体外建立CD4~+记忆性T细胞生成模型和分子机制的初探
摘要
免疫记忆性是机体对外界病原体最有效的抵抗机理,可保证机体再次遇到同一病原体时迅速启动免疫防御机理消灭病原体,也是疫苗作用的物质基础。免疫记忆系统由体液免疫记忆和细胞免疫记忆两部分构成,承担体液免疫系统记忆功能的为记忆性B细胞(memory B cells,Bm),承担细胞免疫系统记忆功能的为记忆性T细胞(memory T cells,Tm)。人Tm的特征性表面标志是CD45RO~+,在小鼠体内根据其归巢部位的不同和表达趋化因子受体不同,Tm分为效应型记忆性T细胞(CD44~+CD62L-CCR7-)和中枢型记忆性T细胞(CD44~+CD62L~+CCR7~+)。对于记忆T细胞来源存在线性(或定向)分化和非线性(或不对称)分化两种模式。线性分化模式认为:初始T细胞先分化为能分泌细胞因子的效应细胞,然后其中很少的一部分转化为记忆细胞。另一种观点认为Tm来源于一类较迟到达免疫应答晚期阶段的前体细胞。非线性分化模式则认为一部分活化的初始T细胞不经过效应T细胞阶段直接分化为记忆细胞。最近的研究表明,记忆细胞的来源更倾向于线性分化模式。
体内研究证明,与CD8~+T细胞不同,IL-7对CD4~+Tm的形成起了至关重要的作用。IL-7-/-小鼠和IL-7R表达突变的小鼠在抗原刺激后不能形成CD4~+Tm。在CD4~+T细胞反应性增殖的高峰期增强IL-7信号,可以促进TCR转基因小鼠的效应CD4~+T细胞增殖以及上调Bcl-2的表达,阻止其在收缩期死亡,从而增加CD4~+Tm。在T细胞收缩期注入IL-2,IL-7或IL-15都可以使CD8~+T细胞收缩期减缓。在收缩期体内注入IL-2或IL-15后,主要积聚的是KLRG1~(hi)CD127~(lo)的短期存活效应和记忆细胞,而注入IL-7则主要积聚KLRG1~(lo)CD127~(hi)的长期存活记忆细胞。但体内研究只能证明细胞因子对CD4~+Tm分化的影响,并不能了解诱导CD4~+Tm分化的分子机理。因此人们将CD4~+Tm分离出来,研究CD4~+Tm自稳性存活和再刺激后增殖的分子机理。研究的靶细胞主要有两类:小鼠自发分化的记忆表型T细胞和抗原特异性T细胞。在正常生理条件下,两类CD4~+Tm的自稳均需要IL-7和IL-15。记忆表型CD4~+Tm的快速增殖还需要MHC-II类分子,抗原特异性CD4~+Tm的快速增殖不需要MHC-II类分子而主要依赖IL-7。IL-7R高表达于静息T细胞,当T细胞被激活后,IL-7Ra (CD127)迅速下调,仅少数可转化为Tm的效应细胞再次表达IL-7Ra。在CD8~+T细胞已经证明,IL-7Ra的表达对检测记忆细胞前体很有作用。
鉴于目前对CD4~+Tm分化主要是体内研究,尚无体外模型。为了能深入研究CD4~+ Tm分化的机理,我们拟首先以流式细胞仪检测CD44和CD62L表达作为指标,用IL-7刺激建立体外诱导CD4~+Tm生成的模型;然后用不同细胞因子或细胞因子组合刺激,观察其诱导CD4~+Tm生成的能力;再通过CFSE标记和抗原再刺激验证体外增殖的细胞确为CD4~+Tm;最后以实时荧光定量RT-PCR和Western-blotting等检测探讨CD4~+Tm形成的分子机制。
第一部分体外建立CD4~+Tm生成模型
目的:最初考虑用OT-II小鼠CD4~+T细胞来建立本模型。但由于OT-II小鼠饲养困难,数量不足,所以拟以FBS作为抗原,用野生型C57BL/6小鼠CD4~+T细胞来建立本模型,并与OVA刺激的OT-II小鼠CD4~+T细胞比较,证明本模型的实用性。用抗原处理的DC和IL-7在体外刺激初始CD4~+T细胞,在不同时间检测T细胞表达CD44和CD62L的情况和再次抗原刺激增殖情况,建立体外诱导CD4~+Tm生成的模型,为探讨体外诱导CD 4~+Tm生成的条件打下基础。方法:无菌条件下分别取OT-II和C57BL/6的股骨和胫骨,提取骨髓细胞在体外分别用FBS或OVA刺激,加入IL-4 (1ng/ml)和GM-CSF(10ng/ml)诱导骨髓来源的成熟DC(mature dendritic cell, OVA-DC或FBS-DC)。无菌条件下分别取OT-II和C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4~+T细胞;将OVA-DC与OT-II CD4~+T细胞1:10混合,FBS-DC与C57BL/6 CD4~+T细胞也以同比例混合,用含IL-7(1ng/ml)的10%FBS 1640培养基培养,96孔板每孔1×105细胞,每3天半量换液一次,分别在第5天、10天、15天、20天、25天、30天时,用流式细胞仪检测CD4~+T细胞表面分子CD44,CD62L的表达情况;同时在第30天收集细胞标记CFSE后,用OVA-DC或FBS-D再刺激,按照DC:T=1:10的比例将细胞悬液铺在96孔板中,72小时后用流式细胞仪检测增殖情况;同时用CFSE标记新鲜分离的OT-II和C57BL/6小鼠脾脏CD4~+T细胞,同样OVA-DC或FBS -DC刺激,48小时后用流式细胞仪检测增殖情况作为对照。结果:初始CD4~+T细胞多为CD44loCD62Lhi,随着培养时间的延长,CD44的表达逐渐升高,CD62L表达降低;OVA-DC刺激OT-II小鼠CD4~+T细胞组高达80%以上,在FBS-DC刺激C57BL/6小鼠CD4~+T细胞组最后CD44~(hi)CD62L~(lo)细胞比例在70%左右。两组CD4~+Tm表达CD44和CD62L的模式相似;增殖实验示培养了30天的抗原特异性细胞在再次接触同种抗原时反应快速,细胞分裂大部分聚集在第三代、四代、五代。提示此种方法诱导生成的细胞在表型和功能上都具备CD4~+Tm的特征。结论:在体外,IL-7可以诱导CD4~+Tm生成。用FBS刺激诱导C57BL/6小鼠CD4~+Tm生成的状况与用OVA刺激诱导OT-II小鼠CD4~+Tm生成的状况相似,说明可以用野生型C57BL/6小鼠CD4~+T细胞代替OT-II小鼠CD4~+T细胞进行CD4~+Tm生成机理的研究,所以我们的后续实验均用FBS作为抗原研究诱导CD4~+Tm生成的条件和机理。
第二部分不同细胞因子对体外诱导CD4~+Tm生成的影响
