【作者】 刘璐；

【导师】 石艳；

【作者基本信息】 山东大学 ， 内科学（血液病）（专业学位）， 2025， 博士

【摘要】 第一部分 ITP患者CD4+T细胞对免疫无能的反应及利用蛋白质组学技术筛选差异蛋白研究背景:原发免疫性血小板减少症(primary immune thrombocytopenia,ITP)的主要特征是血小板自身抗体的产生、血小板计数下降以及出血风险增加。免疫耐受的缺失是ITP发病机制的关键,包括T细胞稳态异常、调节性T细胞缺乏以及效应性T细胞过度激活。人体任何预防自身反应性细胞增殖调控的失败,比如细胞清除、受体编辑、抑制无能细胞的产生都可能导致ITP的发生。T细胞免疫耐受是指在某些情况下,尽管有抗原和共刺激信号的存在,T细胞仍然无法有效激活其免疫反应。这种耐受性可以防止机体免疫系统攻击自身组织,从而避免自身免疫性疾病的发生。它是构成适应性免疫反应的关键要素,调节着机体对抗原的反应能力。这种功能的失能状态通常与免疫系统无法平衡识别自我与非自我抗原有关,从而导致对自身组织的攻击。这一过程对于保持体内免疫稳态至关重要,特别是在预防自身免疫性疾病的过程中。负性共刺激信号对于T细胞无能的形成具有至关重要的作用。共刺激信号为T细胞的正常激活提供了必要的刺激,而负性共刺激信号则通过抑制活化信号的传导来减少T细胞的活化程度,从而诱导无能状态。从机制上讲,细胞毒T淋巴细胞相关抗原4(cytotoxic T lymphocyte-associated antigen-4,CTLA-4)及程序性死亡受体 1(programmed cell death protein,PD-1)是两个主要的负性共刺激受体,它们通过干扰T细胞受体(T cell receptor,TCR)信号和促进细胞内信号抑制通路来实现。研究表明,CTLA-4通过阻断磷脂酰肌醇 3 激酶(phosphatidylinositol 3-kinase,PI3K)与蛋白激酶 B(protein kinase B,AKT)之间的信号传导,减少T细胞对抗原的反应。PD-1则通过抑制TCR介导的信号级联,降低T细胞中钙离子的流动和细胞因子的生成,达到降低T细胞反应强度的效果。实验显示,在PD-1缺失的小鼠中,自身免疫性疾病会显著加重,这进一步表明PD-1在调节自身免疫耐受中的重要性。细胞因子环境也影响T细胞共刺激信号的传导。研究发现,缺乏IL-2的刺激可导致T细胞无能的发生,而补充IL-2能够逆转这一状态。此外,IL-10等抑制性细胞因子的存在也增强了负性共刺激信号的效果,从而促进T细胞的免疫耐受状态。实验中,通过操控这些细胞因子的水平,可以在一定条件下调节T细胞无能的发生与维持。在不同的自身免疫性疾病中,T细胞免疫耐受的表现形式和其在病理过程中的角色可能存在显著差异。在系统性红斑狼疮(systemic lupus erythematosus,SLE)及类风湿性关节炎(rheumatoid arthritis,RA)等疾病中,T细胞免疫耐受可能出现抵抗,表现为T细胞对自身抗原的持续低反应状态,从而导致免疫系统失衡,未能充分抑制炎症反应。而在多发性硬化(multiple sclerosis,MS)中,T细胞无能可能通过抑制病理性T细胞激活而在一定程度上延缓疾病恶化。之前的研究已经证明,CTLA4-Ig能够在体外环境中诱导慢性ITP患者产生血小板特异性的无能T细胞。然而,无能T细胞调节ITP免疫反应的潜在机制尚待研究。本研究体外诱导ITP患者CD4+T细胞无能状态,对其增殖、活化、共刺激分子及细胞因子等相应的变化进行观察。进而利用蛋白质组学技术探索ITP患者蛋白质组的特点和变异信息,筛选ITP患者与健康对照组CD4+T细胞中的差异蛋白,并且寻找参与ITP患者免疫失耐受的蛋白及信号通路。研究目的:1.本研究观察了 CD4+T细胞对无能诱导的反应,分析ITP患者及健康对照组外周血CD4+T细胞在不同刺激条件下的增殖和活化状态。2.对ITP患者及健康对照组外周血CD4+T细胞在不同刺激条件下共刺激分子及细胞因子的水平进行了测定。3.运用串联质谱标签定量蛋白质组学技术,对ITP患者及健康对照组CD4+T细胞差异蛋白进行分析。4.通过蛋白质富集分析来明确差异蛋白在机体中参与的代谢通路。5.通过构建蛋白质交互作用的网路,进一步预测参与ITP患者免疫耐受抵抗的蛋白及信号通路。研究方法:1.样本采集:分别收集30例符合诊断标准的新诊断和复发的ITP患者及同性别、同年龄的健康对照样本29例,分别收集10ml EDTA抗凝的外周血,从中提取出单个核细胞(peripheral blood mononuclear cells,PBMCs)。分离出的细胞再经过磁珠分选出外周血CD4+T细胞。2.体外细胞培养:活化组:CD4+T细胞与抗CD3、抗CD28抗体及IL-2共同孵育18小时(第一次刺激);无能组:CD4+T细胞与离子霉素共同孵育18小时(第一次刺激)。两组洗涤并静置48小时后,加入抗CD3、抗CD28抗体重新刺激96小时(第二次刺激)。静息组:分离后的CD4+T细胞加入含10%胎牛血清(Fetal Bovine Serum,FBS)的RPMI-1640培养基中,与活化及无能组在相同条件孵育、洗涤。3.细胞增殖实验:实验使用了羧基荧光素二醋酸盐琥珀酰亚胺酯(carboxyfluorescein diacetate succinimidyl ester,CFSE)标记静息组、第二次刺激96小时后活化组和无能组的细胞,流式细胞技术检测T细胞的增殖情况。4.细胞活化实验:检测静息组、第二次刺激96小时后活化组和无能组CD4+CD25+T细胞的数量。5.共刺激分子检测:检测静息组、第二次刺激96小时后活化组和无能组中CD4+ICOS+T 细胞、CD4+PD-1+T细胞和 D4+CTLA-4+T 细胞的数量。6.细胞因子检测:采用酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)检测静息组、第二次刺激96小时后活化组和无能组细胞培养液上清中IL-17、TGF-β、IL-6、IL-10 的水平。7.串联质谱标签(Tandem Mass Tag,TMT)定量蛋白质分析:对5例新确诊的ITP患者和5例健康对照者,分别采集10ml EDTA抗凝的静脉血,分离PBMCs并用磁珠分选出外周血CD4+T细胞。随后分离蛋白、烷基化及胰蛋白酶消化,再用TMT标记蛋白标本,经过分离馏分、液质检测后生成质谱检测原始数据。8.生物信息分析:运用GO富集分析对差异蛋白进行功能聚类的分析,同时利用KEGG通路数据库对差异蛋白参与的代谢途径进行富集分析。通过STRING数据库构建蛋白质相互作用网路,寻找差异蛋白及相关的信号通路。9.分析结果验证:根据测序及生信分析结果中筛选出参与ITP患者免疫耐受抵抗的蛋白质。收集ITP患者及健康对照者的外周血PBMCs,分离得到CD4+T细胞,提取RNA及蛋白质,对转录及翻译阶段进行差异表达的验证与分析。研究结果:1.ITP患者活化组和无能组CD4+T细胞的增殖均高于健康对照组。2.采集静息组、第二次刺激96小时后活化组和无能组CD4+T细胞,运用流式细胞技术检测CD4+CD25+T细胞的数量。与健康对照组相比,ITP患者静息和无能组中CD4+CD25+T细胞的比率增加,这表明ITP患者在无能诱导状态下存在异常活化。3.体外研究发现ITP患者在无能诱导状态下存在异常增殖及活化。进一步分析静息组、第二次刺激96小时后活化组和无能组CD4+T细胞表面共刺激分子ICOS、CTLA-4及PD-1的表达。结果提示,ITP患者静息和无能组CD4+PD-1+T细胞的百分比增加;而静息和活化组CD4+CTLA-4+T细胞的百分比降低。各组间CD4+ICOS+T细胞的百分比无明显差异。4.