【作者】 赵毅；

【导师】 蔡真；

【作者基本信息】 浙江大学 ， 内科学， 2011， 博士

【摘要】 造血干细胞移植(hematopoietic stem cell transplantation,HSCT)是当今治疗恶性血液肿瘤的重要手段,移植物抗宿主病(graft-versus-host disease,GVHD)等移植并发症严重影响移植效果和临床的广泛应用。本项目从临床和动物实验模型入手,对造血干细胞移植供受者TLR4(Toll-like receptors, TLRs)基因单核苷酸多态性(Single Nucleotide Polymorphisms,SNP)与移植术后GVHD发病率和严重程度以及术后感染的关系进行了研究。同时也建立了TLR4基因缺陷的小鼠骨髓移植模型,以期研究TLR4分子表达对GVHD发病率和移植生存期的影响,观察TLR4基因缺陷对DC(dendritic cells,DCs)成熟、抗原递呈功能和诱导T细胞异基因免疫反应能力的影响,阐明TLR4在GVHD中的分子机制。本研究将为恶性血液肿瘤移植术后GVHD的预后因素和防治的研究提供新的思路,为造血干细胞移植治疗恶性血液病提供新的理论依据,具有重要的研究意义。关于TLR4对GVHD的影响,国际上有少量报道涉及,而且结论尚有争议。有学者认为TLR4可能减少GVHD的发病风险,而另外学者认为TLR4会增加或不影响GVHD的发生。为明确TLR4在异基因移植后GVHD发生中扮演的角色,我们采用PCR扩增直接测序法和聚合酶链反应-限制性片段多态性分析技术(PolymeraseChain Reaction-Restriction Fragment Length Polymorphism,PCR-RFLP)去对208例在我移植中心行异基因造血干细胞移植术的病人及相配对的亲缘或非亲缘供者进行TLR4基因Asp299Gly和Thr399Ile两个单核苷酸多态性变异位点的基因型测序。Ncol内切酶分析Asp299Gly突变,Hinfl内切酶分析Thr339Ile突变,我们研究发现：在所有被检测的造血干细胞移植供受者人群样本中均没有检测到TLR4的Asp299Gly和Thr399Ile基因多态性存在。我们得出结论：TLR4基因的多态性在不同的地区和人种中分布是不同的,在非洲、欧洲人种中分布相对较高,最高突变报道出现在伊朗西部人群中,而在其他亚洲人种中分布罕见。我们的实验证明了在中国尤其是浙江及周边地区移植人群中TLR4基因Asp299Gly和Thr399Ile的基因多态性非常罕见。由于这个结果使我们无法用统计学方法阐明TLR4基因和GVHD之间的联系,因此,我们试图通过TLR4基因敲除小鼠GVHD模型来证实这个假设：供者或受者的TLR4基因突变可能影响到GVHD的发生。我们通过小鼠移植模型探讨了TLR4基因在骨髓移植后GVHD的发生中扮演的角色。GVHD的发生有3个因素,其中关键因素是供者淋巴细胞识别供者或受者的抗原递呈细胞(antigen -presenting cell, APC)而活化,转移到靶组织器官对受者的组织器官发生攻击,引起组织破坏。树突状细胞作为一类具有最强抗原提呈功能的细胞群体,在识别和递呈抗原启动免疫应答、诱导移植排斥中起重要的作用。人们一直认为,DC是抗原提呈能力最强的APC,是唯一能够激活初始型T细胞的APC,具有激活移植排斥反应的作用。我们利用小鼠同种异基因骨髓移植模型观察TLR4基因缺陷小鼠DC诱导同种移植免疫耐受效果,并分析了相关的免疫机制。通过研究TLR4基因敲除小鼠DC在体内外对供者T细胞成熟、分化和功能的影响以及致耐受作用的不同来了解TLR4在GVHD中所扮演的角色。BALB/c、C57BL/6小鼠是纯系的同种小鼠,这两种小鼠的主要和次要组织相容性抗原(MHC-Ⅰ、Ⅱ)均不相同,可进行同种间移植排斥反应的免疫学研究。TLR4-/-是TLR4基因敲除小鼠其背景与C57BL/6小鼠(TLR4+/+)相同。研究表明,异基因骨髓移植后供者和受者来源的APC均可出现在受者的二级淋巴组织中,供者的T细胞受体(T cell receptor, TCR)可以识别受者APC(直接递呈)或供者APC(间接递呈)呈递的异基因抗原。我们将实验设计为TLR4+/+和TLR4-/-小鼠分别做供者或受者与BALB/c小鼠之间相互进行骨髓移植,了解TLR4-/(?)与TLR4+/+的DC在参与直接递呈和间接递呈识别过程中,两者相比受鼠发生GVHD的不同。通过观察我们发现TLR4-/-小鼠无论作供鼠还是受鼠其移植后的嵌合体与TLR4+/+组相比发生GVHD程度均较轻、体重下降较慢、临床GVHD评分较低、肝脏及小肠等器官、组织的受损程度较小,均预示TLR4-/-小鼠可以诱导机体产生针对特异性抗原的耐受,导致机体同种异体器官移植的免疫耐受。此外我们还观察到TLR4-/-小鼠做供鼠时移植后受鼠BALB/c发生GVHD时间(中位时间16.1天)较TLR4-/-小鼠做受鼠时其嵌合体发生典型GVHD(中位时间12.3天)的时间也要退后一些,解剖发现受鼠肝脏、小肠表面出血点程度更轻；我们认为这可能是移植后受体淋巴组织中供体或受体来源的DC所占比例不同所造成的,当TLR4-/-作为供鼠时,其提供的DC在移植后2周为嵌合小鼠淋巴组织中主要DC,因其递呈异基因抗原使T细胞激活的作用较弱,故诱导发生GVHD的程度要轻。在进一步的体内外实验中我们比较了TLR4-/-小鼠DC的抗原提呈能力、与同种异基因T细胞的混合淋巴细胞反应(mixed lymphocyte reaction,MLR)、抑制T辅助细胞1型(T help cell type 1,Thl)亚群的分化以及体外诱导同种抗原特异性T细胞低反应性等来解释其发生移植免疫耐受的免疫机制。近来的研究认为在T细胞反应的早期,当童贞T细胞识别APCs提呈的抗原时,APCs是否能够表达足够的CD86、CD80、CD40等共刺激分子决定了T细胞是否被完全活化,产生免疫应答,或是未被活化导致凋亡或无能。体外实验我们发现TLR4-/-可以保护非成熟型DC不被外源性脂多糖(lipopolysaccharide,LPS)刺激所激活。我们的FACS数据表明,给予外源性LPS(1μg/ml)刺激24h, TLR4+/+小鼠Day-5-DC表达高水平CD80、CD86、CD40和MHC-Ⅱ类分子,显示出成熟型DC的表型特征,白介素12(Interleukin 12,IL-12)分泌水平明显升高；而TLR4-/-小鼠Day-5-DC则维持在非成熟型状态,DC的表型和IL-12分泌在LPS刺激前后几乎无改变,对T细胞为低刺激活性。IL-12是由DC分泌的重要的免疫调节因子,可以促进干扰素-γ(Interferongamma, IFN-γ)的分泌,引起CD4+T细胞增殖并向Th1细胞分化,IL-12的缺乏则T细胞增殖受抑。TLR4-/-小鼠在LPS刺激后DC的非成熟状态明显抑制了其抗原提呈能力,减弱DC与同种异基因T细胞的混合淋巴细胞反应,引起T细胞增殖的作用较弱,从而减轻GVHD的发生。