IL-4调节人类γδT细胞抗肿瘤免疫功能的作用机制研究
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摘要
γδT细胞是一群不同于αβT细胞的T淋巴细胞,其表面表达由γ链和δ链组成的T细胞受体(T cell receptor, TCR),以主要组织相容性复合物(Major histocompatibility complex, MHC)非限制的方式直接识别并结合抗原分子,在机体抗感染、组织损伤修复及抗肿瘤的天然免疫和适应性免疫应答中发挥着重要的免疫监视及免疫调节作用,被认为是联系天然免疫与适应性免疫的重要桥梁。γδT细胞可通过分泌干扰素(Interferon, IFN)-γ、肿瘤坏死因子(Tumor necrosis factor, TNF)-α、穿孔素-颗粒酶等途径,杀伤上皮组织来源的多种肿瘤细胞,参与人类免疫缺陷病毒(Human immunodeficiency virus, HIV)、Epstein-barr病毒(Epstein-barr virus,EBV)、乙型肝炎病毒(Hepatitis B virus, HBV)等多种病毒感染的免疫监视过程。
白细胞介素-4(IL-4)是具有多种生物学功能的细胞因子,其经典的作用包括刺激B细胞、促使B细胞向浆细胞分化以及诱导B细胞分泌IgE;促使T辅助(Th)细胞向Th2型细胞分化,并抑制Th1型细胞产生。IL-4也是肿瘤免疫中的关键细胞因子之一。尽管早期认为IL-4具有促使机体消除肿瘤的作用,然而更多的研究表明IL-4可能更倾向于抑制机体抗肿瘤的作用。本实验室长期致力于γδT细胞的研究工作,在实验过程中观察到IL-4对于γδT细胞的活化和增殖具有一定的调节作用。因此,本研究试图阐明IL-4和γδT细胞免疫功能之间的联系,从而揭示IL-4在γδT细胞抗肿瘤中的作用机制。
本论文第一部分观察了IL-4对Vδ1T细胞或Vδ2T细胞活化的影响,以及IL-4对γδT细胞活化后增殖的影响。
实验中采用固相化抗Vδ1抗体特异性活化初始Vδ1T细胞,或用帕米磷酸钠或唑来膦酸钠特异性活化初始Vδ2T细胞,在培养过程中,观察IL-4对Vδ1或Vδ2T细胞活化的影响。流式分析结果表明,IL-4不仅能显著抑制抗Vδ1抗体引起的Vδ1T细胞活化,也能抑制帕米磷酸钠引起的Vδ2T细胞活化。然而,当将已活化的YδT细胞继续培养时,我们发现IL-4能促进已经活化的Vδ1或Vδ2T细胞的增殖。因此,这部分实验结果表明,初始γδT细胞在接受TCR信号刺激时如果同时接受IL-4的作用,γδ T细胞的活化将受到抑制；而已经活化的γδT细胞在接受IL-4的作用时,IL-4能促进其增殖。
随后,我们对上述实验现象的作用机制进行了研究。通过与IL-13比较,我们确定IL-4主要通过Ⅰ型IL-4受体发挥作用。通过细胞核内信号分子染色观察到IL-4作用于静息γδT细胞后不仅促使Stat6磷酸化,也促使Stat5磷酸化。这与已知IL-4Rα1介导Stat6磷酸化以及IL-2(γc链)介导Stat5磷酸化的文献报道结果相一致。另一方面,静息γδT细胞在接受TCR活化信号刺激时,能迅速引起SLP76、PLCγ1和Erkl/2的磷酸化以及Ca2+释放,而IL-4则能明显抑制TCR信号通路中关键信号分子SLP76、PLCγ1和Erkl/2的磷酸化以及Ca2+水平,从而抑制TCR活化引起的γδT细胞信号传导。
上述结果表明,IL-4可抑制γδT细胞活化。为了验证IL-4是否通过活化Stat6抑制TCR活化信号,我们采用RNA干扰技术敲低γδT细胞内的Stat6表达,进而采用Phosflow方法观察Stat6-γδ T细胞在接受TCR信号时上述信号分子SLP76、 PLCγ1和Erkl/2的磷酸化水平,并采用激光共聚焦显微技术观察胞内Ca2+变化。结果表明,γδT细胞在敲低Stat6后,IL-4抑制γδT细胞SLP76、PLCyl和Erk1/2磷酸化和Ca2+释放的作用均减弱。为了验证IL-4通过活化Stat6促进γδT细胞增殖的作用,我们同样观察了Stat6-γδT细胞在IL-4作用条件下的增殖情况。实验结果表明,IL-4促进活化γδT细胞增殖的作用因Stat6表达被敲低而被阻断。
在第二部分研究中我们在体外采用抗TCR pan γδ抗体同时活化Vδ1和Vδ2T细胞,进一步观察了IL-4的作用。结果表明,在采用抗TCR pan γδ抗体同时活化Vδ1T和V62T细胞时,IL-4可以造成Vδ1T细胞和Vδ2T细胞之间的比例失衡,向Vδ1T细胞偏倚。这与之前IL-4单独作用于Vδ2T细胞时促使其增殖的实验现象相矛盾。
为了解释这一现象,我们首先观察了V61T细胞是否对Vδ2T细胞具有抑制作用。Transwell结果表明,Vδ1T细胞确实对Vδ2T细胞的增殖具有抑制作用,并且这种抑制作用不依赖于细胞间的直接接触,而是通过可以透过0.4μm隔膜的可溶性因子实现的。为了探明是何种因子发挥了抑制作用,我们在上述实验体系中分别加入了IL-4、IL-10、IL-13和TGF-β的阻断抗体。实验结果表明在加入抗IL-10抗体时能阻断IL-4促进γδT细胞向Vδ1T细胞偏倚的作用,而抗IL-13或抗TGF-β抗体无此作用。随后的实验发现IL-10具有直接抑制Vδ2T细胞活化和增殖的作用,而对于Vδ1T细胞则作用较弱。
