【作者】 刘燕；

【导师】 彭军；

【作者基本信息】 山东大学 ， 内科学（血液病）（专业学位）， 2023， 博士

【摘要】 第一部分:单细胞水平解析ITP中骨髓造血干祖细胞的转录组改变研究背景:原发免疫性血小板减少症(ITP)是一种获得性自身免疫性疾病,以无明确诱因的孤立性外周血血小板计数减少和出血风险增加为主要特点,是临床上最常见的出血性疾病。ITP的主要发病机制已被广泛研究,普遍认为是免疫介导的血小板破坏增加和血小板生成减少。血小板生成是一个复杂的生物过程,涉及到造血干细胞向巨核细胞谱系的定向分化、巨核细胞成熟和血小板释放。此前,包含我们课题组在内的多个团队报道了抗血小板自身抗体、骨髓CD8+T细胞、肿瘤坏死因子相关凋亡诱导配体(TRAIL)损害ITP的巨核细胞生成。最近,Herd等报道了小鼠ITP模型中长期造血干细胞(LT-HSC)的体外激活和增殖水平升高。然而,造血分化是否以及如何参与ITP的发病机制尚不清楚。一方面,血小板的减少和免疫细胞的消耗激活骨髓造血,另一方面,造血干祖细胞(HSPCs)本身可能是自身免疫攻击的目标,因为目标抗原既存在于血小板上,也存在于较不成熟的造血祖细胞上。此外,不同骨髓HSPCs亚群间的细胞串扰可能在诱导巨核生成缺陷中发挥关键作用,ITP中HSPCs间潜在的相互作用仍然是一个亟待深入研究领域。因此,我们提出“ITP骨髓中HSPCs亚群存在异常,导致巨核细胞生成缺陷,血小板生成减少,针对骨髓HSPCs的检查和干预可能成为ITP诊断和治疗的新靶点”的假说。HSPCs具有高度异质性。单细胞技术,特别是转录组测序及其相关生物信息分析策略的进步,使得直接研究异质性细胞群体更加便捷,具有传统实验方法无法比拟的优势。近年来,单细胞转录组测序(scRNA-seq)技术在研究干细胞分化、胚胎发育、肿瘤免疫等方面的研究中都发挥了重要作用。然而,目前仍然没有ITP病理状态下骨髓HSPCs的scRNA-seq的相关报道。在单细胞水平解析骨髓HSPCs转录组学改变,鉴定潜在的致病细胞亚群及分子调控机制,对ITP发病机制的研究十分必要。研究目的:(1)绘制ITP患者骨髓HSPCs单细胞转录组图谱。(2)探索ITP患者骨髓HSPCs单细胞转录组改变。(3)筛选、验证参与巨核细胞生成缺陷的关键骨髓HSPCs亚群和分子机制。研究方法:(1)临床样本采集:采集未经治疗的新诊断ITP患者和健康志愿者的髂骨骨髓液标本。(2)单细胞转录组文库制备及测序:使用流式细胞分选技术分选骨髓CD34+HSPCs,基于10x Genomics平台进行单细胞3’端转录组建库,测序使用Illumina NovaSeq 6000。(3)单细胞转录组数据处理:下机数据使用bcl2fastq进行拆分,Cell Ranger进行参考基因组比对、基因表达水平定量。使用 Seurat、Harmony、ClusterProfiler、GSEA、pySCENIC、Monocle2、Scanpy、CellPhoneDB等工具进行下游分析,以查找ITP骨髓CD34+HSPCs在细胞亚型、基因表达、分化轨迹、转录调控网络、细胞互作等方面的差异。(4)抗体和流式细胞术:细胞表面和核内标记物染色按照说明书进行。采用Kaluza analysis软件进行分析。(5)细胞离体培养和形态观察:使用添加多谱系细胞因子和血清替代物的StemSpan SFEM Ⅱ培养基培养 CD9+Lin-CD34+CD45RA-细胞、CD9-Lin-CD34+CD45RA-细胞、Lin-CD34+CD45RA+CD3 8+CD9+CD10+细胞和 Lin-CD34+CD45RA+CD3 8+CD161+细胞。在37℃、5%二氧化碳的恒温加湿培养箱中培养。细胞形态使用普通光学显微镜、免疫荧光显微镜观察。(6)统计分析:在R软件中使用卡方(χ2)检验、双尾学生t检验、变量转换和方差分析(ANOVA),以及Scheffe事后检验来进行比较分析。研究结果:(1)ITP患者和健康对照组(HC)骨髓CD34+HSPCs的单细胞转录组图谱经过质量控制和批次效应校正后,共计纳入56,312(ITP,n=28,507;HC,n=27,805)个细胞至下游数据分析中,这些细胞进一步被鉴定为15个基因表达模式不同的细胞亚群。包括1个造血干细胞亚群(HSC),1个多能祖细胞亚群(MPP),1个粒细胞-巨噬细胞祖细胞亚群(GMP),1个中性粒细胞祖细胞亚群(NeuP),1个单核细胞-树突细胞祖细胞亚群(MDP),1个嗜酸性粒细胞-嗜碱性粒细胞-肥大细胞祖细胞亚群(EBMP),1个共同淋巴样祖细胞亚群(CLP),3个前B细胞亚群(preB1,preB2和preB3),1个自然杀伤细胞/T细胞祖细胞亚群(NK/Tp),1个巨核细胞-红细胞祖细胞亚群(MEP),2个巨核细胞祖细胞亚群(MkP1和MkP2),1个红细胞祖细胞亚群(EryP)。拟时分析显示ITP组和HC组的骨髓CD34+HSPCs具有相似的分化轨迹,但在转录组尤其是免疫相关基因的表达水平上存在显著差异,这提示在揭露ITP的发病机制时应考虑HSPCs的免疫相互作用。进一步地分析显示两组的巨核细胞/红细胞(Mk/Ery)谱系细胞沿伪时间的分布并不完全一致,这一结果提示与HC组相比,ITP组HSPCs的分化潜能,特别是向巨核细胞和(或)红细胞分化的潜能可能存在异常。(2)ITP中骨髓CD34+HSPCs的转录组改变我们评估了 ITP组在每一个亚群中的转录组改变,总共得到1,166个差异表达基因(DEGs),其中,33.28%的DEGs发生在preB3和NK/Tp这两个亚群,这提示免疫细胞在ITP的发病机制中发挥重要作用。进一步分析显示独特的配体-受体相互作用和转录调控网络的失调可能在这一过程中发挥重要作用。(3)HES1和CD9在ITP中表达下调DEG分析的结果显示,在MkP1中,ITP组最显著上调的基因包括HBB、HBG2和AHSP,它们都与红细胞生成相关;而在MkP2中,ITP组最显著下调的基因包括CD9和TUBB1,它们都与血小板生成相关。这提示在ITP骨髓中巨核细胞生成过程可能存在缺陷。