在体内实验我们对骨髓移植后21天小鼠进行研究,供鼠是TLR4-/-组小鼠脾细胞来源的DC其表面共刺激分子CD80、CD86的表达与供鼠TLR4+/+组相比也明显降低。TLR4基因的缺失可使DC维持非成熟型状态,具备对T细胞的低刺激活性。一般认为CD4+T细胞Th1亚群向Th2亚群漂移,可以改变移植物局部的免疫反应,抑制细胞介导的排斥反应。IL-2和IFN-γ是Th1亚群的特征细胞因子,而IL-10和IL-4是Th2亚群来源的细胞因子；IL-17为第三个T细胞亚群,在预防胞外病原体中扮演重要角色,其缺乏可导致炎症的进展和严重的自身免疫疾病。为了探讨TLR4-/-小鼠的DC与异基因CD4+T细胞在混合淋巴细胞反应中对T细胞分化的影响,我们测定并分析了同种MLR反应体外培养体系上清中Th1和Th2来源的细胞因子,发现TLR4-/-小鼠来源非成熟型DC与新鲜分离BALB/c小鼠的CD4+T细胞作MLR后,代表Th1亚群的细胞因子IFN-γ、IL-2水平明显降低(p<0.01),有意思的是代表Th2亚群的细胞因子IL-10和IL-4水平也表现为下降(p<0.05)。结果说明TLR4-/-小鼠DC可抑制Th1亚群的增生反应,但并未诱导Th1亚群向Th2亚群偏移。此外TLR4+/+小鼠MLR上清中IL-17的水平与TLR4-/-组相比显著升高,说明IL-17对GVHD的进展起促进作用。我们在MLR实验中通过流式和羧基荧光素二醋酸盐琥珀酰亚胺酯(carbox fluorescenceindiacetate succinimidyl ester, CFSE)标记检测发现TLR4-/- DC对异基因T细胞的活化作用减弱,与TLR4+/+组相比T细胞增殖明显减少；本结果说明TLR4基因的缺失使DC在体外同种MLR反应中能够维持处于非成熟型状态,不能充分活化T细胞,从而诱导T细胞对同种抗原的低反应性。血清中Th1亚群分泌的细胞因子与严重的GVHD和死亡率相关。为了解小鼠移植后体内来自Th1亚群和Th2亚群细胞因子水平的变化；推测TLR4-/- DC在体内对T细胞分化的影响；我们收集了移植后7天嵌合体小鼠的血清,供鼠为TLR4-/-的移植后受鼠血清中代表Th1亚群的IL-2水平明显较低；而令人惊讶的是其血清中IFN-γ的水平正好相反,移植后第7天受鼠血清中IFN-γ水平与TLR4+/+小鼠组的比较明显升高,考虑可能是除T细胞以外的其他细胞如自然杀伤细胞(Natural killer cells, NK cells)、APC分泌的较多量IFN-γ所至。这结果也与其他一些学者观点符合：IFN-γ对GVHD起到保护作用,IFN-γ对活化T细胞起负调控作用,抑制细胞分化,促进细胞死亡,保护受体器官不受损坏。研究发现Th2相关的细胞因子可以下调细胞介导的免疫反应,拮抗Th1亚群的细胞因子效应,从而减轻GVHD的发生。我们检测发现代表Th2亚群的细胞因子IL-10水平在供鼠为TLR4-/-的嵌合小鼠血清中表现为明显升高,提示可以抑制小鼠移植后GVHD的发生。此外移植后第7天最大量成熟的DC迁移到淋巴组织,参与到递呈抗原,活化T细胞的过程中,这一定程度解释了实验中发现移植后嵌合体小鼠血清中细胞因子检测为何在第7天水平最高这一现象。影响GVHD的因素很多,在体内这些因素相互关联形成复杂的网络最终影响到GVHD的发生和严重程度,LPS/TLR4的信号传导通路也是其中影响因素之一。我们从这个方面入手了解天然免疫分子TLR4在GVHD中所起的作用,最后得出结论TLR4基因在T细胞对APCs的刺激产生明显的活化和增殖反应中起着重要的桥梁作用,是同种反应性T淋巴细胞启动介导同种免疫应答的关键因素；TLR4基因的缺失可诱导机体产生针对特异性抗原的耐受,导致机体同种异体器官移植的免疫耐受从而显著减少受体GVHD的发生。针对TLR4基因研发新的靶向治疗药物也许可以为今后预防和减轻异基因造血干细胞移植后GVHD的发生提供了一个新的治疗思路。更多还原

【Abstract】 Hematopoietic stem cell transplantation (HSCT) has become one of the main strategies for the treatment of malignant hematological disease. However the full potential of HSCT has not been realized because of transplant related complication. Prominent among these complications is graft-versus-host disease (GVHD) which remains a major cause of morbidity and mortality after HSCT. During the past several years, Toll like receptors 4 (TLR4) has been showed to be a key molecule in the innate immunity and immune tolerance. The role of TLR4 in GVHD after HSCT was still unknown. The objective of this study is intended to elucidate the role and the mechanism of TLR4 in GVHD after HSCT. The project will try to determine the frequency of TLR4 mutations in a large cohort of HSCT recipients and their HLA-identical donors and to test the hypothesis that TLR4 mutations in either the recipient or donor influence the risk of acute GVHD in allogeneic HSCT recipients. To explore the mechanism of TLR4 in GVHD, the HSCT model with TLR4 deficient mouse will be employed. Not only the frequency of GVHD and survival term after HSCT, but also the function of DC and cytotoxicity of T lymphocyte will be investigated in this allograft mouse models. This project will probably acquire new insights into the pathphisology of GVHD and foster the development of new