脾淋巴细胞显著增殖的SEA浓度范围是1-1000ng/ml,选择100ng/ml作为观察SEA体外刺激能力的最佳剂量,其既能刺激T细胞的显著增殖,又不会导致过早的细胞死亡。在体内,根据对T细胞刺激程度和效力维持时间,选择腹腔注射为最佳注射途径;SEA剂量在5-15μg范围内均能促进CD3表达显著上升,最佳剂量为10μg。体内外结果均显示,SEA单次刺激促进了T细胞活化的主要膜表面分子(CD69和CD25)的表达显著升高,使CD4与CD8的表达同步上升,IL-2的合成和释放快速增加。结论:在一定浓度范围内的SEA对体内外的T细胞均具有强烈的活化和增殖作用,其对CD4和CD8亚群具有相同的增殖效果。为下一步研究超抗原诱导T细胞无能的实验提供依据和设计思路。
2.超抗原SEA诱导T细胞无能的体内外研究
目的:建立T细胞无能的小鼠体内和体外模型,观察超抗原SEA反复刺激诱导无能T细胞的规律和特点。方法:①在体外,用MTT法测定间隔48h多次SEA刺激后的脾淋巴细胞的增殖反应;在体内,间隔不同天数反复注射SEA后,通过FACS检测脾淋巴细胞中CD3的表达。②通过FACS分析SEA反复刺激后,体内外淋巴细胞膜表型CD4、CD8、CD69和CD25的变化。③测定细胞培养上清或血清中细胞因子(IL-2、TNF-α、IFN-γ、IL-4和IL-10、)的水平,及细胞内IL-2和IL-10的阳性百分率。④在不同时间点,SEA再刺激体外或体内诱导的无能T细胞,通过MTT法的测定观察其反应性的恢复情况。
结果:①经SEA初次刺激后的体内或体外培养的T细胞,对SEA的再刺激仍显示有继续增殖的能力。在体外,刺激T细胞第二次增殖的SEA最佳浓度为100ng/ml。与SEA初次刺激相比,细胞膜表型CD8表达上升的幅度显著高于CD4,CD69的表达没有明显上升,但仍保持在较高的水平;细胞因子IL-4和IL-10的含量均显著升高。在体内,间隔不同的天数,SEA体内第二次注射均可引起CD3表达升高。间隔3天组中,SEA的再注射主要促进了CD4表达的上升;间隔28天组中CD4和CD8的表达均有上升,且趋势相近。与SEA初次注射相比,血清及细胞内IL-2的水平仍有小幅度的递增,IL-10水平则显著上升。体内外,连续两次SEA刺激均能导致CD25不同程度的表达升高。②在体外,T细胞受间隔48h连续三次SEA刺激后,无明显的增殖反应;CD4和CD8的表达没有继续上升;CD69的表达显著下降;而CD25的表达在末次刺激后48h内保持在较高的水平。在体内,间隔不同天数的SEA连续三次注射后,小鼠脾淋巴细胞中CD3的表达均有不同程度的下降。各组中CD8的表达水平基本相似,CD4和CD25却有明显的不同:间隔3天组中,CD4的表达在末次注射后3天内明显降低;CD25没有上升的趋势,但在末次注射后3天内仍保持一定水平的表达。间隔28天组中,CD4的表达较稳定,没有显著的升降;CD25的表达水平较低。体内外均发现,经SEA多次刺激后,仅IL-10的水平仍继续上升。③体外培养的无能T细胞在7天内几乎没有增殖能力,21天后增殖能力显著提高,接近正常水平。在体内,间隔3天组和间隔28天组中诱导的无能T细胞分别从末次注射后第21天和第28天开始出现较弱的应答反应。结论:①首次在C57BL/6小鼠体内外成功建立了无能T细胞模型。但体内外诱导的无能T细胞经过一段时间的休息,其应答反应是可以逐渐恢复的。②经SEA初次刺激的T细胞,对SEA的再刺激,仍具有潜在的增殖反应。在体外,SEA的第二次刺激主要促进CD8亚群的增殖;在体内,首次证实间隔较短时间的连续两次SEA注射主要促进了CD4亚群的增殖,而间隔较长时间的两次SEA注射则对CD4和CD8亚群产生相似的增殖效果。③在体内实验中,首次证实了注射间隔时间的长短与无能的触发无关,但影响了CD4亚群的克隆清除,即间隔时间越短,被清除的CD4+T细胞越多;间隔时间越长,CD4+T细胞清除的程度越小。④在体内试验中发现,尽管CD25的高表达与无能的诱导密切相关,但不是绝对的,即在CD25低表达的情况下也可触发T细胞无能,并且CD25可能不参与无能状态的维持。⑤体内外试验均证实IL-10水平的升高与诱导T细胞无能密切相关,还可能与无能T细胞的维持有关。
3.超抗原SEA诱导小鼠体内外T细胞无能过程中CD28/CTLA-4的变化
目的:在超抗原SEA诱导小鼠体内外T细胞无能的过程中,观察CD28和CTLA-4变化规律及相互关系。方法:SEA体内或体外单次或多次刺激小鼠脾淋巴细胞后,通过FACS检测细胞膜表面CD28和CD152(CTLA-4)阳性表达率,及胞内CTLA-4的表达。结果:体外和体内的结果相似:SEA初次刺激后,CD28的表达显著上升;而CTLA-4在胞内和膜表面的表达均低。SEA再次刺激后,CD28的表达继续上升;CTLA-4的表达明显增加,胞内CTLA-4的升高尤为显著。SEA连续三次刺激后,CD28的表达显著降低;CTLA-4在膜表面和胞内的表达均开始下降。结论:CTLA-4的负调节作用限制了CD28的表达,抑制T细胞的继续活化,有利于无能的诱导;但CTLA-4在SEA反复刺激后的表达下调,提示其可能不参与无能T细胞的维持。
上述研究显示,超抗原SEA诱导的机体免疫反应是一个有规律的变化过程,在肿瘤免疫治疗中犹如一把双刃剑,当人们正确掌握了它的合适剂量、给药间隔时间以及给药方式的规律,应该说SEA仍然是当前抗肿瘤的一个有力武器。
Superantigen is a powerful immuno-stimulant, which can stimulate T cells bearing certain TCR vβelements when bound as an unprocessed protein outside the antigenic groove of MHC-Ⅱmolecules. Superantigen-activation of lymphocytes results in cytokines production, proliferation and cytotoxicity, which can be used in the tumor therapy. Tumor vaccines have been studied for several years in our lab. We have constraucted the prokaryotic expression vector of