目的:我们选用细胞因子IL-2,IL-4,IL-7,IL-15,IFN-γ,Flt-3L,TGF-β在体外诱导初始CD4~+T细胞,观察T细胞表型CD44和CD62L的表达情况和再次抗原刺激的增殖情况,探讨各种相关细胞因子分别与CD4~+Tm产生的关系。方法:无菌条件下取C57 BL/6小鼠的股骨和胫骨,提取骨髓细胞在体外用FBS刺激,加入IL-4 (1ng/ml)和GM- CSF(10ng/ml)诱导骨髓来源的成熟FBS-DC。无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4~+T细胞;将C57BL/6小鼠的BMDC与CD4~+T细胞1:10混合,分别在含IL-2(50U/ml),IL-7(1ng/ml),IL-15(5ng/ml),IL-4 (10ng/ml),IFN-γ(10 ng/ ml),Flt-3L(5ng/ml),TGF-β(1ng/ml),IL-7~+IL-2和IL-7~+IL-15 10%FBS1640培养基中培养,96孔板每孔1×105细胞,每3天半量换液一次,分别在第5天、10天、15天、20天、25天、30天时,用流式细胞仪检测CD4~+T细胞表面分子CD44,CD62L的表达情况;同时在第30天时分别收集存活下来的细胞标记CFSE后,用FBS-DC再刺激,按照DC:T=1:10的比例将细胞悬液铺在96孔板中,72小时后用流式细胞仪检测增殖情况。结果:单独用细胞因子IL-4,IL-15,IFN-γ,Flt-3L,TGF-β等培养的体系中,细胞不到20天全部死亡,而细胞因子IL-2,IL-7,IL-7~+IL-2和IL-7~+IL-15培养体系细胞存活下来;随着培养时间的延长,CD44的表达逐渐升高,CD62L表达降低;另外细胞因子IL-2和IL-7~+IL-2培养体系组其CD44~（hi）CD62L~(lo)细胞的产生时间在10天左右,而且所占比例远远高于其他组;增殖实验结果显示,与其他组相比,IL-2和IL-7~+IL-2组的细胞增殖能力显著减弱,其相互之间无明显差别;IL-7~+IL-15组与单独IL-7组无明显差别。提示IL-2只能有效地活化CD4~+ T细胞,其诱导生成与CD4~+ Tm表型相符的细胞,但不具备再次免疫应答时快速增殖的性能;同时在和IL-7共培养体系中,有抑制IL-7的功能,导致不能有效诱导CD4~+Tm生成。结论: IL-4,IL-15, IFN-γ,Flt-3L,TGF-β等细胞因子单独在体外不能诱CD4~+Tm的生成,同时在IL-7~+IL-15培养组与单独IL-7培养组无差别;IL-2不能诱导生成CD4~+ Tm,并且抑制了IL-7诱导生成CD4~+Tm的功能。查阅文献,未见关于IL-2能抑制IL-7诱导CD4~+Tm生成的报道。由于IL-15同样使用IL-2R和γc作为受体的一部分,且IL-15并不抑制IL-7诱导的CD4~+Tm生成,说明IL-2的抑制作用可能不是由于通过竞争γc,而与其他信号途径的调节有关。
第三部分CD4~+Tm体外生成的分子机制初探
目的:检测细胞因子IL-7在体外诱导生成CD4~+Tm细胞有关信号分子和凋亡蛋白的表达情况,初步探讨IL-7诱导生成CD4~+Tm的机制。方法:无菌条件下取C57BL/6小鼠的股骨和胫骨,提取骨髓细胞在体外用FBS刺激,加入IL-4(1ng/ml)和GM-CSF(10ng/ml)诱导骨髓来源的成熟FBS-DC;无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4~+T细胞;将C57BL/6小鼠的BMDC与CD4~+T细胞1:10混合,用分别含IL-7(1ng/ml),IL-2(50U/ml),IL-7~+IL-2的10%FBS1640培养基培养,96孔板每孔1×105细胞,每3天半量换液一次,采用实时荧光定量RT-PCR分别在第5d,10d,15d,20d,25d,30d检测细胞因子IL-4,IFN-γ,Bcl-2和Bax mRNA表达水平;另外在培养的第30d用Western-blotting检测STAT5/p-STAT5(Y694),AKT/p-AK T,pro-Caspase-3/ActiveCaspase-3, Bcl-2等分子的蛋白表达水平;同时用刚分离出来的初始CD4~+T细胞做相同的检测。结果:与初始CD4~+T细胞组相比,IL-7培养体系的IL-4,IFN-γ,Bcl-2的mRNA表达水平都显著增高,同时Bcl-2,p-STAT5的蛋白水平增高,Bax,Active Caspase-3的表达降低。提示IL-7可能通过JAK/STAT信号途径作用于抗凋亡蛋白Bcl-2和促凋亡蛋白。结论: IL-7通过JAK/STAT5信号途径作用于活化的初始CD4~+T细胞,上调抗凋亡蛋白Bcl-2和下调促凋亡蛋白,抑制Caspase-3的活化,从而维持已生成的CD4~+Tm存活。IL-2抑制IL-7诱导CD4~+Tm生成的作用可能与其活化AKT有关。
Immunological memory is the most effective mechanism for the body to resistance to external pathogens, which can rapidly eliminate of pathogens when the body re-encounter the same pathogen, but also it is the material basis for the vaccine effect. Immunological memory system consists of humoral Immunological memory and cellular Immunological memory , The memory B cells (memory B cells, Bm) which bear the memory function of humoral Immunological memory and the memory T cells (memory T cells, Tm) which bear the memory function of cellular Immunological memory. In human, the characteristic surface markers of Tm is the CD45RO~+, but in mouse Tm include effect-type memory T cells (CD44~+CD62L-CCR7-) and central-type memory T cells (CD44~+CD62L~+CCR7~+) in according to its different homing parts and chemokine receptors. Memory T cell is born of a linear (or