利用ELISA方法检测静息组、第二次刺激96小时后活化组和无能组细胞培养液上清中IL-17、TGF-β、IL-6、IL-10的水平。与健康对照组相比较,ITP患者静息及无能组中IL-17、TGF-β水平升高;而各组间IL-6和IL-10水平没有显著差异。5.基于TMT定量蛋白质分析的结果发现,与健康对照相比,共有220种差异表达的蛋白质,其中148种蛋白质表达量显著增加,72种蛋白质表达量显著减少。6.在参与生物学过程的蛋白质分析中,差异表达的蛋白质主要富集在刺激反应、免疫反应、核小体DNA结合和翻译延伸等方面。7.ITP患者与健康对照者的差异蛋白利用STRING数据库构建了一个蛋白质-蛋白质相互作用的网络。将CBL确定为磷酸肌醇-3-激酶(PI3K的调节亚基)的调控蛋白,表明CBL-PI3K-AKT通路可能参与了 ITP患者CD4+T细胞功能障碍的调节。8.进一步分析蛋白质组学结果,与健康对照者的蛋白表达相比,ITP患者CD4+T细胞中CBL的表达显著降低的同时,PI3K的表达显著增加。而参与T细胞无能诱导调节的其他泛素连接酶,如GRAIL和itch的表达在健康对照者和ITP患者之间无统计学差异。另外,KEGG富集分析和GSEA分析表明,PI3K-AKT通路在ITP患者CD4+T细胞中表达上调。9.为了进一步验证蛋白质组学的结果,分别检测静息、第一次刺激后活化及无能三个亚组中CBLB的表达,结果发现静息及无能组ITP患者CD4+T细胞中CBLB的mRNA及蛋白质水平均明显低于健康对照者。研究结论:1.ITP患者CD4+T细胞与健康对照者相比,无能诱导状态下存在异常的增殖及活化,提示ITP患者对无能诱导存在抵抗。2.ITP患者无能诱导抵抗的同时伴随着共刺激分子及细胞因子表达的改变。3.在对蛋白质组学结果进行质控筛选后,发现ITP患者与健康对照者蛋白质表达存在差异。4.通过测序和生信分析揭示ITP患者CD4+T细胞中CBL表达降低而PI3K表达增加,且PI3K-AKT通路表达上调。5.体外研究结果验证了ITP患者的CD4+T细胞中CBLB表达降低,CBLB-PI3K-AKT通路可能参与了ITP患者CD4+T细胞免疫耐受功能的调控。第二部分 CBLB通过PI3K信号通路调节ITP患者CD4+T细胞免疫无能的机制研究研究背景:原发免疫性血小板减少症(ITP)以血小板数量持续减低为主要表现。“免疫失耐受”可能是其发病机理的关键因素。正常个体的外周血中也可检测到部分自身反应性T细胞,其作用主要是针对血小板糖蛋白,但其通常处于免疫耐受的状态。免疫反应中,T细胞的活化需要双重信号,其中第一信号是通过T细胞受体(T cell receptor,TCR)对MHC-抗原肽复合物的识别,进而传递出针对特异性抗原的识别信号。第二信号则是由抗原呈递细胞(Antigen-presenting cells,APC)上的共刺激因子与T细胞表面的共刺激分子之间的相互作用所产生。当共刺激分子所给予的协同刺激信号缺乏时,T细胞将无法实现激活,进而处于一种无能状态,甚至可能导致细胞凋亡的发生。有研究表明,CTLA-4Ig可以有效地阻断共刺激信号,从而抑制血小板反应性T细胞的增殖。此时,当再次受到血小板抗原刺激,可以诱导ITP患者T细胞产生免疫耐受。CBL蛋白家族包括CBL、CBLB和CBLC蛋白。CBL蛋白通过靶向蛋白质进行泛素化或与靶蛋白相互作用来调节免疫细胞中的多种信号通路。CBLB是一种RING指E3泛素连接酶,是适应性免疫反应的关键调节剂。CBLB对于构建T细胞激活的阈值并通过不同机制来调控外周T细胞的耐受至关重要。CBLB的缺失使得T细胞增殖可以不依赖CD28共刺激因子和IL-2,表明其参与了 CD28共刺激信号通路。CBLB亦可通过调控PI3K的p85调节亚基的泛素化,作为CD28信号通路的负调节因子来作用于Vav。此外,异常的CBLB表达会损害外周耐受,在多种自身免疫性疾病中发挥重要作用。然而,CBLB在ITP发病机制中的作用尚未有明确认识。在第一部分的研究中,我们发现ITP患者CD4+T细胞在体外对无能诱导存在抵抗,并且伴随着异常的共刺激分子及细胞因子的表达。蛋白质组学发现ITP患者的CD4+T细胞中CBL表达降低,而PI3K表达增加,同时PI3K-AKT信号通路活性增强。体外实验结果进一步证实了ITP患者CD4+T细胞中CBLB表达的变化。我们猜测CBLB-PI3K-AKT通路在ITP患者T细胞免疫失耐受中发挥重要作用。研究目的:1.探究CBLB是否通过PI3K信号通路调节ITP患者CD4+T细胞的免疫无能。2.探究ITP患者CD4+T细胞中CBLB表达降低的原因,阐述低剂量地西他滨对CBLB甲基化及表达水平的调控作用。研究方法:1.样本采集:分别招募了33例新发和复发的ITP患者,以及30例性别和年龄均相匹配的健康对照样本。分别收集10ml EDTA抗凝的外周血,从中提取出单个核细胞(PBMCs)。分离出的细胞再经过磁珠分选出外周血CD4+T细胞。2.体外细胞培养:无能组:CD4+T细胞与离子霉素共同培养18小时(第一次刺激)。随后,洗涤并静置48小时,而后加入抗CD3、抗CD28抗体重新刺激96小时(第二次刺激)。静息组:分离后的CD4+T细胞加入含10%胎牛血清(Fetal Bovine Serum,FBS)的RPMI-1640培养基中,与无能组相同条件孵育、洗涤。3.细胞增殖实验:实验使用羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)对无能组第二次刺激后收集得到的CD4+T细胞进行标记,随后流式细胞技术检测T细胞的增殖情况。4.相关性分析:利用Wes全自动蛋白表达分析系统检测参与细胞增殖实验的同一患者无能组第一次刺激后CBLB的表达,并与T细胞增殖指数进行相关性分析。5.细胞活化实验:构建CBLB重组腺病毒并转染ITP患者和正常对照者CD4+T细胞,在第二次刺激后收集细胞,使用流式细胞术检测ITP患者与健康对照者CD4+CD25+T细胞的数量。6.蛋白免疫印迹(Western blot):将静息组、第一次刺激并静置后无能组的细胞用抗CD3、抗CD28抗体进行短时间(0、5及10分钟)的再次刺激,收集细胞沉淀,利用Wes全自动蛋白表达分析系统检测ITP患者及健康对照者CD4+T细胞中CBLB、AKT和p-AKT的表达情况。ITP患者和正常对照者CD4+T细胞转染CBLB重组腺病毒后,在上述实验条件相同的时间点,检测CD4+T细胞中CBLB、AKT和p-AKT的表达情况。7.DNA甲基化测序:对7例ITP患者及5例健康对照者的CD4+T细胞中CBLB基因启动子和5’端非翻译区(5’-untranslated region,UTR)的甲基化水平。8.地西他滨治疗前后差异蛋白表达:收集小剂量地西他滨治疗有效的ITP患者治疗前后外周血PBMCs,并用磁珠分选出外周血CD4+T细胞。利用Wes全自动蛋白表达分析系统检测CBLB、p-AKT及AKT的表达。研究结果:1.ITP患者无能诱导后CBLB表达与细胞增殖指数之间呈负相关。为了进一步确定ITP患者CD4+T细胞中CBLB与无能诱导的关系,我们分析了同一ITP患者无能诱导后CBLB的表达与其细胞增殖指数之间的关系。结果表明,无能诱导后CBLB表达与增殖指数之间呈负相关。2.ITP患者及健康对照者CD4+T细胞转染CBLB重组腺病毒后,再次给予无能诱导。结果显示,两组间CD4+CD25+T细胞的百分比没有显著差异。这一结果提示过表达CBLB可以改善ITP患者无能诱导时的过度活化。3.ITP患者CD4+T细胞在静息和无能组中CBLB的表达显著降低。无能组中,健康对照者CD4+T细胞p-AKT/AKT比值显著降低,而ITP患者CD4+T细胞p-AKT/AKT比值呈现上升趋势。4.与转染阴性对照载体的CD4+T细胞相比,过表达CBLB的ITP患者及健康对照者的CD4+T细胞在经历无能诱导后,p-AKT/AKT比值显著降低。5.ITP患者的CD4+T细胞中CBLB基因的S2区域5’UTR的甲基化程度显著高于健康对照者。6.低剂量地西他滨治疗有效的ITP患者,治疗后CD4+T细胞CBLB的表达显著增加。说明低剂量地西他滨对CBLB的甲基化和表达有调控作用。研究结论:1.CBLB通过PI3K-AKT信号通路调节ITP患者T细胞的免疫无能。2.CBLB基因5’UTR甲基化水平升高在ITP患者CBLB表达降低中发挥作用。3.低剂量地西他滨能够帮助ITP患者的CD4+T细胞恢复CBLB的表达。更多还原