therapies to control GVHD after the hematopoietic stem cell transplantation.A few of controversial studies have not well identified the role of TLR4 activation on GVHD after HSCT. One retrospective study indicated a reduced risk of acute GVHD associated with TLR4 mutations in humans, whereas another study reported an increased risk for severe GVHD and intestinal GVHD or no significant influence of TLR4 on the incidence of GVHD. In order to clarify the role of TLR4 in the occurrence of acute GVHD after HSCT,we collected 208 samples from HSCT recipients and their HLA-identical donors to test the hypothesis that TLR4 polymorphils either in either the recipients or donors influence the risk of acute GVHD in allogeneic HSCT recipients. We examined the Asp299Gly and Thr339Ile polymorphism in all samples from post transplant GVHD patients and their donors using the TaqMan and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. The products of PCR were sequenced to identify and characterize the SNPs, at the same time,.we used two restriction enzymes to randomly test samples, NcoⅠfor the PCR-RFLP assay of the Asp299Gly allele and Hinfl for the Thr339Ile allele. No homozygous or heterozygous variant alleles of the Asp299Gly or Thr339Ile polymorphisms were detected in any samples of our study.Our results demonstrate that the frequencies of variant alleles of TLR4 polymorphisms were variable among different ethnic groups. The frequency of TLR4 Asp299Gly polymorphism is higher in African and in Europeans.The highest mutation rate was reported amongst western Iranians. However the G allele or T allele of the Asp299Gly or Thr399Ile polymorphisms were very rare in other Asians populations.Our results demonstrated that the TLR4 Asp299Gly and Thr399Ile polymorphisms might be very rare in Chinese population, especially in transplant population of Zhejiang. The results of this study cannot confirm the role of TLR4 mutation in the pathogenesis of GVHD in humans.In followed studies, we evaluated the role of TLR4 in GVHD associated with allogeneic bone marrow transplantation by TLR4 knockout mice and found a obvious conclusion.We confirmed the hypothesis that TLR4 mutations in either the recipient or donor mice influence the risk of acute GVHD in allogeneic HSCT recipient mouse.In this study, we have found that TLR4 plays a critical role in the occurrence of acute GVHD using murine GVHD models. Donor T cell being activated by APCs is the most important one among the three stages described in acute GVHD, and then migrate to target organs that caused the recipient’s target tissue damage. Dendritic cells (DCs), as immune cells, can recognize and present alloantigens to initiate the adaptive immune response and induce transplant rejection. People always believe that, DC is a kind of APC which having strongest antigen-presenting ability, and can unique activate naive T cell and leading to acute graft rejection mainly。