directional) differentiation and asymmetric division mode. The linear differentiation model: the initial T cells can be differentiated into effector cells, and then a very small part of them differentiate into memory cells. Another view is that Tm come from precursor cells arrivallig at the late stages of immune response last. Differentiation pattern of non-linear part that the initial cells directly differentiate into memory cells . Recent studies have shown that memory cells are more likely the source of the linear differentiation model.
In vivo studies have shown that IL-7 played a crucial role in formation of CD4~+Tm. L-7-/- mice and IL-7R mutant mice can not from CD4~+Tm. Enhanced IL-7 signaling in the peak proliferation of CD4~+T cells can increase the population of CD4~+Tm in TCR transgenic mice by promoting the proliferation of effective CD4~+T cell,increasing expression of Bcl-2 and preventing death in systole. In the time of T cell contraction to inject IL-2, IL-7 or IL-15 can slow CD8~+T cell contraction. Inject IL-2 or IL-15 at systolic in vivo, the major accumulation of short-term survival effect and memory cells KLRG1~(hi)CD127~(lo), whereas inject IL-7 the mainly major accumulation of long-term survival of memory cells KLRG1~(lo)CD127~(hi). The studies in vivo only show the effect of cytokines on the differentiation of CD4~+Tm, but not show the molecular mechanism of differentiation induce CD4~+Tm. So people isolate CD4~+Tm, to study the molecular mechanism of the stability, re-stimulated survival and proliferation. Two types: spontaneous differentiation of phenotype memory T cell and antigen-specific T cells. Under normal physiological conditions, two types of CD4~+Tm self-stability require IL-7 and IL-15.The rapid proliferation of the phenotype memory CD4~+Tm need MHC-II molecules, whereas the rapid proliferation of the antigen-specific CD4~+Tm main dependent IL-7. IL-7R(CD127) is high expression in the resting T cells, whereas is down quickly in the activated T cells. Only a few can be transformed into Tm cells and expressed IL-7Ra again. It has proven that the expression of IL-7Ra can be used to detection of memory cell precursors in the CD8 ~+ T cells.
Given the current differentiation of CD4~+Tm mainly studies in vivo. In order to further study the mechanism of differentiation of CD4~+Tm, we first establish model of CD4~+Tm which stimulated with IL-7 in vitro and intend the expression of CD44 and CD62L as an indicator by flow cytometry, Then use different combinations of cytokines or cytokine stimulate to observe the capacity of generation CD4~+Tm; and then through the antigen re-stimulation and CFSE labeling to verify CD4~+Tm by proliferation; Finally, to investigate the molecular mechanism of CD4~+Tm by real time quantitative RT-PCR and Western -blotting.