为了验证Vδ1T细胞是抑制性细胞因子IL-10的主要来源,我们通过磁珠分选技术分离获得Vδ1和Vδ2T细胞,对两类细胞在活化前和活化后的IL-10分泌水平进行分析,并对两类细胞进行胞内染色。结果表明,Vδ1T细胞在活化后分泌高水平的IL-10,而Vδ2T细胞的IL-10分泌量极低。通过流式技术进行胞内染色的结果同样提示,Vδl T细胞胞内表达有IL-10,而在Vδ2T细胞中则未检测到IL-10。此外,研究中发现IL-4能诱导活化的Vδ2T细胞进一步表达IL-10受体CD210,而作用于活化的Vδ1T细胞时,CD210表达则无明显变化。同时IL-4促使活化的Vδ1T细胞表达IL-4受体CD124,而作用于Vδ2T细胞时,CD124表达无明显变化。这些数据提示,IL-4导致γδT细胞向Vδ1T细胞偏倚的原因可能是由Vδ1T细胞分泌的IL-10所致。
为了观察γδT细胞向Vδ1T细胞偏倚带来的生物学意义,我们通过磁珠分选技术分离获得了Vδ1T细胞和Vδ2T细胞,对两类细胞在活化前和活化后的表面分子、细胞因子和转录因子进行分析,并对这两类细胞的杀伤功能进行了研究。结果表明,Vδ1T细胞和Vδ2T细胞的细胞因子分泌谱存在明显差异。在检测的10种细胞因子中,其中包括了Th1、Th2、Th17特异性细胞因子,Vδ1T细胞在活化后除了分泌高水平的IL-10以外,仅分泌少量IFNγ(约400pg/mL)和TNFa(约70pg/mL),其他细胞因子则均低于检测限。而Vδ2T细胞在活化后分泌高水平的IFNγ(3ng/mL)和TNFα(约600pg/mL),以及一定量的IL-4(90pg/mL)和IL-5(300pg/mL)。
在细胞表型方面,Vδ2T细胞在活化后均表达杀伤性NKG2D,而Vδ1T细胞在活化时部分细胞不表达NKG2D,并且IL-4能明显抑制Vδ1T细胞NKG2D的表达。为了确定Vδ1和Vδ2T细胞的特征性转录因子,对核内三种特异性T细胞转录因子Gata3、Foxp3以及T-bet进行了染色,结果表明两种细胞均表达T-bet,尤其是Vδ2T细胞表达高水平的T-bet。但两种细胞均不表达Gata3和Foxp3。
在比较Vδ1T细胞和Vδ2T细胞杀伤肿瘤细胞的作用时,发现两种细胞在杀伤MGC803、 K562和G401肿瘤细胞时,Vδ1T细胞的作用均显著弱于Vδ2T细胞。
综上所述,IL-4主要通过两种方式对γδT细胞的免疫应答发挥负向调节作用：第一、抑制γδT细胞活化；第二、促使γδT细胞向Vδ1T细胞偏倚,后者通过分泌大量IL-10间接抑制具有强细胞毒活性的Vδ2T细胞的增殖。此外,IL-4还抑制Vδ1T细胞表达杀伤性受体NKG2D。因此,Vδ1T细胞偏倚将导致γδT细胞介导的免疫功能减弱。
本研究揭示了IL-4调节γδT细胞免疫应答的作用机制,不仅有利于对γδT细胞生物学作用的全面理解,而且有助于指导基于γδT细胞的生物学治疗。
γδ T cells, a group of T-lymphocytes that are different from αβ T cells, can express on its surface the T-cell receptor (TCR) composed of y chain and δ chain. γδ T cells can directly recognize and bind the antigen molecules in a major histocompatibility complex (MHC)-unrestricted fashion, and play an important role of immune surveillance and immune regulation in the innate immunity and adaptive immunity of anti-infection, tissue repair and anti-tumor. These cells have been recognized as an important bridge between innate immunity and adaptive immunity. y8T cells can kill a variety of tumor cells from the epithelial tissue through the secretion of interferon-y (IFNy), tumor necrosis factor-a (TNFa) and perforin-granzyme, and get involved in the immune surveillance of infection caused by various virus such as human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), hepatitis B virus (HBV), etc.