TLR signaling leads to the induction of costimulatory molecules and cytokines necessary for activation and differentiation of T lymphocytes. In an initial effort to identify the effect of DC from TLR4 knockout (TLR4-/-) mice on allograft immune tolerance, we used the mice model of allogeneic Bone Marrow Transplantation (BMT), and analyzed the related immunological mechanisms. DC can affect donor T cells’ proliferation, differentiation and induce graft rejection. We have clearly demonstrated that TLR4 plays an essential role in the course of mice GVHD.BALB/c and C57BL/6 mice are purebred and congenic mice strains with different major and minor histocompatibility antigen (MHC-Ⅰ、Ⅱ) that are available for graft rejection study. TLR4-/- refers to TLR4 knockout mice which have the same background of C57BL/6 mice (TLR4+/+). It was reported that both host-derived and donor-derived APCs were present in secondary donor lymphoid tissue after allo-BMT. both host-derived and donor-derived APCs are present in secondary lymphoid organs. The T-cell receptor (TCR) of the donor T cells can not only recognize alloantigens on host APCs (direct presentation) but also donor APCs (indirect presentation).To gain insight into the effect of TLR4-/- derived DC in GVHD between direct presentation and indirect presentation, we designed mice allo-BMT model with TLR4+/+ and TLR4-/- mice as the donor, and injected with bone marrow cells and splenocytes obtained from BALB/c recipient. In subsequent reverse direction experiments, in which BALB/c as the recipient, then TLR4+/+ and TLR4-/- mice as the donor. We want to learn the different expression of GVHD on TLR4-/- compare with TLR4+/+ recipient mice when TLR4-/- derived DC on direct and indirect presenting alloantigens. The results was demonstrated that TLR4-/- mice as either donor or recipient, the chimera have slightly GVHD and lower clinic score, less weight loss, minor liver and small intestine damage. These indicated that TLR4-/- mice can induce tolerance against specific antigens, resulting immune tolerance in allograft rejection. In addition, we also observed that TLR4-/- mice as the donor compared with TLR4-/- mice as the recipient, the chimera showed later time on the occurrence of GVHD(median time:16.1 days vs 12.3 days), rare bleeding blot on surface of liver and small intestine. We believed that it was due to different proportion of both host-derived and donor-derived DC on recipient lymphoid tissues after transplantation. With TLR4-/- mice as the donor, provided DC were the major DC in the chimera lymphoid tissue after allo-BMT 14 days. Because of its weaker ability on activating T cells by presenting allogeneic antigen, the degree of GVHD occurred is slightly.In next study, we compared the antigen presenting ability of TLR4-/- mice derived DC, the MLR response with allogeneic T cell both in vivo and vitro, inhibited the differentiation of Thl subsets and induced hyporesponsiveness of alloantigen-specific T cells in vitro to explain the immunological mechanisms of transplant tolerance. Recent studies suggest that DCs express the highest concentration of MHC Class II molecules, such as CD86, CD80, and CD40 on their surface, play a major role in the activation of naive T lymphocytes. However, this is not enough to cause activation and maturation of T cells, anergizing and leading to apoptosis, if the antigen presenting cell can not express enough costimulatory molecules。