Part I: The establishment of CD4~+Tm generated in vitro
Objective: First consider the use of CD4~+T cells from OT-II mice to create the model. However, due to OT-II mice feeding difficulties, insufficient numbers, so we intend to use FBS as antigen, with CD4~+T cells from wild-type C57BL/6 mice to create the model, simultaneously, with OT-II CD4~+T cells compared to prove the usefulness of C57BL/6 mice model. DC treated with antigen and IL-7 stimulate initial CD4~+T cells in vitro, detect the expression of CD44 and CD62L at different times and re-proliferation condition after antigen stimulation again, the establishment CD4~+Tm generated model to lay the foundation for investigatig the conditions of induction CD4~+Tm generating in vitro. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS or OVA stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced bone marrow-derived mature DC (mature dendritic cell, OVA-DC or FBS-DC). Take spleen from OT-II and C57BL/6 mouse, isolate mononuclear cells and separate CD4~+T cells by immunomagnetic beas under sterile conditions; the OVA-DC and OT-II CD4~+T cells mixed 1:10, FBS-DC and C57BL/6 CD4~+T cells mixed in the same proportion, cultured in medium containing IL-7 (1ng/ml) of 10% FBS-1640. chang half of the medium every 3 days, check the expression of CD44, CD62L by flow cytometry in the 5d, 10d, 15d, 20d, 25d, 30d respectively; At the same time on the 30d, collect cells labeled with CFSE, then stimulate with OVA-DC or FBS-DC for 72 hours, detect cell proliferation by flow cytometry. At the same time, with the CFSE labeled naive OT-II and C57BL/6 CD4~+T cells, then OVA-DC or FBS-DC stimulate for 72 hours, then detect the proliferation by flow cytometry as a control. Results: The naive CD4~+T cells (0d) mostly express CD44~(lo)CD62L~(hi), as the culture time, the expression of CD44 gradually increased, CD62L decreased; in the OVA-DC stimulate CD4~+T cells from OT-II mice group the ratio of CD44~(hi)CD62L~(lo) to 80% at last. The expression pattern of CD4~+Tm is similar in two groups. Proliferation experiments show cultured 30d cells fast response to the same antigen, mostly gather in the third,fourth,fifth or more generates. The results suggest that the phenotype and function of induced cells by this method is in accordance with CD4~+Tm characteristics. Conclusion: In vitro, IL-7 can induce CD4~+ Tm generation. The expression pattern and function of two groups CD4~+Tm are similar, so that, wild-type C57BL/6 mice can instead of OT-II mouse to study the mechanism of CD4~+Tm formation .
Part II: The influence of different cytokines on induction of CD4~+Tm in vitro
Objective: To explore the relationship between a variety of cytokines and CD4~+Tm , we use cytokine IL-2, IL-4, IL-7, IL-15, IFN-γ, TGF-βand Flt-3L to induce of the initial CD4~+T cells in vitro, check the expression of CD44 and CD62L and proliferation re-antigens stimulate. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced FBS-DC. Take spleen from C57BL/6, isolate mononuclear cells and separate CD4~+T cells by immunomagnetic beas under sterile conditions; the FBS-DC and C57BL/6 CD4~+T cells mixed 1:10, respectively, with IL-2 (50U/ml), IL-7 (1ng/ml), IL-15 (5ng/ml), IL-4 (10ng/ml), IFN-γ(10ng/ml), Flt-3L (5ng/ml),TGF-β(1ng/ml), IL-7+IL-2 and IL-7+IL-15 of 10% FBS-1640 culture medium, change half of the medium every 3 days, detect the expression of CD44, CD62L by flow cytometry in the 5d, 10d, 15d, 20d, 25d, 30d respectively; At the same time on the 30th day, collect cells labeled with CFSE, then stimulate with FBS-DC for 72 hours, detect cell proliferation by flow cytometry. Results: Respectively in IL-4, IL-15, Flt-3L, IFN-γand TGF-βculture system alone, cell death of less than 20d, while IL-2, IL-7, IL-7~+IL-2, IL-7~+IL-15 cell culture system survive; as the culture time, CD44 expression gradually increase, CD62L expression decrease; Another cytokine IL-2 and IL-7~+IL-2 training system group the time of its CD44~(hi)CD62L~(lo) cell generation in about 10 days, but the proportion is much higher than other groups. Compared with other groups, proliferation experiments show that IL-2 and IL-7+IL-2 groups of cell proliferation are significantly reduced, no significant difference between them; IL-7+IL-15 group compared with IL-7 alone is no significant difference. The results suggest that IL-2 only can effectively activate CD4~+T cells, which induce the same phenotype as CD4~+Tm cell, but they do not have the capability of rapid proliferation; while in IL-7 co-culture system, it can inhibit the function of IL-7. Conclusion: IL-15 do not induce generation of CD4~+Tm alone in vitro, and can not influence the capability of IL-7. IL-2 do not induce CD4~+Tm, but inhibit the function of IL-7. Literature review, no reports about IL-2 can inhibit IL-7 the induction of CD4~+Tm generated. Because IL-15 also uses IL-2R andγc as part of receptor, and IL-15 do not inhibit IL-7 induced CD4~+Tm generation, indicating that the inhibitory effect of IL-2 may not be through a competitiveγc, but their signal condition.
Part III: The mechanisms of CD4~+Tm generated in vitro
Objective: To preliminary study the mechanism of CD4~+Tm induced by IL-7 in vitro, detect related signaling molecules, transcription molecules and the apoptosis-related protein of CD4~+Tm induced by IL-7. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced FBS-DC. Take spleen from C57BL/6, isolate mononuclear cells and separate CD4~+T cells by immunomagnetic beas under sterile conditions; the FBS-DC and C57BL/6 CD4~+T cells mixed 1:10, respectively, with IL-7 (1ng/ml), IL-7+IL-2 of 10% FBS-1640 culture medium, changed half of the medium every 3days, detect of IL-4, IFN-γ, Bcl-2, Bax by Real-time quantitative RT-PCR in the 5d, 10d, 15d, 20d, 25d, 30d respectively and STAT5 /p-STAT5(Y694), AKT/p-AKT, pro-Caspase-3/Active Caspase-3, Bcl-2 by Western -blotting after 30 days. Results: Compared with the naive CD4~+T cells group, IL-7 culture system, IL-4, IFN-γ, p-STAT5, Bcl-2 are significantly higher, Active Caspase-3 is lower. IL-7 may prompt anti-apoptotic protein Bcl-2 through the JAK/STAT. Conclusion: IL-7 plays important role in the activation of naive CD4~+T cells, increasing the expression of Bcl-2 by JAK/STAT5, thereby inhibit the activation of caspase-3, so that, part of the activated CD4~+T cells survive for long term, then formation of CD4~+Tm.