【Abstract】 Part Ⅰ:The study on the response of CD4+T cells in ITP to immune anergy and the screening of differential proteins using proteomicsBackground:Immune thrombocytopenia(ITP)is an autoimmune disorder marked by the presence of platelet autoantibodies,decreased platelet counts,and an elevated risk of bleeding.The breakdown of immune tolerance plays a pivotal role in the pathogenesis of ITP.This process includes abnormalities in T cell homeostasis,a deficiency of regulatory T cells,and the hyperactivation of effector T cells.Any failure of the body’s regulatory mechanisms to prevent the expansion of autoreactive cells-such as processes involving cell clearance,receptor editing,induced unresponsiveness,and exogenous cell suppression-can contribute to the occurrence of ITP.T cell immune tolerance refers to the phenomenon when T cells cannot effectively activate their immune response despite the presence of antigens and co-stimulatory signals under certain conditions.This tolerance helps prevent the immune system from attacking the body’s own tissues,which avoids the onset of autoimmune diseases.The dysfunction often stems from an imbalance to distinguish between self and non-self-antigens in the immune system,which can lead to attacks on the body’s own tissues.This process is crucial for maintaining immune homeostasis,especially in the prevention of autoimmune diseases.Negative co-stimulatory signals play a critical role in T cell anergy.Co-stimulatory signals are essential for normal T cell activation,while negative co-stimulatory signals reduce T cell activation by inhibiting the transmission of activation signals,thereby inducing anergy.Cytotoxic T lymphocyte-associated antigen-4(CTLA-4)and programmed cell death protein-1(PD-1)are two major receptors of negative co-stimulatory that inhibit T cell activation by interfering with T cell receptor(TCR)signaling and promoting intracellular signaling inhibition pathways.Research indicates that CTLA-4 reduces T cell responses to antigens by inhibiting the phosphatidylinositol 3-kinase(PI3K)and protein kinase B(AKT)signaling pathway.On the other hand,PD-1reduces T cell response intensity by inhibiting TCR-mediated signaling cascades,decreasing calcium flux,and cytokine production in T cells.Studies demonstrate that autoimmune diseases worsen significantly in mice lacking PD-1,highlighting its crucial role in maintaining self-tolerance in autoimmunity.The cytokine also affects the transmission of T cell co-stimulatory signals.Studies show that insufficient IL-2 stimulation leads to T cell anergy.In contrast,providing IL-2 can reverse this condition.Additionally,inhibitory cytokines like IL-10 amplify the effects of negative signals,promoting T cell immune tolerance.By manipulating the levels of these cytokines in experiments,the occurrence and maintenance of T cell anergy can be regulated under certain conditions.The manifestation of T cell immune tolerance and its role in the pathological process may be different in autoimmune diseases.In systemic lupus erythematosus(SLE)and