CTLA4Ig对NK细胞功能调节及其在大面积烧伤治疗中作用的实验研究

发布时间：2018-03-13 14:17

本文选题：NK细胞　切入点：CTLA4Ig　出处：《第三军医大学》2009年硕士论文　论文类型：学位论文

【摘要】： 大面积烧伤后,患者免疫应答低下(1, 2),容易发生感染,另一方面机体却对皮肤移植物产生强有力的排斥反应,因此在不增加患者感染几率的前提下提高异体皮的存活时间(即所谓免疫耐受的诱导),是大面积烧伤治疗的核心问题。 NK细胞作为天然免疫系统最重要的细胞在机体杀伤肿瘤细胞和抵抗病原体(病毒、细菌等)感染中发挥着重要作用。NK细胞的活化与否受控于NK细胞表面表达的抑制性受体(如NKG2A)和激活性受体(如NKG2D)的状态。然而,在大面积烧伤后,患者NK细胞毒性功能显著下降(26, 27),是机体容易发生感染的重要原因之一。研究发现,院内获得性绿脓杆菌感染是烧伤感染和败血症发生的重要原因(3)。绿脓杆菌分泌的外毒素可抑制NK细胞毒性和IFN-γ的分泌(4),且对现在临床使用的大部分抗生素均耐药(5)。激活性受体NKG2D在NK细胞清除肺绿脓杆菌感染中起了关键作用(6, 7),那么NK细胞功能的增强是否会降低机体播散性感染发生的几率呢? 在免疫耐受诱导的研究中,20年前发现的CTLA4以及在此基础上制备的重组融合蛋白CTLA4Ig最令人瞩目。由于CTLA4Ig能高亲和力地结合抗原递呈细胞上的CD80/CD86,阻断了共刺激信号,使T细胞不能接受第二信号,从而导致T细胞不应答,所以CTLA4Ig是T细胞活化中重要的负调节分子(8)。目前,CTLA4Ig的衍生物-Abatacept和Belatacept分别被用于治疗自身免疫性疾病和控制器官移植免疫排斥反应(9-12)。我们的前期研究结果(13)和文献报道(14)也证实注射CTLA4Ig或创面局部应用CTLA4Ig重组腺病毒均可延长异体皮移植存活期,因此,CTLA4Ig如果可以应用于烧伤患者将会抑制移植物排斥反应,延长移植物的存活时间。那么,在烧伤情况下使用CTLA4Ig会加重烧伤后的病情和增加并发症及死亡率吗? Abatacept是被美国FDA批准用于临床治疗类风湿关节炎的药物,由人CTLA4分子胞外区和突变的IgG1 Fc段融合而成。奇怪的是,这一T细胞强有力的抑制剂应用于人体后,其肿瘤形成、病毒及细菌感染等副作用的发生率却比预期大大降低(15-17)。亦有研究证实,小鼠全身应用两倍剂量于人的药物浓度时,并不影响其抵抗肺结核感染的能力,但这一有悖于常理的现象的机制和意义还是未知数(18)。Ursula Grohmann发现,CTLA4Ig与DCs表面B7分子结合后,向DCs胞内传递激活信号(19)。该研究说明,CTLA4Ig对表达CD80/CD86分子的细胞有调节作用。另有报道,天然免疫系统中最重要的细胞-NK细胞表面也表达CD86分子,且在某些条件下可表达CD80分子(20, 21)。Beissert S发现,长期暴露于紫外线中的CTLA4Ig转基因小鼠,其皮肤肿瘤结节数量明显少于野生型小鼠(22),事实上,NK细胞在皮肤癌细胞杀伤中发挥中重要作用(23)。综上所述,CTLA4Ig可通过阻断T细胞激活的共刺激信号通路抑制皮肤移植免疫排斥反应.其全身应用并不会显著增加机体感染和肿瘤形成的风险.同时,CTLA4Ig可能对表面表达CD80/CD86分子且在控制感染和肿瘤形成的其重要作用的细胞(如NK细胞)有调节作用(23).因此,我们提出如下科学问题:CTLA4Ig是否因为与NK细胞表面B7分子结合增强NK细胞毒性而造成机体清除皮肤癌细胞(或其他肿瘤细胞)能力的增强?其机制如何?这些作用可否解释CTLA4Ig在活体内使用后并不显著降低整体抵抗力的现象? 本实验通过体内、体外试验验证CTLA4Ig对NK细胞功能是否具有调节功能,通过小鼠感染、烫伤和肿瘤模型验证上述调节作用,并初步探讨其分子机制。从而深化对CTLA4Ig免疫调节作用的认识,并为CTLA4Ig应用于大面积烧伤治疗提供理论基础。我们将本研究获得的主要结果和结论归纳如下: 一、CTLA4Ig及其衍生物Abatacept是NK细胞强有力的激活剂: 1、CTLA4Ig在体外可明显增强人PBMC和NK细胞功能: 为了研究CTLA4Ig体外对人PBMC和NK细胞功能有无调节作用,我们利用流式细胞技术检测加入不同浓度的CTLA4Ig后的人PBMC和NK细胞毒性。在本实验系统中,我们发现:1)浓度大于1μg/ml的CTLA4Ig作用于人PBMC和NK细胞后,其杀伤K562细胞的能力(即NK细胞毒性)明显增强;2)加入CTLA4Ig与加入IL-2后对人PBMC和NK细胞毒性调节无明显差异;3)在本实验浓度范围内,人PBMC和NK细胞毒性并没有随CTLA4Ig浓度增加而发生明显变化。上述结果说明CTLA4Ig在体外可明显增强人PBMC和NK细胞毒性,CTLA4Ig与IL-2激活NK细胞毒性的能力相当,且在我们的实验体系中CTLA4Ig对NK细胞毒性调节作用与其浓度无关。 2、CTLA4Ig(Abatacept)在体内可显著增强小鼠NK细胞毒性: 为了研究Abatacept在抑制T细胞活化的同时却并未显著增加肿瘤或感染发生的几率的现象是否与Abatacept对NK细胞功能调节有关,我们将高于临床治疗类风湿关节炎剂量的Abatacept经小鼠尾静脉注射入小鼠体内,比较其脾脏NK细胞毒性与未注射小鼠是否有差异。