因此,我们聚焦于影响分化潜能的DEGs,并选择HES1和CD9进行进一步验证。多参数流式细胞术实验的结果显示,ITP中HES1+细胞和CD9+细胞的数量显著减少,与单细胞转录组数据的分析结果一致。(4)CD9+Lin-CD34+CD45RA-HSPCs在体外具有巨核细胞生成倾向,并在ITP中存在显著缺陷细胞离体培养实验表明,CD9+Lin-CD34+CD45RA-HSPCs具有向巨核细胞分化的趋势,但这种趋势在ITP中没有观察到,培养体系中反而产生了更多的红细胞。在HC组中,CD9+Lin-CD34+CD45RA-HSPCs培养体系中分化产生的巨核细胞的比例约比CD9-Lin-CD34+CD45RA-HSPCs 的高 3 倍,而在 ITP 组中,CD9+Lin-CD34+CD45RA-HSPCs培养体系中分化产生的巨核细胞的比例下降到HC组的 1/4,与CD9-Lin-CD34+CD45RA-HSPCs的基本相当,这些结果表明ITP中CD9+HSPCs向巨核细胞分化的过程存在异常,细胞分化潜能可能受损。此外,我们证明CD9可用于富集具有巨核细胞分化偏倚的HSPCs,而且CD9+Lin-CD34+CD45RA-HSPCs有可能成为ITP诊断和治疗新靶点。(5)与ITP来源的pre-B细胞共培养显著降低了 CD9+Lin-CD34+CD45RA-HSPCs后代中巨核细胞的比例分选ITP患者和健康志愿者的骨髓pre-B细胞,分别与健康志愿者的骨髓CD9+Lin-CD34+CD45RA-HSPCs进行Transwell细胞共培养,结果显示ITP来源的pre-B细胞使CD9+Lin-CD34+CD45RA-HSPCs后代中巨核细胞的比例显著下降,提示ITP中的骨髓巨核细胞生成缺陷可能与异常的pre-B细胞相关。(6)MkP的细胞异质性及ITP中MkP亚群的转录组变化对ITP和HC所有样本中的MkP(MkP1和MkP2)细胞进行汇集和再分类分析,我们发现MkP可以进一步地分为7个具有不同基因表达模式和功能的亚群。ITP相关的DEGs具有MkP亚型特异性,其中98.5%的上调基因和82.4%的下调基因集中在1个具有免疫调节和血小板生成双重功能的亚群中,这提示在MkP中,该亚群与ITP发病机制最具相关性。在该亚群中,与血小板生成相关的基因,例如GP9、PF4、CD9、PPBP等,表达是显著下调的,而与红细胞生成相关的基因,例如HBB、HBD、CA1、AHSP等,表达则是显著上调的,这与前述MkP1和MkP2的分析结果一致。富集分析结果显示,该亚群血小板活化和细胞免疫通路显著下调,RNA剪接和HSC分化调控显著上调研究结论:通过本研究,我们提供了一个全面的骨髓CD34+HSPCs单细胞转录组图谱来描述未经治疗的新诊断ITP患者的转录组改变。我们的分析显示,ITP骨髓中存在一个独特的免疫生态系统,其特征是造血过程中免疫相关基因和通路的增强,pre-B和NK/Tp亚群中大量的转录组改变,HSPCs亚群间免疫串扰的激活,以及MkP亚群中伴随血小板生成潜能下调的免疫调节功能减弱。功能试验结果表明,ITP患者Lin-CD34+CD45RA-HSPCs中HES1+细胞和CD9+细胞数量均减少,CD9+Lin-CD34+CD45RA-HSPCs的巨核细胞分化潜能受损,而这一损伤可能与异常的pre-B细胞相关。我们在单细胞水平解析了 ITP患者骨髓CD34+HSPCs的异质性和疾病特征,为ITP研究提供了新的病理生理学见解和潜在治疗靶点。第二部分:HDAC3 rs2530223单核苷酸多态性与ITP易感性和严重程度增加相关研究背景:原发免疫性血小板减少症(ITP)是一种获得性自身免疫性出血性疾病,定义为血小板计数<100×109/L且无其他原因引起的血小板减少症。ITP是临床上最常见的出血性疾病,约占所有出血性事件的30%,在成人中常表现为慢性疾病。普遍认为体液免疫和细胞免疫参与了其发病过程,包括T细胞的异常分化和应答,以及浆细胞分泌抗巨核细胞和血小板的自身抗体。使用糖皮质激素和其他药物治疗ITP仍有约50%的患者治疗无效或在短期内复发。因此,有必要进一步探讨ITP的危险因素,以确定新的潜在治疗靶点。组蛋白去乙酰化酶(HDAC)超家族在哺乳动物中由基因组编码的11个HDAC亚型组成。我们以前报道过,低剂量的西达本胺可以恢复ITP患者的免疫耐受,而西达本胺是一种选择性HDAC抑制剂。在成年哺乳动物中,HDAC3在应对环境挑战、调控昼夜节律、调节营养与代谢途径、限制自身免疫、维持体内稳态等方面都发挥作用。HDAC3通过系统性免疫反应调节免疫功能、影响细胞稳态,从而调节自身免疫和炎症反应。例如,HDAC3是介导炎症巨噬细胞免疫耐受和反应的一个很有价值的蛋白质靶标,巨噬细胞中HDAC3的缺失表现出抗炎组蛋白乙酰化基因表达模式,具有抗炎症的保护作用。T细胞的HDAC3缺失会阻碍其向双阳性阶段的转变,并表现出T细胞功能和细胞周期相关基因的表达水平下调。此外,调节性T细胞(Treg)中的HDAC3缺乏会扰乱免疫抑制性Treg的HDAC3依赖性功能,从而导致炎症性肠病(IBD)和其他致命的自身免疫病。因此,HDAC3作为一个动态染色质调节因子调控自身免疫,其在ITP中的作用还有待进一步阐明。遗传学研究表明许多遗传变异,尤其是单核苷酸多态性(SNP)与自身免疫性疾病有关。已有研究表明HDAC3rs2530223与糖尿病及肿瘤免疫有关。考虑到HDAC3在免疫调节中发挥重要功能,我们假设HDAC3多态性在ITP发病机制中有重要作用。研究目的:探究HDAC3 rs2530223多态性与中国汉族人群ITP易感性、严重程度以及治疗反应之间的关系。研究方法:(1)临床样本采集:收集ITP患者的临床资料和外周静脉血样本,并根据外周血血小板计数、皮质类固醇敏感性、难治性进行分层。收集年龄、性别匹配的健康志愿者的外周静脉血样本作为对照组。所有参与者都属于汉族人群。(2)HDAC3基因多态性测定:分离ITP患者和健康志愿者样本中的外周血单个核细胞(PBMC)并提取其基因组DNA。利用MassARRAY平台对HDAC3 rs2530223多态性进行基因分型。