rheumatoid arthritis(RA),T cell immune tolerance may show resistance.This is characterized by persistently low responses to self-antigens,leading to immune system imbalance and inadequate suppression of inflammatory responses.In multiple sclerosis(MS),anergic T cell may delay disease progression by inhibiting the activation of pathological T cells.Previous study has demonstrated that CTLA4-Ig can induce the generation of platelet-specific anergic T cells from chronic ITP patients in vitro.However,the potential mechanisms by which anergic T cells regulate the immune response in ITP remain to be studied.This study aims to induce an anergic state in CD4+T cells from ITP patients in vitro and observe corresponding changes in proliferation,activation,co-stimulatory molecules,and cytokines.Furthermore,proteomics technology will be used to explore the characteristics and variations of the proteome in ITP patients,screening for differential proteins in CD4+T cells between ITP patients and healthy controls,and identifying proteins and signaling pathways involved in the anergic resistance of ITP patients.Objectives:1.To investigate the response of CD4+T cells to anergy induction,we detected the proliferation and activation of peripheral blood CD4+T cells from ITP patients and healthy controls under different stimulation conditions.2.To assess the levels of co-stimulatory molecules and cytokines in peripheral blood CD4+T cells from ITP patients and healthy controls under different stimulation conditions.3.To analyze the expression of differential proteins in CD4+T cells from ITP patients and healthy controls using tandem mass tag quantitative proteomics.4.To clarify the metabolic pathways involved by analyzing the enrichment of differential proteins in vivo.5.Constructing a protein interaction network,to further predict the proteins and signaling pathways involved in the resistance to immune tolerance in ITP patients.Methods:1.Sample collection:Blood samples will be collected from 30 newly diagnosed and relapsed ITP patients who meet diagnostic criteria,along with 29 healthy controls matched by gender and age.Each sample consist of 10 ml of EDTA-anticoagulated peripheral blood,from which peripheral blood mononuclear cells(PBMCs)will be isolated,and CD4+T cells will be magnetically sorted.2.Cell culture in vitro:For activation assays,CD4+T cells were incubated with anti-CD3 and anti-CD28 antibodies,in the presence of IL-2 for 18 hours(first stimulation).For anergy assays,CD4+T cells were incubated with ionomycin for 18 hours(first stimulation).The cells were washed and reactivated with anti-CD3 and anti-CD28 antibodies for 96 hours(second stimulation).For resting group,CD4+T cells were rested in RPMI-1640 medium containing 10%fetal bovine serum(FBS)and incubated under the same conditions as the activation and anergy assays.3.Proliferation assay:Label the cells from resting,activation and anergy groups after 96 hours of second stimulation with carboxyfluorescein diacetate succinimidyl ester(CFSE).Then,we assess T cell proliferation with flow cytometry.4.Activation assay:CD4+CD25+T cells were detected from resting,activation and anergy groups after 96 hours of second stimulation by Beckman Coulter.5.Co-stimulatory molecule analysis:Analyze co-stimulatory molecules by