SEA and gained SEA protein with biological activity. We have mixed the MAGE-1-HSP70 fusion protein with superantigen SEA as a new complex protein vaccine (MHS), which was encapsulated in nanoemulsion, and evaluated the vaccine’s immune responses and therapeutic effects on tumor. We have also constructed hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 gene driven by AFP gene transcription- regulatory elements, which could inhibit the growth of Hepa1-6 tumor and increase the survival rates of mouse.
Although exposure of mature T cells to superantigen generally induces a marked proliferative response in vivo, this often followed by unresponsiveness of T cells, also called anergy. Anergy is one of mechanisms of tolerance. Such property of superantigen strongly limits its antitumor therapy effect. The mechanisms of superantigen in tolerance have been highly studied. However, the tumor vaccine could not be used at clinic only by the studies of mechanisms, it is also important to know the changes and regulations of the responses of T cells in vitro and in vivo affected by the doses and operation method of superantigen, which have not been reported comprehensively. In this study, we designed the models of anergic T cells in vitro and in vivo, and evaluated the responses and regulations of the induction of T cells induced by SEA stimulations by detecting comprehensively the proliferation and surface molecule expressions and the levels of cytokines.
1. Activation and proliferation of T cells stimulated by SEA in vitro and in vivo
Objective: to evaluate the regulations and characteristics of proliferation of activated T cells after SEA stimulation in vitro and in vivo. Methods:①Proliferation of spleen cells was detected by MTT assay after SEA stimulation.②surface molecule expressions were analyzed by FACS.③The IL-2 levels were measured with CTLL-2 cells and intracellular IL-2 was analyzed after permeabilized using 0.5% saponin before stained. Results: In vitro, the results showed a strong proliferation after a single SEA stimulation. A dose-dependent relationship was clearly observed and 100ng/ml was the optimal dose of SEA, which could accelerate the proliferation of T cells and could not lead to the early death of the cells. In vivo, we selected i.p. as the optimal method of injection and the optimal dose of SEA was 10μg in our study. The data showed both in vitro and in vivo that the expressions of CD69 and CD25 were markedly upregulated and expressions of CD4 and CD8 increased similarly. At the same time, large amounts of IL-2 were released. Conclusion: a significant activation and proliferation of T cells could be stimulated by SEA both in vitro and in vivo, which was similar to CD4+T cells and CD8+T cells.