(3)统计分析:对对照组HDAC3 rs2530223基因型进行哈迪-温伯格平衡定律(HWE)检验。数据统计分析使用SPSS 26.0软件进行,统计方法为卡方(χ2)检验、Fisher精确检验和二元单因素Logistic回归分析。研究结果:(1)研究对象共计招募到209例ITP患者和210例健康志愿者,两组在年龄、性别上无显著性差异(P值分别为0.25和0.25)。ITP患者中,血小板计数<30×109/L的占75.60%(n=158),难治性的占6.22%(n=13),使用皮质类固醇治疗的占80.86%(n=169),其中86例发生皮质类固醇抵抗。健康对照组的HDAC3 SNP rs2530223符合HWE(P=0.41)。(2)HDAC3基因多态性与ITP易感性的关系在隐性、显性、共显性和等位基因模型中,HDAC3rs2530223的基因型频率和等位基因频率与ITP易感性显著相关(P值分别为0.036、0.033、0.035和0.009)。T等位基因使 ITP 易感性增加 1.472 倍(OR 1.472;95%CI 1.100-1.969;P=0.009)。共显性和隐性模型中TT基因型以及显性模型下TC/TT基因型的ITP易感性均显著增加(共显性模型:OR 2.264,95%CI 1.175-4.360,P=0.015;隐性模型:OR 1.512,95%CI 1.028-2.224,P=0.036;显性模型:OR 1.965,95%CI 1.046-3.656,P=0.036)。(3)HDAC3基因多态性与血小板计数的关系将ITP患者分为血小板计数<30×109/L(n=158)和血小板计数≥30×109/L(n=51)两组,在显性和共显性模型中,HDAC3rs2530223基因型频率在两组间存在差异,提示ITP患者HDAC3rs2530223基因型频率与血小板计数具有显著相关性(P值分别为0.008和0.030),而在隐性和等位基因模型下差异无统计学意义(P值分别为0.448和0.068)。在共显性和显性模型中,TC/TT基因型与血小板计数<30×109/L的风险增加相关(OR 3.932;95%CI 1.426-10.842;P=0.008)。(4)HDAC3基因多态性与皮质类固醇敏感性的关系将接受皮质类固醇治疗的ITP患者分为皮质类固醇敏感组(n=83)和皮质类固醇抵抗组(n=86),HDAC3rs2530223的基因型频率和等位基因频率在这两组间没有显著性差异(隐性模型:P=0.494;显性模型:P=0.169;共显性模型:P=0.110;等位基因模型:P=0.968)。(5)HDAC3基因多态性与ITP难治性的关系将对药物治疗和脾切除术有反应的ITP患者归为非难治性组(n=196),其他ITP患者归为难治性组(n=13),HDAC3rs2530223的基因型频率和等位基因频率在这两组间没有显著性差异(隐性模型:P=0.871;显性模型:P=0.606;共显性模型:P=0.753;等位基因模型:P=0.547)。研究结论:我们发现HDAC3 rs2530223单核苷酸多态性与ITP易感性和严重程度增加相关,与ITP难治性或皮质类固醇敏感性无关。HDAC3可能参与了 ITP的分子发病机制。更多还原

【Abstract】 Part I:Deciphering transcriptome alterations in bone marrow hematopoiesis at single-cell resolution in immune thrombocytopeniaBackground:Primary immune thrombocytopenia(ITP)is an acquired autoimmune disorder characterized by reduction in platelet count and increase in the risk of bleeding.It is the most common hemorrhagic disease in clinic practice.Its pathogenesis has been extensively studied,with immune-mediated increase in destruction and decrease in the production of platelets as the accepted mechanisms.Platelet production is a complex biological process that involves hematopoietic stem cell commitment to the megakaryocytic lineage,megakaryocyte maturation,and platelet release.Previously,our group and others have demonstrated that antiplatelet autoantibodies,bone marrow(BM)CD8+ T cells,and tumor necrosis factor-related apoptosisinducing ligand(TRAIL)in BM plasma and megakaryocytes impair megakaryopoiesis in ITP.Recently,Herd et al.reported elevated long-term hematopoietic stem cell(LT-HSC)activation and proliferation in vitro in a murine ITP model.However,whether and how hematopoietic differentiation contributes to the pathogenesis of ITP in humans remain unclear.On