measuring the number of CD4+ICOS+T cells,(D4+PD-1+T cells,and CD4+CTLA-4+T cells in the resting,activation and anergy groups after 96 hours of the second stimulation.6.Cytokine analysis:For cytokine analysis,utilize enzyme-linked immunosorbent assay(ELISA)to measure levels of IL-17,TGF-β,IL-6,and IL-10 in the supernatants of cell cultures from the resting,activation and anergy groups after 96 hours of second stimulation.7.Tandem mass tag(TMT)quantitative protein analysis:Collect samples from 5 newly diagnosed ITP patients and 5 healthy controls,isolating PBMCs and magnetically sorting out CD4+T cells.After that,isolate proteins,alkylate,digest with trypsin,and label the protein samples with TMT.The raw data for mass spectrometry were generated after fractionation and liquid chromatography-mass spectrometry detection.8.Bioinformatics analysis:Functional clustering analysis of all differential proteins based on Gene Ontology(GO)enrichment analysis results.Additionally,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway database enrichment analysis analyze the metabolic pathways involved with differential proteins.Finally,construct a protein-protein interaction network using the STRING database to identify differential proteins and related signaling pathways.9.Verification of analysis results:Identify the proteins involved in the anergic resistance in ITP patients based on the results from sequencing and bioinformatics analysis.Extract CD4+T cells from PBMCs in ITP patients and healthy controls.Extract RNA and proteins for differential expression detection and analysis at the transcriptional and translational levels.Results:1.The proliferation of CD4+T cells from patients with ITP was significantly higher than that of cells from healthy donors under both activation and anergy-inducing conditions.2.CD4+CD25+T cells were detected from resting,activation and anergy groups after 96 hours of second stimulation by flow cytometry.Compared with that of healthy donors,the percentage of CD4+CD25+T cells was significantly increased in the cells from patients with ITP after resting and ionomycin treatment.3.In vitro studies revealed abnormal proliferation and activation in ITP patients under anergy induction.Further detection of the expression of three co-stimulatory molecules(ICOS,CTLA-4,and PD-1)on the surface of CD4+T cells showed an increase in the percentage of CD4+PD-1+T cells in the resting and anergy groups of ITP patients after 96 hours of second stimulation.The percentage of CD4+CTLA-4+T cells decreased in both the resting and activation groups.There were no significant differences in the percentage of CD4+ICOS+T cells among the groups.4.The levels of IL-17,TGF-β,IL-6,and IL-10 were measured in the supernatants of cell cultures from the resting,activation,and anergy groups using the ELISA method.Compared to healthy controls,IL-17 and TGF-β levels were elevated in the resting and anergy groups of ITP patients.However,there were no significant differences in IL-6 and IL-10 levels between healthy controls and ITP patients.5.According to the results of the TMT quantitative protein analysis,220 differentially expressed proteins were identified in ITP patients compared to healthy controls,including 148 proteins that were upregulated and 72 proteins that were downregulated.6.In