2. Anergy of T cells induced by SEA in vitro and in vivo.
Objective: to construct the model of anergy of T cells in vitro and in vivo, and evaluate the characteristics of anergic T cells induced by repeated SEA stimulations. Methods:①In vitro, Proliferation of spleen cells was detected by MTT assay after repeated SEA stimulations at 48h interval. In vivo, the expressions of CD3 were analyzed by FACS after repeated SEA injections at different time-intervals.②The expressions of CD4、CD8、CD69、CD25 were analyzed by FACS.③IL-2 and TNF-αlevels were measured with CTLL-2 cells and L929 cells, and IL-4、IL-10 and IFN-γproductions were measured with sandwich ELISA kits. Intracellular IL-2 and IL-10 were analyzed after permeabilized using 0.5% saponin before stained.④The responsiveness of anergic T cells induced in vitro and in vivo was measured by MTT. Results:①There was a maintained expansion in response to SEA twice stimulations. In vitro, 100ng/ml was the optimal dose of SEA. The twice stimulations induced a different increase between the two T subsets and the up-regulation of CD8+ T cells appeared a dominant position. The expressions of CD25 were going up, while CD69 expressions didn’t appear upregulation. Productions of IL-4 and IL-10 increased markedly. In vivo, the effects on expression of CD4+ and CD8+ T cells were different at different time-intervals between SEA injections. The 3-days intervals led to an obvious increase of CD4, not to the increase of CD8; while the increase of CD4 and CD8 was both seen markedly at 28 days intervals. The expressions of CD25 were seen markedly regulated in both time-intervals. The maintained level of IL-2 was seen and productions of IL-10 increased markedly.②the unresponsiveness could be induced after three SEA stimulations in vitro and in vivo. In vitro, there was no proliferation of T cells after third SEA stimulation. No up-regulations of CD4 and CD8 expressions were seen and the expression of CD69 decreased significantly. The CD25 expression only kept high levels during 48h, and then gradually decreased. Only IL-10 productions still increased. In vivo, It was CD4+T cells that were reduced markedly after three SEA injections at 3 days interval. The expression of CD25 was not increased, which kept high levels during 3 days after last injections. At 28 days interval, the expression of CD4 was not seen markedly up or down, and the CD25 expression was low. Only IL-10 production increased markedly after injections.③The responsiveness of anergic T cells induced in vitro could be seen after 21 days in culture. In vivo, faint responsiveness of anergic T cells induced by SEA injections at 3 days interval and at 28 days interval could be seen respectively after 21 days and 28 days. Conclusion:①the model of T cells anergy were constructed successfully in vitro and in vivo. The responsiveness of anergic T cells could be restored gradually after some times.②there were still expansion of SEA-reactive T cells after SEA re-stimulation. In vitro, the second stimulation mainly resulted in the proliferation of CD8+T cells. In vivo, it was firstly proved that only the proliferation of CD4+T cells was induced by twice SEA injections at a short interval, while the proliferation of both subsets was induced by twice SEA injections at a long interval.③there was no relationship between the induction of anergy and the time-interval. However, the time of interval affected the clonal deletion. the time of interval was shorter, the more amount of CD4+T cells were deleted..④Although the high expression of CD25 was related with the induction of anergy, the unresponsivnession of T cells could be induced in the state of low expression of CD25.⑤IL-10 was related to the induction and maintenance of anergy.
3. CD28/CTLA-4 expressions during induction of T-cell anergy in vitro and in vivo.
Objective: to evaluate characteristics of CD28 and CTLA-4 expressions in induction of T cells anergy in vitro and in vivo. Methods: the spleen cells were stimulated with SEA single or repeatedly in vitro and in vivo. Cells were collected and analyzed for the expressions of CD28 and CTLA-4. Results: Expression of CD28 was significantly upregulated after single SEA treatment, with low expression of CTLA-4. Twice SEA stimulations led to increases of CTLA-4, while upregulation of CD28 was also seen. The expression of CTLA-4 and CD28 were gradually down after three SEA stimulations. Conclusion: The downregulation of CD28 and upregulation of CTLA-4 accelerated the induction of anergy, and CTLA-4 might have no relation with the maintenance of anergy.
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