结果显示注射Abatacept 24h和48h后小鼠脾脏NK细胞毒性较未注射小鼠脾脏NK细胞毒性明显上升。这一结果说明,Abatacept注射小鼠体内后,CTLA4分子可显著增强小鼠NK细胞的毒性。这可能是Abatacept应用并未显著增加肿瘤或感染发生的几率的重要原因。 3、CTLA4Ig增强人NK细胞毒性的作用是CTLA4分子依赖的: 为了明确CTLA4Ig对NK细胞毒性增强的效应是否全部由ADCC引起,我们进行如下实验:首先在体外实验系统中引入可溶性hIgG1或模拟膜表达的hIgG1作为对照,通过比较加入CTLA4Ig与引入FcR信号后的NK细胞毒性,明确ADCC作用在CTLA4Ig对NK细胞调节中的作用。结果显示加入CTLA4Ig后NK细胞毒性增加的比率远高于引入FcR。因此,CTLA4Ig对NK细胞毒性的增强效应主要是由CTLA4分子介导的;为了进一步说明上述问题,我们选用表面仅表达少量低亲和力FcR-CD16,且无CD32和CD64的表达(25)的NK92细胞株作为效应细胞。在NK92细胞和K562共培养体系中加入CTLA4Ig后,NK92细胞的毒性仍然明显增强,证实CTLA4分子在增强NK细胞毒性作用中起绝对的主导作用。因此我们认为体外实验中CTLA4Ig增强人NK细胞毒性的作用是CTLA4分子依赖的。二、CTLA4Ig(Abatacept)可增强机体抗肿瘤的能力: NK细胞在机体抗肿瘤形成中起着至关重要的作用,因此本实验探讨CTLA4Ig对NK细胞毒性增强作用是否有利于机体控制肿瘤形成。我们的实验发现注射超过临床剂量的Abatacept不仅没有因为抑制T细胞激活促进肿瘤形成和生长,相反地,极大地减少了B16细胞肺转移。不仅如此,荷瘤小鼠的生存时间亦明显延长。提示Abatacept可增强机体抗肿瘤(B16)的能力。三、我们对CTLA4Ig(Abatacept)在大面积烧伤治疗中的新认识: 我们的前期研究结果(13)和文献报道(14)都证实注射CTLA4Ig或创面局部应用CTLA4Ig重组腺病毒均可延长异体皮移植存活期,这就意味着CTLA4Ig如果可以应用于烧伤患者将会抑制移植物排斥反应,延长移植物的存活时间。因此,我们利用绿脓杆菌感染模型和烫伤模型评价CTLA4Ig用于烧伤患者治疗皮肤移植排斥反应的安全性,我们的研究首次发现使用Abatacept不仅可以显著提高Balb/c小鼠清除绿脓杆菌的能力而且还能增强烫伤小鼠脾脏细胞的毒性,与体外的实验结果一致。这一研究发现提示CTLA4Ig未来可用于大面积严重烧伤患者以诱导对皮肤移植物的免疫耐受,增强机体天然免疫力。这将是严重烧伤疾病治疗的重大突破。四、CTLA4Ig通过与NK细胞表面CD80/CD86分子结合上调激活性受体NKG2D和NKp44表达,增强NK细胞毒性功能: 1,CTLA4Ig通过与NK细胞表面CD80/CD86分子的结合促进NK细胞的毒性功能:本实验研究抗CD80抗体和抗CD86抗体对NK细胞有无调节作用。结果发现:抗CD80抗体和抗CD86抗体在体外可明显增强人PBMC对K562细胞的毒性,且与CTLA4Ig组NK细胞毒性无明显差异。结合上述实验结果我们认为,CTLA4Ig通过与NK细胞表面CD80/CD86分子的结合促进NK细胞的毒性功能。 2,CTLA4Ig可上调NK细胞表面激活性受体NKG2D、NKp44的表达:我们采用流式细胞技术探讨CTLA4Ig对NK细胞表面NKG2D和NKp44表达的调节作用。结果发现体外加入CTLA4Ig后,人NK细胞表面激活性受体NKG2D、NKp44表达明显上调,且程度与IL-2相似。FcR受体与NK细胞结合不会导致NKG2D、NKp44表达上调。这一结果说明NK细胞表面激活性受体NKG2D、NKp44的在CTLA4Ig增强NK细胞毒性功能的调节作用中可能发挥了重要作用。总结: 一、CTLA4在体内、体外均可增强NK细胞毒性; 二、CTLA4Ig通过与NK细胞表面CD80/CD86分子结合上调NK细胞表面NKG2D和NKp44的表达,增强NK细胞功能; 三、CTLA4Ig及其衍生物(如Abatacept)在全身应用,可增强小鼠抗肿瘤和抗感染的能力,不增加患者罹患肿瘤及感染的几率,扩大了其临床应用的适应症; 四、CTLA4Ig全身应用不仅可治疗皮肤移植物排斥反应,且可增加烫伤小鼠NK细胞毒性,减少播散性感染的发生,为CTLA4Ig应用于治疗烧伤患者提供了新思路; 五、我们发现CTLA4对于获得性免疫系统(T细胞)和天然免疫系统(NK细胞)截然不同的作用,这对我们深入了解免疫系统的调节和内环境的稳定具有重大意义,将为临床肿瘤生物治疗和大面积烧伤救治提供全新的理论基础和研究方向。
[Abstract]:Burn patients with low immune response (1, 2), prone to infection, on the other hand, the body has produced strong rejection of skin grafts, thus improving skin allograft survival time in order not to increase the probability of infection of patients with lower (i.e. so-called induced immune tolerance), is the core issue of treatment burns.