体内研究证明,与CD8~+T细胞不同,IL-7对CD4~+Tm的形成起了至关重要的作用。IL-7-/-小鼠和IL-7R表达突变的小鼠在抗原刺激后不能形成CD4~+Tm。在CD4~+T细胞反应性增殖的高峰期增强IL-7信号,可以促进TCR转基因小鼠的效应CD4~+T细胞增殖以及上调Bcl-2的表达,阻止其在收缩期死亡,从而增加CD4~+Tm。在T细胞收缩期注入IL-2,IL-7或IL-15都可以使CD8~+T细胞收缩期减缓。在收缩期体内注入IL-2或IL-15后,主要积聚的是KLRG1~(hi)CD127~(lo)的短期存活效应和记忆细胞,而注入IL-7则主要积聚KLRG1~(lo)CD127~(hi)的长期存活记忆细胞。但体内研究只能证明细胞因子对CD4~+Tm分化的影响,并不能了解诱导CD4~+Tm分化的分子机理。因此人们将CD4~+Tm分离出来,研究CD4~+Tm自稳性存活和再刺激后增殖的分子机理。研究的靶细胞主要有两类:小鼠自发分化的记忆表型T细胞和抗原特异性T细胞。在正常生理条件下,两类CD4~+Tm的自稳均需要IL-7和IL-15。记忆表型CD4~+Tm的快速增殖还需要MHC-II类分子,抗原特异性CD4~+Tm的快速增殖不需要MHC-II类分子而主要依赖IL-7。IL-7R高表达于静息T细胞,当T细胞被激活后,IL-7Ra (CD127)迅速下调,仅少数可转化为Tm的效应细胞再次表达IL-7Ra。在CD8~+T细胞已经证明,IL-7Ra的表达对检测记忆细胞前体很有作用。
鉴于目前对CD4~+Tm分化主要是体内研究,尚无体外模型。为了能深入研究CD4~+ Tm分化的机理,我们拟首先以流式细胞仪检测CD44和CD62L表达作为指标,用IL-7刺激建立体外诱导CD4~+Tm生成的模型;然后用不同细胞因子或细胞因子组合刺激,观察其诱导CD4~+Tm生成的能力;再通过CFSE标记和抗原再刺激验证体外增殖的细胞确为CD4~+Tm;最后以实时荧光定量RT-PCR和Western-blotting等检测探讨CD4~+Tm形成的分子机制。
第一部分体外建立CD4~+Tm生成模型
目的:最初考虑用OT-II小鼠CD4~+T细胞来建立本模型。但由于OT-II小鼠饲养困难,数量不足,所以拟以FBS作为抗原,用野生型C57BL/6小鼠CD4~+T细胞来建立本模型,并与OVA刺激的OT-II小鼠CD4~+T细胞比较,证明本模型的实用性。用抗原处理的DC和IL-7在体外刺激初始CD4~+T细胞,在不同时间检测T细胞表达CD44和CD62L的情况和再次抗原刺激增殖情况,建立体外诱导CD4~+Tm生成的模型,为探讨体外诱导CD 4~+Tm生成的条件打下基础。方法:无菌条件下分别取OT-II和C57BL/6的股骨和胫骨,提取骨髓细胞在体外分别用FBS或OVA刺激,加入IL-4 (1ng/ml)和GM-CSF(10ng/ml)诱导骨髓来源的成熟DC(mature dendritic cell, OVA-DC或FBS-DC)。无菌条件下分别取OT-II和C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4~+T细胞;将OVA-DC与OT-II CD4~+T细胞1:10混合,FBS-DC与C57BL/6 CD4~+T细胞也以同比例混合,用含IL-7(1ng/ml)的10%FBS 1640培养基培养,96孔板每孔1×105细胞,每3天半量换液一次,分别在第5天、10天、15天、20天、25天、30天时,用流式细胞仪检测CD4~+T细胞表面分子CD44,CD62L的表达情况;同时在第30天收集细胞标记CFSE后,用OVA-DC或FBS-D再刺激,按照DC:T=1:10的比例将细胞悬液铺在96孔板中,72小时后用流式细胞仪检测增殖情况;同时用CFSE标记新鲜分离的OT-II和C57BL/6小鼠脾脏CD4~+T细胞,同样OVA-DC或FBS -DC刺激,48小时后用流式细胞仪检测增殖情况作为对照。结果:初始CD4~+T细胞多为CD44loCD62Lhi,随着培养时间的延长,CD44的表达逐渐升高,CD62L表达降低;OVA-DC刺激OT-II小鼠CD4~+T细胞组高达80%以上,在FBS-DC刺激C57BL/6小鼠CD4~+T细胞组最后CD44~(hi)CD62L~(lo)细胞比例在70%左右。两组CD4~+Tm表达CD44和CD62L的模式相似;增殖实验示培养了30天的抗原特异性细胞在再次接触同种抗原时反应快速,细胞分裂大部分聚集在第三代、四代、五代。提示此种方法诱导生成的细胞在表型和功能上都具备CD4~+Tm的特征。结论:在体外,IL-7可以诱导CD4~+Tm生成。用FBS刺激诱导C57BL/6小鼠CD4~+Tm生成的状况与用OVA刺激诱导OT-II小鼠CD4~+Tm生成的状况相似,说明可以用野生型C57BL/6小鼠CD4~+T细胞代替OT-II小鼠CD4~+T细胞进行CD4~+Tm生成机理的研究,所以我们的后续实验均用FBS作为抗原研究诱导CD4~+Tm生成的条件和机理。
第二部分不同细胞因子对体外诱导CD4~+Tm生成的影响
目的:我们选用细胞因子IL-2,IL-4,IL-7,IL-15,IFN-γ,Flt-3L,TGF-β在体外诱导初始CD4~+T细胞,观察T细胞表型CD44和CD62L的表达情况和再次抗原刺激的增殖情况,探讨各种相关细胞因子分别与CD4~+Tm产生的关系。方法:无菌条件下取C57 BL/6小鼠的股骨和胫骨,提取骨髓细胞在体外用FBS刺激,加入IL-4 (1ng/ml)和GM- CSF(10ng/ml)诱导骨髓来源的成熟FBS-DC。无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4~+T细胞;将C57BL/6小鼠的BMDC与CD4~+T细胞1:10混合,分别在含IL-2(50U/ml),IL-7(1ng/ml),IL-15(5ng/ml),IL-4 (10ng/ml),IFN-γ(10 ng/ ml),Flt-3L(5ng/ml),TGF-β(1ng/ml),IL-7~+IL-2和IL-7~+IL-15 10%FBS1640培养基中培养,96孔板每孔1×105细胞,每3天半量换液一次,分别在第5天、10天、15天、20天、25天、30天时,用流式细胞仪检测CD4~+T细胞表面分子CD44,CD62L的表达情况;同时在第30天时分别收集存活下来的细胞标记CFSE后,用FBS-DC再刺激,按照DC:T=1:10的比例将细胞悬液铺在96孔板中,72小时后用流式细胞仪检测增殖情况。