Interleukin-4(IL-4), a cytokine with a variety of biological functions, has the main biological effects of stimulating B cells, promoting the differentiation of B cell into plasma cells, and inducing the excretion of IgE by B cells. IL-4can promote the differentiation of Tho cells into Th2cell, and inhibit the production of Th1cells. IL-4is also one of the key cytokines in the process of tumor immunity. It has been previously considered that IL-4can promote the elimination of tumor, but more studies have shown that IL-4may be more likely to inhibit the anti-tumor effect of the body. Our laboratory has been committed to research work of γδ T cells for a long time. It has been observed in the experimental process that IL-4can play a regulatory role in the activation and proliferation of y8T cells. Therefore, this study was designed to clarify the relationship between IL-4and the immune function of γδ T cells, and thus to reveal the effect of IL-4on the anti-tumor process of γδ T cells.
The first part of this paper was to observe the effect of IL-4on the activation of V81T cells or V82T cells induced by TCR activation signals, and on the activated y8T cell.
In this experiment, the naive Vδ1T cells were specifically activated by the immobilized anti-V81antibody, or the naive V82T cells were specifically activated by pamidronate disodium or zoledronic acid. The effect of IL-4on the activation of V81T cells or V82T cells was observed during the culture of cells. Flow cytometry analysis indicated that IL-4can not only significantly inhibit the activation of V81T cells induced by the anti-Vδ1antibody, but also inhibit the activation of V82T cells induced by pamidronate disodium. However, when the activated γδ T cells were further cultured, IL-4was found to be capable of promoting the proliferation of activated Vδ1T cells or Vδ2T cells. Therefore, according to this part of the experimental results, the activation of γδ T cells caused by the stimulation of TCR signals would be inhibited if the naive γδ T cells received the treatment of IL-4simultaneously, while the proliferation of activated γδ T cells would be promoted by IL-4.
Then, we studied the mechanism of the phenomenon of the above experiment. Compared with IL-13, we determined that the IL-4played its role through type Ⅰ IL-4receptor. Not only Stat6phosphorylation but also Stat5phosphorylation was observed in IL-4treated γδ T cells through staining of nucleus signaling molecules. It is consistent with the known conclusion that the IL-4Rα1mediated the phosphorylation of Stat6and IL-2(γc chain) mediated the phosphorylation of Stat5. On the other hand, resting γδ T cells under TCR activation signal stimulation can quickly trigger the phosphorylation of SLP76, PLCy1and ERK1/2and Ca2+release, while IL-4could significantly inhibit the key signaling molecules SLP76, PLCγ1and Erk1/2phosphorylation in TCR signaling pathway, as well as Ca2+levels, thereby inhibiting the TCR activation of γδ T cell signaling.
These results suggest that IL-4can inhibit the activation of γδ T cells. For the purpose to verify whether the inhibitory effect of IL-4on TCR activation signal was mediated through the activation of Stat6, the Stat6expression of the γδ T cells was inhibited using RNA interference, then we observed the phosphorylation of SLP76, PLCγ1and ERK1/2and the Ca2+release in Stat6-γδ Tcells when received TCR stimulation and examined the intracellular Ca2+changes using laser scanning confocal microscopy. The results showed that the inhibitory effect of IL-4on the phosphorylation of SLP76, PLCγ1and ERK1/2. as well as Ca2+release, was suppressed in Stat6-γδ T cells. In order to verify the role of IL-4to promote the proliferation of γδ T cells by activation of the Stat6, we also observed the proliferation of Stat6-γδ T cells under the condition of IL-4treatment. The experimental results showed that the effect of IL-4in promoting the proliferation was blocked due to the inhibition of Stat6expression.