the analysis of proteins involved in biological processes,differentially expressed proteins were mainly enriched in terms of stimulus response,immune response,nucleosome DNA binding,and translation elongation.7.A protein-protein interaction network was constructed using STRING database,and CBL was identified as a hub for phosphoinositol-3-kinase—the regulatory subunit of PI3K—suggesting that the CBL-PI3K-AKT pathway may be involved in the dysfunction of CD4+T cells in patients with ITP.8.Quantitative protein analysis revealed that the expression of CBL was significantly decreased and PI3K expression significantly increased in patients with ITP compared with expression in healthy controls.Additionally,gene related to anergy in lymphocytes and itchy homolog are other ubiquitin ligases that participate in the anergy-induced genetic program.The expression of gene related to anergy in lymphocytes and itchy homolog showed no significant difference between healthy controls and patients with ITP in proteomic analysis.Strikingly,KEGG and gene set enrichment analysis(GSEA)pathway analysis identified that the PI3K-AKT pathway was highly upregulated in CD4+T cells from patients with ITP.9.To further validate the proteomics results,we analyzed CBLB expression under rest,activation,and anergy conditions.After resting and anergy induction,CBLB messenger RNA and protein levels were significantly lower in CD4+T cells from patients with ITP than in those from healthy controls.Conclusions:1.CD4+T cells show abnormal proliferation and activation under anergy induction compared to healthy controls,indicating that anergy resistance in ITP.2.Changes in the levels of co-stimulatory molecules and cytokines accompany the resistance to anergy induction observed in ITP patients.3.After quality control of the proteomics,the results revealed differences in protein expression between ITP patients and healthy controls.4.Sequencing and bioinformatics analysis revealed that CBL expression decreased while PI3K expression increased in CD4+T cells of ITP patients.It also indicated the upregulation of the PI3K-AKT pathway.5.The expression of CBLB was reduced in vitro.It suggested that the CBLB-PI3K-AKT pathway may be involved in the dysfunction of CD4+T cells in patients with ITP.Part Ⅱ:The study on mechanisms of CBLB modulates T-cell anergic resistance via PI3K pathway in ITPBackground:Immune thrombocytopenia(ITP)is an acquired autoimmune disease characterized by persistently low platelet counts.The pathogenesis of ITP is still unclear."Resistance of immune tolerance" is suggested a key theory.Autoreactive T cells that target platelet glycoproteins are present in the peripheral blood of healthy individuals,but they usually remain in a state of immune tolerance.Activating T cell requires double signaling.The first signal comes from the T cell receptor(TCR)recognizing the MHC-antigen peptide complex,further transmitting the antigen-specific recognition signal.The second signal arises from the interaction between co-stimulatory factors from antigen-presenting cells(APCs)and those on T cells.Without these co-stimulatory signals,T cells cannot be activated without these co-stimulatory signals,leading to either a state of anergy or apoptosis.Research indicates that blocking these signals with CTLA-4Ig