结果:单独用细胞因子IL-4,IL-15,IFN-γ,Flt-3L,TGF-β等培养的体系中,细胞不到20天全部死亡,而细胞因子IL-2,IL-7,IL-7~+IL-2和IL-7~+IL-15培养体系细胞存活下来;随着培养时间的延长,CD44的表达逐渐升高,CD62L表达降低;另外细胞因子IL-2和IL-7~+IL-2培养体系组其CD44~（hi）CD62L~(lo)细胞的产生时间在10天左右,而且所占比例远远高于其他组;增殖实验结果显示,与其他组相比,IL-2和IL-7~+IL-2组的细胞增殖能力显著减弱,其相互之间无明显差别;IL-7~+IL-15组与单独IL-7组无明显差别。提示IL-2只能有效地活化CD4~+ T细胞,其诱导生成与CD4~+ Tm表型相符的细胞,但不具备再次免疫应答时快速增殖的性能;同时在和IL-7共培养体系中,有抑制IL-7的功能,导致不能有效诱导CD4~+Tm生成。结论: IL-4,IL-15, IFN-γ,Flt-3L,TGF-β等细胞因子单独在体外不能诱CD4~+Tm的生成,同时在IL-7~+IL-15培养组与单独IL-7培养组无差别;IL-2不能诱导生成CD4~+ Tm,并且抑制了IL-7诱导生成CD4~+Tm的功能。查阅文献,未见关于IL-2能抑制IL-7诱导CD4~+Tm生成的报道。由于IL-15同样使用IL-2R和γc作为受体的一部分,且IL-15并不抑制IL-7诱导的CD4~+Tm生成,说明IL-2的抑制作用可能不是由于通过竞争γc,而与其他信号途径的调节有关。
第三部分CD4~+Tm体外生成的分子机制初探
目的:检测细胞因子IL-7在体外诱导生成CD4~+Tm细胞有关信号分子和凋亡蛋白的表达情况,初步探讨IL-7诱导生成CD4~+Tm的机制。方法:无菌条件下取C57BL/6小鼠的股骨和胫骨,提取骨髓细胞在体外用FBS刺激,加入IL-4(1ng/ml)和GM-CSF(10ng/ml)诱导骨髓来源的成熟FBS-DC;无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,磁珠分选CD4~+T细胞;将C57BL/6小鼠的BMDC与CD4~+T细胞1:10混合,用分别含IL-7(1ng/ml),IL-2(50U/ml),IL-7~+IL-2的10%FBS1640培养基培养,96孔板每孔1×105细胞,每3天半量换液一次,采用实时荧光定量RT-PCR分别在第5d,10d,15d,20d,25d,30d检测细胞因子IL-4,IFN-γ,Bcl-2和Bax mRNA表达水平;另外在培养的第30d用Western-blotting检测STAT5/p-STAT5(Y694),AKT/p-AK T,pro-Caspase-3/ActiveCaspase-3, Bcl-2等分子的蛋白表达水平;同时用刚分离出来的初始CD4~+T细胞做相同的检测。结果:与初始CD4~+T细胞组相比,IL-7培养体系的IL-4,IFN-γ,Bcl-2的mRNA表达水平都显著增高,同时Bcl-2,p-STAT5的蛋白水平增高,Bax,Active Caspase-3的表达降低。提示IL-7可能通过JAK/STAT信号途径作用于抗凋亡蛋白Bcl-2和促凋亡蛋白。结论: IL-7通过JAK/STAT5信号途径作用于活化的初始CD4~+T细胞,上调抗凋亡蛋白Bcl-2和下调促凋亡蛋白,抑制Caspase-3的活化,从而维持已生成的CD4~+Tm存活。IL-2抑制IL-7诱导CD4~+Tm生成的作用可能与其活化AKT有关。
Immunological memory is the most effective mechanism for the body to resistance to external pathogens, which can rapidly eliminate of pathogens when the body re-encounter the same pathogen, but also it is the material basis for the vaccine effect. Immunological memory system consists of humoral Immunological memory and cellular Immunological memory , The memory B cells (memory B cells, Bm) which bear the memory function of humoral Immunological memory and the memory T cells (memory T cells, Tm) which bear the memory function of cellular Immunological memory. In human, the characteristic surface markers of Tm is the CD45RO~+, but in mouse Tm include effect-type memory T cells (CD44~+CD62L-CCR7-) and central-type memory T cells (CD44~+CD62L~+CCR7~+) in according to its different homing parts and chemokine receptors. Memory T cell is born of a linear (or directional) differentiation and asymmetric division mode. The linear differentiation model: the initial T cells can be differentiated into effector cells, and then a very small part of them differentiate into memory cells. Another view is that Tm come from precursor cells arrivallig at the late stages of immune response last. Differentiation pattern of non-linear part that the initial cells directly differentiate into memory cells . Recent studies have shown that memory cells are more likely the source of the linear differentiation model.