In the second part of the experiment, to further observe the role of IL-4, the naive Vδ1T cells and Vδ2T cells were activated by the immobilized anti-TCR pan γδ antibody in vitro. The results showed that IL-4promoted Vδ1T cell growth, and inhibited Vδ2T cell growth, resulting in the balance between Vδ1T cells and Vδ2T cells to Vδ1T cells bias. But this is contradictiory with the previous results that IL-4promoted the proliferation of Vδ2T cells.
To explain this phenomenon, we first observed whether Vδ1T cells can inhibit Vδ2T cells. Transwell results showed that Vδ1T cells did inhibit Vδ2T cell proliferation, and this inhibition was not dependent on the direct cell-cell contact but rather achieved through a certain soluble factors. In order to investigate which factors play the inhibitory role, anti-IL-4antibody, anti-IL-10antibody, anti-IL-13antibody or anti-TGF-beta antibody was added in the above-mentioned experimental system. The results of cytokine blocking experiments showed that the addition of anti-IL-10antibody can block IL-4to promote γδ T cells balance to Vδ1T cells bias, while the addition of anti-IL-13or anti-TGF-beta antibody had no effect. And the inhibitory effect of IL-10on the activation and proliferation of Vδ2T cells was observed while the effect of IL-10on Vδ1T cells is very weak.
In order to verify Vδ1T cell is the main source of inhibitory cytokine IL-10, Vδ1and Vδ2T cells were separated by magnetic bead separation technology. And the secretion levels and intracellular staining of IL-10before and after activation was examined. The results showed that V81T cells after activation secreted high level of IL-10, while the IL-10secretion from Vδ2T cell is minimal. Intracellular staining results through flow cytometry also suggested that IL-10was expressed in Vδ1T cell, while IL-10expression in Vδ2T cells was not detected. Furthermore, the study found that IL-4can induce the further expression of CD210on Vδ2T cells, while the CD210expression on Vδ1T cells did not change significantly after IL-4treatment. IL-4induced the further expression of CD124on activated V81T cells while CD124expression on Vδ1T cells was not significantly changed after IL-4treatment. The data indicated that the effect of IL-4-induced Vδ1T cells bias was mediated by IL-10, which was secreted by Vδ1T cells.
In order to confirm the biological significance caused by Vδ1T cells bias, Vδ1T cells and Vδ2T cells were obtained by magnetic bead separation technology. And the surface molecules, cytokines and transcription factors, as well as the killing function of these two kinds of cells, were analyzed before and after activation. The results showed that there were significant differences of cytokine secretion spectrum between Vδ1T cells and V82T cell. In the10kinds of cytokines detected, including Thl, Th2, Th17specific cytokines, Vδ1T cells secreted high level of IL-10and only a small amount of IFNy (400pg/mL) and TNFa (～70pg/mL), and other cytokines were below the detection limit. V82T cells secreted high level of IFNy (3ng/mL) and TNFa (-600pg/mL), and a certain amount of IL-4(90pg/mL) and IL-5(300pg/mL) when activated, but almost no IL-10was detected.
For cell phenotype, all V82T cells expressed NKG2D receptor while there were some NKG2DV81T cells after activation. Especially IL-4significantly inhibited the expression of NKG2D on V81T cells. In order to determine the characteristic transcription factor for V81T cells and V82T cells, three-specific Th cell transcription factors Gata3, Foxp3and T-bet were stained. The data showed that both cells expressed T-bet, especially V82T cells expressed high level of T-bet. However, both types of cells did not express Gata3and Foxp3.
It was demonstrated that V81T cells were significantly weaker than V82T cells in killing tumor cells MGC803, K562and G401.
Thus, we demonstrated that IL-4played a major role in the negative regulation for y8T cell immune response. First, IL-4inhibited the activation of y8T cells; secondly, IL-4polarized γδ T cells toward V81T cells, which in turn inhibited the growth of V82cells through the secretion of large amount of IL-10. In addtion, IL-4also can downregulate in vitro the expression of NK.G2D of Vδ1T cell. Therefore, V81T cells bias weakened the overall immune response mediated by y8T cells.
This study revealed the mechanism of action that IL-4regulated y8T cell immune response, not only conducive to a comprehensive understanding on the biological role of γδ T cells, but also helpful to guide the γδ T cell-based biological therapy.