can inhibit the proliferation of T cells that respond to platelets.Upon re-exposure to platelet antigen,immune tolerance is induced in T cells of ITP patients.The casitas B-lineage lymphoma(CBL)protein family consists of CBL,CBLB,and CBLC proteins.CBL proteins regulate multiple signaling pathways in immune cells by targeting proteins for ubiquitination or by interacting with target proteins.In particular,CBLB,a RING(really interesting new gene)finger E3 ubiquitin ligase,is a critical regulator of adaptive immune responses.CBLB is essential for establishing a threshold of T-cell activation and regulating peripheral T-cell tolerance through various mechanisms.The loss of CBLB uncouples the requirement of CD28 costimulation for T-cell proliferation and interleukin(IL)-2 production,suggesting its involvement in the CD28 costimulatory signaling pathway.CBLB also acts as a potent negative regulator of the CD28 signaling cascade to Vav through its ability to ubiquitinate the p85 regulatory subunit of phosphoinositide 3-kinase(PI3K).Furthermore,the dysregulation of CBLB expression disrupts peripheral tolerance and significantly contributes to the pathogenesis of autoimmune disorders,including rheumatoid arthritis,systemic lupus erythematosus,and experimental autoimmune encephalomyelitis.However,the role of CBLB in the pathogenesis of ITP remains unclear.In the first part of the study,we found that anergy resistance in CD4+T cells from patients with ITP in vitro,accompanied by abnormal expression of costimulatory molecules and cytokines.Proteomic analysis revealed decreased CBL expression and increased PI3K expression in CD4+T cells from ITP patients,with upregulation of the PI3K-AKT pathway.In vitro studies confirmed the reduced expression of CBLB in CD4+T cells from ITP patients.We hypothesize that the CBLB-PI3K-AKT pathway plays an important role in anergic resistance of T cells in ITP patients.Objectives:1.To investigate whether CBLB affects the immune anergy of CD4+T cells in ITP patients through the PI3K pathway.2.To explore the reasons for the decreased expression of CBLB in CD4+T cells of ITP patients and to clarify the regulatory effect of low-dose decitabine on CBLB methylation and expression.Methods:1.Sample collection:Blood samples will be collected from 33 newly diagnosed and relapsed ITP patients who meet diagnostic criteria,along with 30 healthy controls matched by gender and age.Each sample consist of 10 ml of EDTA-anticoagulated peripheral blood,from which peripheral blood mononuclear cells(PBMCs)will be isolated,and CD4+T cells will be magnetically sorted.2.Cell culture in vitro:For anergy assays,CD4+T cells were incubated with ionomycin for 18 hours(first stimulation).The cells were washed and reactivated with anti-CD3 and anti-CD28 antibodies for 96 hours(second stimulation).For resting group,CD4+T cells were rested in RPMI-1640 medium containing 10%fetal bovine serum(FBS)and incubated under the same conditions as anergy assays.3.Proliferation assay:Label the cells from anergy groups after 96 hours of second stimulation with carboxyfluorescein diacetate succinimidyl ester(CFSE).Then,we assess T cell proliferation with flow cytometry.4.Correlation analysis:After the first stimulation,the automated protein expression analysis system of Wes was employed