the one hand,decrease in platelet count and depletion of immune cells activated hematopoiesis,while on the other hand,hematopoietic stem and progenitor cells(HSPCs)themselves could be targets of autoimmune attack because of the target antigens present on both platelets and the more immature hematopoietic progenitors.In addition,the cellular crosstalk among different subclusters of HSPCs might play critical roles in inducing defective megakaryopoiesis.However,comprehensive documentation of the underlying molecular interactions in HSPCs in ITP remains an open area of investigation.Therefore,we proposed the hypothesis that abnormalities of HSPCs in ITP BM lead to megakaryocytogenesis defects and reduced platelet production,and the examination and intervention of BM HSPCs may become a new target for the diagnosis and treatment of ITP.HSPCs are highly heterogeneous.Single cell technology,especially transcriptome sequencing and its bioinformation analysis strategies,which has incomparable advantages over traditional experimental methods,has made it convenient to directly study heterogeneous cell populations.In recent years,single-cell RNA sequencing(scRNA-seq)has played an important role in the research of stem cell differentiation,embryonic development,and tumor immunity.However,there are still no reports on scRNA-seq of BM HSPCs in ITP.It is necessary to analyze the transcriptome changes of BM HSPCs at the single-cell level and identify the potential pathogenic subpopulations and molecular mechanisms,so as to study the pathogenesis of ITP.Objective:(1)To construct the single-cell transcriptome profile of BM HSPCs in ITP.(2)To investigate the single-cell transcriptome alterations of BM HSPCs in ITP.(3)To determine the cellular and molecular basis for megakaryopoiesis defect in ITP.Methods:(1)Clinical sample collection:Iliac BM samples were obtained from newly diagnosed treatment-naive ITP patients and healthy donors.(2)Single cell RNA library preparation and sequencing:BM CD34+HSPCs were purified using fluorescence-activated cell sorting(FACS).ScRNA-seq libraries were performed using the Chromium Single Cell 3 Solution V3(10x Genomics)and sequenced using Illumina NovaSeq 6000.(3)Processing of single-cell RNA-seq data:The raw files were split using bcl2fastq,read alignment and gene expression quantification processed using Cell Ranger(10x Genomics).In order to find the changes of ITP BM CD34+HSPCs in cell subtypes,gene expression,pseudotime trajectories,transcriptional regulatory networks and cell-cell interactions,Seurat,Harmony,ClusterProfiler,GSEA,pySCENIC,Monocle2,Scanpy,CellPhoneDB and other tools were used for downstream analysis.