白细胞介素-4(IL-4)是具有多种生物学功能的细胞因子,其经典的作用包括刺激B细胞、促使B细胞向浆细胞分化以及诱导B细胞分泌IgE;促使T辅助(Th)细胞向Th2型细胞分化,并抑制Th1型细胞产生。IL-4也是肿瘤免疫中的关键细胞因子之一。尽管早期认为IL-4具有促使机体消除肿瘤的作用,然而更多的研究表明IL-4可能更倾向于抑制机体抗肿瘤的作用。本实验室长期致力于γδT细胞的研究工作,在实验过程中观察到IL-4对于γδT细胞的活化和增殖具有一定的调节作用。因此,本研究试图阐明IL-4和γδT细胞免疫功能之间的联系,从而揭示IL-4在γδT细胞抗肿瘤中的作用机制。
本论文第一部分观察了IL-4对Vδ1T细胞或Vδ2T细胞活化的影响,以及IL-4对γδT细胞活化后增殖的影响。
实验中采用固相化抗Vδ1抗体特异性活化初始Vδ1T细胞,或用帕米磷酸钠或唑来膦酸钠特异性活化初始Vδ2T细胞,在培养过程中,观察IL-4对Vδ1或Vδ2T细胞活化的影响。流式分析结果表明,IL-4不仅能显著抑制抗Vδ1抗体引起的Vδ1T细胞活化,也能抑制帕米磷酸钠引起的Vδ2T细胞活化。然而,当将已活化的YδT细胞继续培养时,我们发现IL-4能促进已经活化的Vδ1或Vδ2T细胞的增殖。因此,这部分实验结果表明,初始γδT细胞在接受TCR信号刺激时如果同时接受IL-4的作用,γδ T细胞的活化将受到抑制；而已经活化的γδT细胞在接受IL-4的作用时,IL-4能促进其增殖。
随后,我们对上述实验现象的作用机制进行了研究。通过与IL-13比较,我们确定IL-4主要通过Ⅰ型IL-4受体发挥作用。通过细胞核内信号分子染色观察到IL-4作用于静息γδT细胞后不仅促使Stat6磷酸化,也促使Stat5磷酸化。这与已知IL-4Rα1介导Stat6磷酸化以及IL-2(γc链)介导Stat5磷酸化的文献报道结果相一致。另一方面,静息γδT细胞在接受TCR活化信号刺激时,能迅速引起SLP76、PLCγ1和Erkl/2的磷酸化以及Ca2+释放,而IL-4则能明显抑制TCR信号通路中关键信号分子SLP76、PLCγ1和Erkl/2的磷酸化以及Ca2+水平,从而抑制TCR活化引起的γδT细胞信号传导。
上述结果表明,IL-4可抑制γδT细胞活化。为了验证IL-4是否通过活化Stat6抑制TCR活化信号,我们采用RNA干扰技术敲低γδT细胞内的Stat6表达,进而采用Phosflow方法观察Stat6-γδ T细胞在接受TCR信号时上述信号分子SLP76、 PLCγ1和Erkl/2的磷酸化水平,并采用激光共聚焦显微技术观察胞内Ca2+变化。结果表明,γδT细胞在敲低Stat6后,IL-4抑制γδT细胞SLP76、PLCyl和Erk1/2磷酸化和Ca2+释放的作用均减弱。为了验证IL-4通过活化Stat6促进γδT细胞增殖的作用,我们同样观察了Stat6-γδT细胞在IL-4作用条件下的增殖情况。实验结果表明,IL-4促进活化γδT细胞增殖的作用因Stat6表达被敲低而被阻断。
在第二部分研究中我们在体外采用抗TCR pan γδ抗体同时活化Vδ1和Vδ2T细胞,进一步观察了IL-4的作用。结果表明,在采用抗TCR pan γδ抗体同时活化Vδ1T和V62T细胞时,IL-4可以造成Vδ1T细胞和Vδ2T细胞之间的比例失衡,向Vδ1T细胞偏倚。这与之前IL-4单独作用于Vδ2T细胞时促使其增殖的实验现象相矛盾。
为了解释这一现象,我们首先观察了V61T细胞是否对Vδ2T细胞具有抑制作用。Transwell结果表明,Vδ1T细胞确实对Vδ2T细胞的增殖具有抑制作用,并且这种抑制作用不依赖于细胞间的直接接触,而是通过可以透过0.4μm隔膜的可溶性因子实现的。为了探明是何种因子发挥了抑制作用,我们在上述实验体系中分别加入了IL-4、IL-10、IL-13和TGF-β的阻断抗体。实验结果表明在加入抗IL-10抗体时能阻断IL-4促进γδT细胞向Vδ1T细胞偏倚的作用,而抗IL-13或抗TGF-β抗体无此作用。随后的实验发现IL-10具有直接抑制Vδ2T细胞活化和增殖的作用,而对于Vδ1T细胞则作用较弱。
为了验证Vδ1T细胞是抑制性细胞因子IL-10的主要来源,我们通过磁珠分选技术分离获得Vδ1和Vδ2T细胞,对两类细胞在活化前和活化后的IL-10分泌水平进行分析,并对两类细胞进行胞内染色。结果表明,Vδ1T细胞在活化后分泌高水平的IL-10,而Vδ2T细胞的IL-10分泌量极低。通过流式技术进行胞内染色的结果同样提示,Vδl T细胞胞内表达有IL-10,而在Vδ2T细胞中则未检测到IL-10。此外,研究中发现IL-4能诱导活化的Vδ2T细胞进一步表达IL-10受体CD210,而作用于活化的Vδ1T细胞时,CD210表达则无明显变化。同时IL-4促使活化的Vδ1T细胞表达IL-4受体CD124,而作用于Vδ2T细胞时,CD124表达无明显变化。这些数据提示,IL-4导致γδT细胞向Vδ1T细胞偏倚的原因可能是由Vδ1T细胞分泌的IL-10所致。