We also realized that TLR4-/- could protect immature DC from mature by exogenous LPS in vitro. Our data from FACS demonstrated that Day-5-DCs from TLR4+/+ mice were stimulated by exogenous LPS (1μg/ml) for 24h, expressed high levels of CD80, CD86, CD40 and MHC-Ⅱmolecules, showing mature DC phenotypes, increased IL-12 secretion levels. While TLR4-/-derived Day-5-DC were still maintain immature phenotype and IL-12 secretion of DC have no difference with or without LPS stimulation, making T cell unreactive to activation.Interleukin 12(IL-12), as mainly produced by dendritic cells in response to lipopolysaccharide, can induce interferon-γ(IFN-γ) production and trigger CD4+ T cells to differentiate into Thl phenotype, The proliferation of T cells will inhibited by lacking of IL-12. The immature phenotype of TLR4-/- derived DCs significantly decreased the ability of antigen-presenting after LPS treated, reduced MLR between DC and allogeneic T cells, induced weaker T cell proliferation, leading to reduced the occurrence of GVHD. In vivo experiment, the expression of costimulatory molecules CD80 and CD86 in DCs obtained from spleen cells of TLR4-/-→BALB/c mice after BMT 21 day were obviously decreased compared to TLR4+/+→BALB/c, TLR4 gene deficiency will help DC maintain immature with low sensitivity of T cells.It has been suggested that the Thl/Th2 polarization of T helper cell subsets plays an important role in the development of GVHD. Thl switch to Th2 cells can change the local graft immune response, inhibit cell-mediated rejection. The Thl response is characterized by the production of IFN-y, IL-2 and characteristics of the Th2 response is the production of IL-4 and IL-10. IL-17 plays a critical role in prevention of extracellular pathogens as the third subset of T cell, which deficiency may lead to the progression of inflammation and severe autoimmune diseases.To determine the differentiate effect on allogeneic CD4+ T cells with TLR4-/-mice derived DC on MLR, we have tested and analyzed the expression of Th cytokines in the supernatant of MLR culture media. We concluded that the levels of cytokines produced by Thl subsets, IFN-y, IL-2, significantly decreased after immature DC from TLR4-/-mice and CD4+ T cell from BALB/C mice in MLR (p<0.01). While the levels of cytokines produced by Th2 subsets, IL-10, IL-4, also significantly decreased at the same time, interestingly (p<0.05). Results indicate that DC from TLR4-/- mice, can inhibit the proliferation of Thl subsets, without inducing Thl switch to Th2 subsets. Besides the expression of IL-17 in the supernatant of MLR from