NK cell as the most important natural immune system to kill tumor cells and resistance to pathogens in the body (viruses, bacteria) play an important role in the activation of.NK cells is controlled by the NK cell surface expression of inhibitory receptors in infection (such as NKG2A) and activated receptor (NKG2D) status. However, in after large area burns, significantly decreased NK cell toxicity in patients with (26, 27), the function is one of the important reasons of the body prone to infection. The study found that hospital acquired Pseudomonas aeruginosa infection is an important cause of burn infection and septicemia (3). The secretion of Pseudomonas aeruginosa exotoxin secretion can inhibit NK cell toxicity IFN- and gamma (4), and most of the clinical use of antibiotics now were resistant (5). The activation of receptor NKG2D in NK cell clearance plays a key role in pulmonary Pseudomonas aeruginosa infection (6, 7), then whether the enhanced function of NK cells decreased What is the probability of an organism's disseminated infection?
In the study of immune tolerance, found 20 years ago CTLA4 and on the basis of preparation of the recombinant fusion protein CTLA4Ig is the most remarkable. Because the CTLA4Ig high affinity binding antigen presenting cells on CD80/CD86, blocking the costimulatory signal to T cells can not accept the second signal, which causes the T cells response. So CTLA4Ig is an important negative regulator of T cell activation (8). At present, the derivatives of CTLA4Ig -Abatacept and Belatacept were used for the treatment of autoimmune disease and organ transplantation rejection (9-12). The results of a previous study we have reported in the literature (13) and (14) also confirmed that the injection of CTLA4Ig or wound local application of CTLA4Ig recombinant adenovirus can prolong allograft survival, therefore, if CTLA4Ig can be applied to burn patients will inhibit graft rejection, prolong the survival time of the graft. So, in the case of burn, can the use of CTLA4Ig aggravate the condition after the burn and increase the complications and mortality?