为了观察γδT细胞向Vδ1T细胞偏倚带来的生物学意义,我们通过磁珠分选技术分离获得了Vδ1T细胞和Vδ2T细胞,对两类细胞在活化前和活化后的表面分子、细胞因子和转录因子进行分析,并对这两类细胞的杀伤功能进行了研究。结果表明,Vδ1T细胞和Vδ2T细胞的细胞因子分泌谱存在明显差异。在检测的10种细胞因子中,其中包括了Th1、Th2、Th17特异性细胞因子,Vδ1T细胞在活化后除了分泌高水平的IL-10以外,仅分泌少量IFNγ(约400pg/mL)和TNFa(约70pg/mL),其他细胞因子则均低于检测限。而Vδ2T细胞在活化后分泌高水平的IFNγ(3ng/mL)和TNFα(约600pg/mL),以及一定量的IL-4(90pg/mL)和IL-5(300pg/mL)。
在细胞表型方面,Vδ2T细胞在活化后均表达杀伤性NKG2D,而Vδ1T细胞在活化时部分细胞不表达NKG2D,并且IL-4能明显抑制Vδ1T细胞NKG2D的表达。为了确定Vδ1和Vδ2T细胞的特征性转录因子,对核内三种特异性T细胞转录因子Gata3、Foxp3以及T-bet进行了染色,结果表明两种细胞均表达T-bet,尤其是Vδ2T细胞表达高水平的T-bet。但两种细胞均不表达Gata3和Foxp3。
在比较Vδ1T细胞和Vδ2T细胞杀伤肿瘤细胞的作用时,发现两种细胞在杀伤MGC803、 K562和G401肿瘤细胞时,Vδ1T细胞的作用均显著弱于Vδ2T细胞。
综上所述,IL-4主要通过两种方式对γδT细胞的免疫应答发挥负向调节作用：第一、抑制γδT细胞活化；第二、促使γδT细胞向Vδ1T细胞偏倚,后者通过分泌大量IL-10间接抑制具有强细胞毒活性的Vδ2T细胞的增殖。此外,IL-4还抑制Vδ1T细胞表达杀伤性受体NKG2D。因此,Vδ1T细胞偏倚将导致γδT细胞介导的免疫功能减弱。
本研究揭示了IL-4调节γδT细胞免疫应答的作用机制,不仅有利于对γδT细胞生物学作用的全面理解,而且有助于指导基于γδT细胞的生物学治疗。
γδ T cells, a group of T-lymphocytes that are different from αβ T cells, can express on its surface the T-cell receptor (TCR) composed of y chain and δ chain. γδ T cells can directly recognize and bind the antigen molecules in a major histocompatibility complex (MHC)-unrestricted fashion, and play an important role of immune surveillance and immune regulation in the innate immunity and adaptive immunity of anti-infection, tissue repair and anti-tumor. These cells have been recognized as an important bridge between innate immunity and adaptive immunity. y8T cells can kill a variety of tumor cells from the epithelial tissue through the secretion of interferon-y (IFNy), tumor necrosis factor-a (TNFa) and perforin-granzyme, and get involved in the immune surveillance of infection caused by various virus such as human immunodeficiency virus (HIV), Epstein-Barr virus (EBV), hepatitis B virus (HBV), etc.