(4)Antibodies and flow cytometry:Cell surface and intranuclear marker staining was performed according to the manufacturer’s protocol.Flow cytometric analysis(FCA)was performed using the Kaluza Analysis software.(5)In vitro culture and morphological observation:CD9+Lin-CD34+CD45RA-HSPCs,CD9-Lin-CD34+CD45RA-HSPCs,Lin-CD34+CD45RA+CD38+CD9+CD10+HSPCs,and Lin-CD34+CD45RA+CD38+CD161+HSPCs were cultured with StemSpan SFEM II medium added multi-lineage supplemented factors and serum substitute.All cultures were incubated at 37℃ in a humidified chamber in the presence of 5%carbon dioxide.Cell morphology was observed using light microscopy and immunofluorescence microscopy.(6)Statistical analysis:The chi-square(χ2)test,two-tailed Student’s t-test,variable transformation,and analysis of variance(ANOVA),followed by a Scheffe’s post-hoc test were used in R for comparing the experimental groups.Results:(1)Single-cell transcriptomes of BM CD34+HSPCs from ITP patients and healthy controlsAfter quality control and batch effect correction,56,312(ITP,n=28,507;HC,n=27,805)single-cell profiles were included in the downstream analyses.We manually annotated the cell clusters into 15 different cell types with distinct gene expression patterns.These populations included hematopoietic stem cells(HSC),multipotent progenitors(MPP),granulocytemacrophage progenitors(GMP),neutrophil progenitors(NeuP),monocytedendritic-cell progenitors(MDP),eosinophil-basophil-mast-cell progenitors(EBMP),common lymphoid progenitors(CLP),three pre-B cell populations(preB1,preB2,and preB3),natural killer/T cell progenitors(NK/Tp),megakaryocyte-erythroid progenitors(MEP),two MkP populations(MkPl and MkP2),and erythroid progenitor cell populations(EryP).We further analyzed the trajectories in ITP and HC samples separately and obtained similar transition trajectories.However,the immune and transcriptional states differed considerably between ITP and HC,suggesting that immunological interactions within HSPCs should be considered for understanding the pathogenesis of ITP.In addition,the detailed distributions of megakaryocyte and erythrocyte(Mk/Ery)lineage cells along the pseudotime were not entirely consistent,implying that the differentiation potential of HSPCs in ITP might be affected.