Abatacept is approved by the FDA for the clinical treatment of rheumatoid arthritis, the extracellular domain of human CTLA4 gene and mutation of IgG1 Fc fused together. Strangely, this powerful T cell inhibitor used in the human body, the formation of tumors, the incidence of viral and bacterial infections and other side effects were less than expected greatly reduced (15-17). Some studies have confirmed that the drug concentration in systemic application of two times the dose to the human, does not affect its ability to resist TB infection, but this abnormal phenomenon of the mechanism and significance is still unknown (18).Ursula Grohmann and DCs CTLA4Ig found that the surface of B7 molecules, to intracellular delivery of DCs activation signal (19). The study shows that the regulatory role of CTLA4Ig on the expression of CD80/CD86 molecules in cells. Another report, the most important cell surface of -NK cells in the innate immune system is the expression of CD86, and in a The expression of CD80 may be some conditions (20, 21).Beissert S found that CTLA4Ig transgenic mice chronically exposed to ultraviolet radiation, the skin tumor nodule number was significantly less than the wild-type mice (22), in fact, NK cells play an important role in the destruction of skin cancer cells (23). In summary, by CTLA4Ig blocking the activation of T cell costimulatory signal pathway and inhibition of skin allograft rejection. The systemic application does not significantly increase the risk of infection and tumor formation. At the same time, CTLA4Ig may affect the surface expression of CD80/CD86 molecules and in the control of infection and tumor formation in the role cells (such as NK cells (23) Regulation). Therefore, we put forward the following scientific questions: whether CTLA4Ig and NK because of the combination of cell surface B7 molecules with enhanced NK cell cytotoxicity caused by scavenging of skin cancer cells (or other tumor cells) to enhance the ability of? What is the mechanism? Can these effects explain the fact that CTLA4Ig does not significantly reduce the overall resistance after use in living bodies?
Through the experiments in vivo, in vitro experiments of CTLA4Ig on NK cell function has the function of regulation, the infected mice, verify the regulation effect of scald and tumor model, and to explore its molecular mechanism. In order to deepen the understanding of immune regulation of CTLA4Ig, and provide a theoretical basis for the application of CTLA4Ig in large area burn treatment. We will mainly the results and conclusions of this study are summarized as follows:
First, CTLA4Ig and its derivative, Abatacept, are powerful activators in NK cells.
1, CTLA4Ig can significantly enhance the function of human PBMC and NK cells in vitro.
In order to study the CTLA4Ig in vitro has no moderating effect on human PBMC and NK cell function, we use PBMC and NK cells by flow cytometry with different concentrations of CTLA4Ig. In the experiment system, we found that: 1) the concentration of CTLA4Ig is greater than 1 g/ml in PBMC and NK cells. The ability to kill K562 cells (NK cells toxicity) significantly enhanced; 2) CTLA4Ig and entering IL-2 on PBMC and NK cells regulate no significant difference; 3) in the experimental concentration range, PBMC and NK cell toxicity did not change significantly with the increase of CTLA4Ig concentration. The results show that CTLA4Ig PBMC and NK can significantly enhance the cytotoxicity in vitro, the ability of CTLA4Ig and activation of IL-2 NK cell toxicity, and in the CTLA4Ig experimental system in our independent regulation of its concentration on NK cells.
2, CTLA4Ig (Abatacept) can significantly enhance the toxicity of NK cells in mice in vivo.
In order to study the probability of Abatacept in inhibition of T cell activation but did not significantly increase the tumor or infection phenomenon with Abatacept on NK cell function regulation, we will be higher than the treatment of rheumatoid arthritis clinical dose of Abatacept mice through caudal vein injection into mice, the spleen NK cell toxicity and no injection in mice there are differences. The results showed that the injection of Abatacept 24h and 48h NK cells of spleen toxicity in mice were injected mouse spleen NK cells increased significantly. This result shows that the injection of Abatacept mice after CTLA4 can significantly enhanced NK cell toxicity in mice. This may be an important reason for the application of Abatacept did not significantly increase the risk of tumor or infection happen.