In vivo studies have shown that IL-7 played a crucial role in formation of CD4~+Tm. L-7-/- mice and IL-7R mutant mice can not from CD4~+Tm. Enhanced IL-7 signaling in the peak proliferation of CD4~+T cells can increase the population of CD4~+Tm in TCR transgenic mice by promoting the proliferation of effective CD4~+T cell,increasing expression of Bcl-2 and preventing death in systole. In the time of T cell contraction to inject IL-2, IL-7 or IL-15 can slow CD8~+T cell contraction. Inject IL-2 or IL-15 at systolic in vivo, the major accumulation of short-term survival effect and memory cells KLRG1~(hi)CD127~(lo), whereas inject IL-7 the mainly major accumulation of long-term survival of memory cells KLRG1~(lo)CD127~(hi). The studies in vivo only show the effect of cytokines on the differentiation of CD4~+Tm, but not show the molecular mechanism of differentiation induce CD4~+Tm. So people isolate CD4~+Tm, to study the molecular mechanism of the stability, re-stimulated survival and proliferation. Two types: spontaneous differentiation of phenotype memory T cell and antigen-specific T cells. Under normal physiological conditions, two types of CD4~+Tm self-stability require IL-7 and IL-15.The rapid proliferation of the phenotype memory CD4~+Tm need MHC-II molecules, whereas the rapid proliferation of the antigen-specific CD4~+Tm main dependent IL-7. IL-7R(CD127) is high expression in the resting T cells, whereas is down quickly in the activated T cells. Only a few can be transformed into Tm cells and expressed IL-7Ra again. It has proven that the expression of IL-7Ra can be used to detection of memory cell precursors in the CD8 ~+ T cells.
Given the current differentiation of CD4~+Tm mainly studies in vivo. In order to further study the mechanism of differentiation of CD4~+Tm, we first establish model of CD4~+Tm which stimulated with IL-7 in vitro and intend the expression of CD44 and CD62L as an indicator by flow cytometry, Then use different combinations of cytokines or cytokine stimulate to observe the capacity of generation CD4~+Tm; and then through the antigen re-stimulation and CFSE labeling to verify CD4~+Tm by proliferation; Finally, to investigate the molecular mechanism of CD4~+Tm by real time quantitative RT-PCR and Western -blotting.
Part I: The establishment of CD4~+Tm generated in vitro
Objective: First consider the use of CD4~+T cells from OT-II mice to create the model. However, due to OT-II mice feeding difficulties, insufficient numbers, so we intend to use FBS as antigen, with CD4~+T cells from wild-type C57BL/6 mice to create the model, simultaneously, with OT-II CD4~+T cells compared to prove the usefulness of C57BL/6 mice model. DC treated with antigen and IL-7 stimulate initial CD4~+T cells in vitro, detect the expression of CD44 and CD62L at different times and re-proliferation condition after antigen stimulation again, the establishment CD4~+Tm generated model to lay the foundation for investigatig the conditions of induction CD4~+Tm generating in vitro. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS or OVA stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced bone marrow-derived mature DC (mature dendritic cell, OVA-DC or FBS-DC). Take spleen from OT-II and C57BL/6 mouse, isolate mononuclear cells and separate CD4~+T cells by immunomagnetic beas under sterile conditions; the OVA-DC and OT-II CD4~+T cells mixed 1:10, FBS-DC and C57BL/6 CD4~+T cells mixed in the same proportion, cultured in medium containing IL-7 (1ng/ml) of 10% FBS-1640. chang half of the medium every 3 days, check the expression of CD44, CD62L by flow cytometry in the 5d, 10d, 15d, 20d, 25d, 30d respectively; At the same time on the 30d, collect cells labeled with CFSE, then stimulate with OVA-DC or FBS-DC for 72 hours, detect cell proliferation by flow cytometry. At the same time, with the CFSE labeled naive OT-II and C57BL/6 CD4~+T cells, then OVA-DC or FBS-DC stimulate for 72 hours, then detect the proliferation by flow cytometry as a control. Results: The naive CD4~+T cells (0d) mostly express CD44~(lo)CD62L~(hi), as the culture time, the expression of CD44 gradually increased, CD62L decreased; in the OVA-DC stimulate CD4~+T cells from OT-II mice group the ratio of CD44~(hi)CD62L~(lo) to 80% at last. The expression pattern of CD4~+Tm is similar in two groups. Proliferation experiments show cultured 30d cells fast response to the same antigen, mostly gather in the third,fourth,fifth or more generates. The results suggest that the phenotype and function of induced cells by this method is in accordance with CD4~+Tm characteristics. Conclusion: In vitro, IL-7 can induce CD4~+ Tm generation. The expression pattern and function of two groups CD4~+Tm are similar, so that, wild-type C57BL/6 mice can instead of OT-II mouse to study the mechanism of CD4~+Tm formation .