(2)Transcriptional changes in BM CD34+HSPCs in ITPWe assessed transcriptome alterations in each HSPC subset of ITP,yielding 1,166 differentially expressed genes(DEGs)in total.Among these DEGs,the expression of 33.28%was altered in preB3 and NK/Tp,suggesting that immune cells play an important role in the pathogenesis of ITP.And further analysis revealed that the unique cell-cell interactions and dysregulation of transcriptional regulatory networks may play important roles in this process.(3)HES1 and CD9 were downregulated in ITP patientsDEG analysis indicated that the top upregulated genes in MkPl of ITP included HBB,HBG2,and AHSP,which were associated with erythropoiesis,while the top downregulated genes in MkP2 of ITP included CD9 and TUBB1,which are well-established genes related to thrombopoiesis,suggesting a bias toward erythrocytes in megakaryopoiesis in ITP.We therefore focused on DEGs that affected the differentiation potential,and HES1 and CD9 were selected for further verification.Multi-parameter flow cytometric analysis showed reduction in the number of HES1+ cells and CD9+cells in ITP,confirming the results of our single-cell transcriptome analysis.(4)CD9+Lin-CD34+CD45RA-HSPCs were biased toward megakaryopoiesis in vitro and were defective in ITPLiquid culture assays demonstrated that CD9+Lin-CD34+CD45RA-HSPCs tended to differentiate into megakaryocytes;however,this tendency was not observed in ITP patients and more erythrocytes were produced.The percentage of megakaryocytes differentiated from CD9+Lin-CD34+CD45RA-HSPCs was 3-fold higher than that of the CD9-counterparts from healthy controls,whereas in ITP patients,the percentage decreased to only 1/4th of that in the healthy controls and was comparable to that from the CD9-HSPCs,implying that differentiation of CD9+HSPCs toward the megakaryopoietic lineage was impaired in ITP.CD9 can be used to enrich Mk-biased HSPCs,and CD9+Lin-CD34+CD45RA-HSPCs have the potential to be novel diagnostic and therapeutic targets in ITP.(5)Co-culture with pre-B cells from ITP markedly decreased the generation of megakaryocytes from CD9+Lin-CD34+CD45RA-HSPCsPre-B cells from healthy donors and ITP patients were co-cultured with CD9+Lin-CD34+CD45RA-HSPCs from healthy donors.And the proportion of megakaryocytes in the CD9+Lin-CD34+CD45RA-HSPC progeny decreased significantly in the group co-cultured with pre-B cells from ITP.The data demonstrated that the defective megakaryopoiesis in ITP might be related to aberrant pre-B cells.