TLR4+/+mice was significantly higher than TLR4-/-mice, which means IL-17 has promoted effect to the GVHD progression. In MLR experiments we found TLR4-/- derived DC had attenuated effect on allogeneic T cell activation compared to TLR4+/+ group, detected by FCS and CFSE. The results indicate that TLR4 gene deficiency in MLR response in vitro will keep DC in immature state, leading to alloantigen hyporesponsiveness of T cells without fully activation.The cytokines produced by Thl subsets are associated with the severity GVHD and mortality. To investigate the variance level of cytokines produced by Th1/Th2 subset after transplantation in murine BMT model, to speculate on the direction of allogeneic T cells differentiation under the influence of TLR4-/- derived DC in vivo. We used ELISA method to detect the level of cytokines in the serum of day 7 post-transplantation chimeric mice, found the levels of IL-2 as representation of Thl subset from TLR4-/-donor mice were significantly lower. The interesting thing is the level of IFN-y in reverse, which in the serum of TLR4-/-→BALB/c chimeric mice is significantly higher than TLR4+/+→BALB/c mice. It is probability because of other cells such as NK, APC in addition to T cells also secreted more amount of IFN-γ. This result consistent with other scholars view that IFN-γcan reduce the occurrence of GVHD, negatively regulate alloreactive T cells via inhibiting cell differentiation, promoting cell death, and to prevent host tissue from damage. Studies found that Th2 cytokines can be associated with down-regulation cell-mediated immune responses, to antagonistic the effect of Thl cytokines, and to reduce GVHD. TLR4-/- mice as donor, the serum of recipient BALB/c mice were analyzed on day 7 post-transplant for cytokine, the production of IL-10 as the characteristic of the Th2 response showed significantly increased, suggesting that mice can inhibit the occurrence of GVHD after transplantation. In addition, the maximum number of mature DCs emigrates to host lymphoid tissue on day 7 after transplantation. To some extent, this explained why the level of serum cytokines from chimeric mice after transplantation is highest on day 7.There are many factors affect on the GVHD, which have formed a complex network of immune interactions in vivo, As a result, it does ultimately affect the occurrence and severity of GVHD. Signal transduction pathway of LPS/TLR4 plays an important role in immune response, which is mediated and priming by allogeneic T lymphocytes. Based on these, we take our interest in the role of TLR4 in GVHD and finally concluded T cells from TLR4-/- knockout mice can not be effectively activated and then proliferate under the stimulation by APCs. On the contrary, it can induce tolerance for specific antigen, leading to immune tolerance of allogeneic organ transplantation and thus significantly reduce GVHD of host. In future, the development of new targeted therapy for TLR4 gene will provides a new treatment idea.更多还原