Interleukin-4(IL-4), a cytokine with a variety of biological functions, has the main biological effects of stimulating B cells, promoting the differentiation of B cell into plasma cells, and inducing the excretion of IgE by B cells. IL-4can promote the differentiation of Tho cells into Th2cell, and inhibit the production of Th1cells. IL-4is also one of the key cytokines in the process of tumor immunity. It has been previously considered that IL-4can promote the elimination of tumor, but more studies have shown that IL-4may be more likely to inhibit the anti-tumor effect of the body. Our laboratory has been committed to research work of γδ T cells for a long time. It has been observed in the experimental process that IL-4can play a regulatory role in the activation and proliferation of y8T cells. Therefore, this study was designed to clarify the relationship between IL-4and the immune function of γδ T cells, and thus to reveal the effect of IL-4on the anti-tumor process of γδ T cells.
The first part of this paper was to observe the effect of IL-4on the activation of V81T cells or V82T cells induced by TCR activation signals, and on the activated y8T cell.
In this experiment, the naive Vδ1T cells were specifically activated by the immobilized anti-V81antibody, or the naive V82T cells were specifically activated by pamidronate disodium or zoledronic acid. The effect of IL-4on the activation of V81T cells or V82T cells was observed during the culture of cells. Flow cytometry analysis indicated that IL-4can not only significantly inhibit the activation of V81T cells induced by the anti-Vδ1antibody, but also inhibit the activation of V82T cells induced by pamidronate disodium. However, when the activated γδ T cells were further cultured, IL-4was found to be capable of promoting the proliferation of activated Vδ1T cells or Vδ2T cells. Therefore, according to this part of the experimental results, the activation of γδ T cells caused by the stimulation of TCR signals would be inhibited if the naive γδ T cells received the treatment of IL-4simultaneously, while the proliferation of activated γδ T cells would be promoted by IL-4.
Then, we studied the mechanism of the phenomenon of the above experiment. Compared with IL-13, we determined that the IL-4played its role through type Ⅰ IL-4receptor. Not only Stat6phosphorylation but also Stat5phosphorylation was observed in IL-4treated γδ T cells through staining of nucleus signaling molecules. It is consistent with the known conclusion that the IL-4Rα1mediated the phosphorylation of Stat6and IL-2(γc chain) mediated the phosphorylation of Stat5. On the other hand, resting γδ T cells under TCR activation signal stimulation can quickly trigger the phosphorylation of SLP76, PLCy1and ERK1/2and Ca2+release, while IL-4could significantly inhibit the key signaling molecules SLP76, PLCγ1and Erk1/2phosphorylation in TCR signaling pathway, as well as Ca2+levels, thereby inhibiting the TCR activation of γδ T cell signaling.
These results suggest that IL-4can inhibit the activation of γδ T cells. For the purpose to verify whether the inhibitory effect of IL-4on TCR activation signal was mediated through the activation of Stat6, the Stat6expression of the γδ T cells was inhibited using RNA interference, then we observed the phosphorylation of SLP76, PLCγ1and ERK1/2and the Ca2+release in Stat6-γδ Tcells when received TCR stimulation and examined the intracellular Ca2+changes using laser scanning confocal microscopy. The results showed that the inhibitory effect of IL-4on the phosphorylation of SLP76, PLCγ1and ERK1/2. as well as Ca2+release, was suppressed in Stat6-γδ T cells. In order to verify the role of IL-4to promote the proliferation of γδ T cells by activation of the Stat6, we also observed the proliferation of Stat6-γδ T cells under the condition of IL-4treatment. The experimental results showed that the effect of IL-4in promoting the proliferation was blocked due to the inhibition of Stat6expression.
In the second part of the experiment, to further observe the role of IL-4, the naive Vδ1T cells and Vδ2T cells were activated by the immobilized anti-TCR pan γδ antibody in vitro. The results showed that IL-4promoted Vδ1T cell growth, and inhibited Vδ2T cell growth, resulting in the balance between Vδ1T cells and Vδ2T cells to Vδ1T cells bias. But this is contradictiory with the previous results that IL-4promoted the proliferation of Vδ2T cells.