to detect CBLB expression under the anergy induction and to analyze the relationship with the T cell proliferation index.5.Activation assay:CD4+T cells were transfected with adenovirus vectors overexpressing CBLB.CD4+CD25+T cells were detected from resting,activation and anergy groups after 96 hours of second stimulation by Beckman Coulter.6.Western blot:Cells from the resting and the anergy groups were restimulated with anti-CD3 and anti-CD28 antibodies for short durations(0,5,and 10 minutes)after the first stimulation.The cells were then collected for protein analysis to detect CBLB,AKT,and p-AKT expression in ITP patients and healthy controls.After transfecting CD4+T cells from ITP patients and healthy controls with CBLB recombinant adenovirus,the expression of CBLB,AKT,and p-AKT were detected at the same time points under the same experimental conditions as above.7.DNA methylation sequencing:The methylation levels of the CBLB gene promoter and 5’ untranslated region(5’UTR)are detected in CD4+T cells from 7 ITP patients and 5 healthy controls.8.Effects of decitabine on protein expression:PBMCs were collected from ITP patients who responded to low-dose decitabine treatment before and after treatment.The CD4+T cells were then collected to detect CBLB,p-AKT,and AKT expression.Results:1.A significantly negative correlation was found between the CBLB expression and division index after anergy induction in ITP patients.To further determine the relationship between CBLB and anergy induction in CD4+T cells in ITP,we analyzed the expression of CBLB after anergy induction and its correlation with the cell proliferation index in the same ITP patient.The results indicate a negative correlation between CBLB expression and proliferation index after anergy induction.2.After transfecting CD4+T cells from ITP patients and healthy controls with CBLB recombinant adenovirus.After anergy restimulation,we found that the percentage of CD4+CD25+T cells was not significantly different between ITP and control groups overexpressing CBLB.The results indicated that the cellular hyperactivation state was corrected by overexpressing CBLB.3.CBLB expression after resting and anergy induction were significantly reduced in CD4+T cells of patients with ITP.After anergy induction,the p-AKT-to-AKT ratio tended to increase in patients with ITP but decreased remarkably in healthy controls.4.Compared with that of CD4+T cells transfected with a negative control vector,CD4+T cells overexpressing CBLB showed a markedly decreased p-AKT-to-AKT ratio after treatment with ionomycin in both patients with ITP and healthy controls.5.Methylation levels were significantly higher in the 5’ UTR of the S2 region of CBLB in CD4+T cells from patients with ITP than in those from healthy controls.6.In ITP patients who responded to low-dose decitabine treatment,the expression of CBLB was significantly increased after treatment.It indicated the regulatory effect of low-dose decitabine on CBLB methylation and expression.Conclusions:1.CBLB modulates T-cell anergic resistance via PI3K-AKT signaling pathway.2.Elevated methylation levels in the 5’UTR of the CBLB gene are associated with decreased expression of CBLB in ITP patients.3.Low-dose decitabine treatment promoted CBLB expression in CD4+T cells from ITP patients.更多还原