Part II: The influence of different cytokines on induction of CD4~+Tm in vitro
Objective: To explore the relationship between a variety of cytokines and CD4~+Tm , we use cytokine IL-2, IL-4, IL-7, IL-15, IFN-γ, TGF-βand Flt-3L to induce of the initial CD4~+T cells in vitro, check the expression of CD44 and CD62L and proliferation re-antigens stimulate. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced FBS-DC. Take spleen from C57BL/6, isolate mononuclear cells and separate CD4~+T cells by immunomagnetic beas under sterile conditions; the FBS-DC and C57BL/6 CD4~+T cells mixed 1:10, respectively, with IL-2 (50U/ml), IL-7 (1ng/ml), IL-15 (5ng/ml), IL-4 (10ng/ml), IFN-γ(10ng/ml), Flt-3L (5ng/ml),TGF-β(1ng/ml), IL-7+IL-2 and IL-7+IL-15 of 10% FBS-1640 culture medium, change half of the medium every 3 days, detect the expression of CD44, CD62L by flow cytometry in the 5d, 10d, 15d, 20d, 25d, 30d respectively; At the same time on the 30th day, collect cells labeled with CFSE, then stimulate with FBS-DC for 72 hours, detect cell proliferation by flow cytometry. Results: Respectively in IL-4, IL-15, Flt-3L, IFN-γand TGF-βculture system alone, cell death of less than 20d, while IL-2, IL-7, IL-7~+IL-2, IL-7~+IL-15 cell culture system survive; as the culture time, CD44 expression gradually increase, CD62L expression decrease; Another cytokine IL-2 and IL-7~+IL-2 training system group the time of its CD44~(hi)CD62L~(lo) cell generation in about 10 days, but the proportion is much higher than other groups. Compared with other groups, proliferation experiments show that IL-2 and IL-7+IL-2 groups of cell proliferation are significantly reduced, no significant difference between them; IL-7+IL-15 group compared with IL-7 alone is no significant difference. The results suggest that IL-2 only can effectively activate CD4~+T cells, which induce the same phenotype as CD4~+Tm cell, but they do not have the capability of rapid proliferation; while in IL-7 co-culture system, it can inhibit the function of IL-7. Conclusion: IL-15 do not induce generation of CD4~+Tm alone in vitro, and can not influence the capability of IL-7. IL-2 do not induce CD4~+Tm, but inhibit the function of IL-7. Literature review, no reports about IL-2 can inhibit IL-7 the induction of CD4~+Tm generated. Because IL-15 also uses IL-2R andγc as part of receptor, and IL-15 do not inhibit IL-7 induced CD4~+Tm generation, indicating that the inhibitory effect of IL-2 may not be through a competitiveγc, but their signal condition.
Part III: The mechanisms of CD4~+Tm generated in vitro
Objective: To preliminary study the mechanism of CD4~+Tm induced by IL-7 in vitro, detect related signaling molecules, transcription molecules and the apoptosis-related protein of CD4~+Tm induced by IL-7. Methods: Take the femur and tibia from C57BL/6 mouse under sterile conditions, extraction of bone marrow cells treated with FBS stimulation in vitro, by adding IL-4 (1ng/ml) and GM-CSF (10ng/ml) induced FBS-DC. Take spleen from C57BL/6, isolate mononuclear cells and separate CD4~+T cells by immunomagnetic beas under sterile conditions; the FBS-DC and C57BL/6 CD4~+T cells mixed 1:10, respectively, with IL-7 (1ng/ml), IL-7+IL-2 of 10% FBS-1640 culture medium, changed half of the medium every 3days, detect of IL-4, IFN-γ, Bcl-2, Bax by Real-time quantitative RT-PCR in the 5d, 10d, 15d, 20d, 25d, 30d respectively and STAT5 /p-STAT5(Y694), AKT/p-AKT, pro-Caspase-3/Active Caspase-3, Bcl-2 by Western -blotting after 30 days. Results: Compared with the naive CD4~+T cells group, IL-7 culture system, IL-4, IFN-γ, p-STAT5, Bcl-2 are significantly higher, Active Caspase-3 is lower. IL-7 may prompt anti-apoptotic protein Bcl-2 through the JAK/STAT. Conclusion: IL-7 plays important role in the activation of naive CD4~+T cells, increasing the expression of Bcl-2 by JAK/STAT5, thereby inhibit the activation of caspase-3, so that, part of the activated CD4~+T cells survive for long term, then formation of CD4~+Tm.
引文
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[40] Hans Dooms, Kristen Wolslegel, Patricia Lin, and Abul K. Abbas. Interleukin-2 enhances CD4 T cell memory by promoting the generation of IL-7Rα–expressing cells.J Exp Med,2007, 204(3):547-557
[41] MacLeod MK, McKee A, Crawford F, White J, Kappler J, Marrack P. CD4 memory T cells divide poorly in response to antigen because of their cytokine profile. Proc Natl Acad Sci USA, 2008 ,105(38):14521-6.
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