To explain this phenomenon, we first observed whether Vδ1T cells can inhibit Vδ2T cells. Transwell results showed that Vδ1T cells did inhibit Vδ2T cell proliferation, and this inhibition was not dependent on the direct cell-cell contact but rather achieved through a certain soluble factors. In order to investigate which factors play the inhibitory role, anti-IL-4antibody, anti-IL-10antibody, anti-IL-13antibody or anti-TGF-beta antibody was added in the above-mentioned experimental system. The results of cytokine blocking experiments showed that the addition of anti-IL-10antibody can block IL-4to promote γδ T cells balance to Vδ1T cells bias, while the addition of anti-IL-13or anti-TGF-beta antibody had no effect. And the inhibitory effect of IL-10on the activation and proliferation of Vδ2T cells was observed while the effect of IL-10on Vδ1T cells is very weak.
In order to verify Vδ1T cell is the main source of inhibitory cytokine IL-10, Vδ1and Vδ2T cells were separated by magnetic bead separation technology. And the secretion levels and intracellular staining of IL-10before and after activation was examined. The results showed that V81T cells after activation secreted high level of IL-10, while the IL-10secretion from Vδ2T cell is minimal. Intracellular staining results through flow cytometry also suggested that IL-10was expressed in Vδ1T cell, while IL-10expression in Vδ2T cells was not detected. Furthermore, the study found that IL-4can induce the further expression of CD210on Vδ2T cells, while the CD210expression on Vδ1T cells did not change significantly after IL-4treatment. IL-4induced the further expression of CD124on activated V81T cells while CD124expression on Vδ1T cells was not significantly changed after IL-4treatment. The data indicated that the effect of IL-4-induced Vδ1T cells bias was mediated by IL-10, which was secreted by Vδ1T cells.
In order to confirm the biological significance caused by Vδ1T cells bias, Vδ1T cells and Vδ2T cells were obtained by magnetic bead separation technology. And the surface molecules, cytokines and transcription factors, as well as the killing function of these two kinds of cells, were analyzed before and after activation. The results showed that there were significant differences of cytokine secretion spectrum between Vδ1T cells and V82T cell. In the10kinds of cytokines detected, including Thl, Th2, Th17specific cytokines, Vδ1T cells secreted high level of IL-10and only a small amount of IFNy (400pg/mL) and TNFa (～70pg/mL), and other cytokines were below the detection limit. V82T cells secreted high level of IFNy (3ng/mL) and TNFa (-600pg/mL), and a certain amount of IL-4(90pg/mL) and IL-5(300pg/mL) when activated, but almost no IL-10was detected.
For cell phenotype, all V82T cells expressed NKG2D receptor while there were some NKG2DV81T cells after activation. Especially IL-4significantly inhibited the expression of NKG2D on V81T cells. In order to determine the characteristic transcription factor for V81T cells and V82T cells, three-specific Th cell transcription factors Gata3, Foxp3and T-bet were stained. The data showed that both cells expressed T-bet, especially V82T cells expressed high level of T-bet. However, both types of cells did not express Gata3and Foxp3.
It was demonstrated that V81T cells were significantly weaker than V82T cells in killing tumor cells MGC803, K562and G401.
Thus, we demonstrated that IL-4played a major role in the negative regulation for y8T cell immune response. First, IL-4inhibited the activation of y8T cells; secondly, IL-4polarized γδ T cells toward V81T cells, which in turn inhibited the growth of V82cells through the secretion of large amount of IL-10. In addtion, IL-4also can downregulate in vitro the expression of NK.G2D of Vδ1T cell. Therefore, V81T cells bias weakened the overall immune response mediated by y8T cells.
This study revealed the mechanism of action that IL-4regulated y8T cell immune response, not only conducive to a comprehensive understanding on the biological role of γδ T cells, but also helpful to guide the γδ T cell-based biological therapy.
引文
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3. Kabelitz, D., D. Wesch, and W. He, Perspectives of gammadelta T cells in tumor immunology. Cancer Res,2007.67(1):p.5-8.
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5. Gao, Y., et al., Gamma delta T cells provide an early source of interferon gamma in tumor immunity. J Exp Med,2003.198(3):p.433-42.
6. Ottones, F., et al., V gamma 9V delta 2 T cells impair intracellular multiplication of Brucella suis in autologous monocytes through soluble factor release and contact-dependent cytotoxic effect. J Immunol,2000.165(12):p. 7133-9.
7. Dieli, F., et al., Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes. J Infect Dis, 2001.184(8):p.1082-5.
8. Oliaro, J., et al., Vgamma9Vdelta2 Tcells use a combination of mechanisms to limit the spread of the pathogenic bacteria Brucella. J Leukoc Biol,2005.77(5): p.652-60.
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