3, the role of CTLA4Ig in enhancing the cytotoxicity of NK cells is dependent on CTLA4 molecules:
In order to clarify whether the effect of CTLA4Ig on NK cells enhanced all caused by ADCC, we performed the following experiments: firstly, soluble hIgG1 or analog film is introduced in vitro expression of hIgG1 by NK cells as control, toxicity CTLA4Ig and introduction of FcR signal, clear the role of ADCC in the regulation of CTLA4Ig on NK cells effect of ratio. The results showed that after adding CTLA4Ig NK increased cytotoxicity is far higher than the introduction of FcR. so the enhancement effects of CTLA4Ig on NK cells is mainly mediated by CTLA4 molecules; in order to further explain the above questions, we select the surface expressed low affinity FcR-CD16, and no expression of CD32 and CD64 (25). NK92 cells were used as effector cells. Co cultured in NK92 cells and K562 into CTLA4Ig system, NK92 cell toxicity still significantly enhanced, confirmed that CTLA4 molecules in enhanced NK cytotoxicity Sex plays an absolute leading role. Therefore, we believe that the effect of CTLA4Ig on enhancing the toxicity of human NK cells in vitro is dependent on CTLA4 molecules.
Two, CTLA4Ig (Abatacept) can enhance the body's ability to antitumor:
NK cells in tumor formation plays an important role, so the experiment of CTLA4Ig on NK cell toxicity enhancement effect is beneficial to the body control. Tumor formation were found than the clinical dose of Abatacept injection did not inhibit T cell activation because promote tumor formation and growth, on the contrary, greatly reduced B16 cells of lung metastasis. Moreover, the survival time of tumor bearing mice was prolonged. Abatacept can enhance the anti-tumor ability (B16).
Three, we have a new understanding of CTLA4Ig (Abatacept) in the treatment of large area burns:
The results of our previous study (13) and (14) reported in the literature have confirmed that prolonged injection CTLA4Ig or wounds CTLA4Ig recombinant adenovirus can be allograft survival, this means that if CTLA4Ig can be applied to burn patients will inhibit graft rejection, prolong the survival time of the graft. Therefore, we use the green aeruginosa infection model and scald model evaluation of CTLA4Ig for the safety of the treatment of burn patients with skin graft rejection, our study is the first to find that Abatacept not only can significantly improve the clearance of Pseudomonas aeruginosa in Balb/c mice but also enhance the ability of scalded mice spleen cell toxicity in vitro, consistent with the experimental results. The research findings suggest that CTLA4Ig in the future can be used for patients with severe burns to the skin to induce graft immune tolerance, enhance the body's natural immunity. It will be strictly A major breakthrough in the treatment of severe burn diseases.
Four, CTLA4Ig increases the expression of activator NKG2D and NKp44 by binding to CD80/CD86 molecules on the surface of NK cells to enhance the toxic function of NK cells:
1, CTLA4Ig promotes the toxicity function of NK cells by binding to cell surface NK CD80/CD86 molecule: the experimental study of anti CD80 antibody and anti CD86 antibody on NK cells have no effect. The results showed that the anti CD80 antibody and anti CD86 antibody can significantly enhance the toxicity of PBMC on K562 cells in vitro, and no obvious difference group CTLA4Ig and NK cell toxicity. It indicated that CTLA4Ig promoted the toxic function of NK cells by binding to cell surface NK CD80/CD86 molecules.
In 2, CTLA4Ig in NK cells was up-regulated by activating receptor NKG2D, NKp44 expression: We used to study the role of CTLA4Ig expression on NK cell surface NKG2D and NKp44 flow cytometry. Results showed that in vitro after joining CTLA4Ig, the surface of human NK cell activating receptor NKG2D, NKp44 achieve a significant increase, and the degree of similarity and IL-2.FcR receptor and NK cell binding does not lead to NKG2D, the expression level of NKp44 increased. These results show that the surface of NK cell activating receptor NKG2D, NKp44 in CTLA4Ig enhancement may play an important role in regulating cellular toxicity of NK function.
Summary:
1. In vivo, CTLA4 can enhance the toxicity of NK cells in vitro.
Two, CTLA4Ig enhanced the function of NK cells by binding up the expression of NKG2D and NKp44 on the surface of NK cells by binding to the surface CD80/CD86 molecules on the surface of NK cells.
Three, CTLA4Ig and its derivatives (such as Abatacept) can enhance the ability of anti-tumor and anti infection in mice, do not increase the risk of tumor and infection, and expand their indications for clinical application.
Four, CTLA4Ig can not only treat skin graft rejection, but also increase NK cytotoxicity and reduce the incidence of disseminated infection, which provides a new idea for CTLA4Ig in the treatment of burn patients.
Five, we found that CTLA4 for the acquired immune system (T cells) and natural immune system (NK cells) from the role of our in-depth understanding of regulation and internal environment of the immune system stability is of great significance, will burn for clinical tumor therapy and treatment of large area provides new theoretical basis and research direction.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392;R644

【共引文献】