(6)Cellular heterogeneity in MkP and transcriptional changes in MkP subclusters of ITPWe pooled MkP from all samples of ITP and HC,and these cells were further divided into seven subclusters with different gene expression patterns and functions.The ITP-associated DEGs were MkP subtype-specific,and 98.5%of the upregulated genes and 82.4%of the downregulated genes in these subtype-specific genes occurred in the subcluster possessing dual functions of immunomodulation and platelet generation,suggesting that this subcluster was most associated with ITP.We found that thrombopoiesis-related genes,such as GP9,PF4,CD9,and PPBP,were downregulated significantly in this subcluster,meanwhile,erythropoiesisrelated genes,such as HBB,HBD,CA1,and AHSP,were upregulated significantly,consistent with the previous finding in MkP1 and MkP2.Enrichment analysis revealed that platelet activation and cellular immunity pathways were significantly downregulated in this subcluster of ITP,while RNA splicing and regulation of HSC differentiation were significantly upregulated.Conclusion:Here,we provided a comprehensive single-cell transcriptomic atlas of HSPCs to characterize transcriptome alterations in newly diagnosed treatment-naive ITP.Our analysis revealed a distinct immune ecosystem in ITP BM,characterized by enhancement of immunerelated genes and pathways during hematopoiesis,abundant transcriptome changes in pre-B and NK/Tp subpopulations,activation of immune crosstalk within HSPC clusters,and weakened immunomodulatory functions of an MkP subcluster,along with downregulation of plateletgeneration potential.Functional assays demonstrated that the number of CD9+cells and HES1+ cells in Lin-CD34+CD45RA-HSPCs decreased in ITP,and the differentiation of CD9+Lin-CD34+CD45RA-HSPCs toward the megakaryopoietic lineage was impaired,which might be related to aberrant pre-B cells.In summary,this is the first study to show the significant heterogeneity and disease characteristics of BM CD34+ HSPCs from ITP patients at single-cell resolution using transcriptomic profiling,revealing new insights regarding the pathophysiology of ITP.Part Ⅱ:HDAC3 single-nucleotide polymorphism rs2530223 is associated with increased susceptibility and severity of primary immune thrombocytopeniaBackground:Primary immune thrombocytopenia(ITP)is an acquired autoimmune bleeding disorder,defined as a platelet count<100×109/L without other causes of thrombocytopenia.ITP is the most common clinical hemorrhagic disease,accounting for～30%of all hemorrhagic events and is often a chronic disorder in adults.For years,it is well noted that humoral and cellular immune responses are participating in its pathogenesis,including abnormal T cell differentiation and responses,and autoantibody secretion of plasma cells targeting megakaryocytes and platelets.Although glucocorticoids and other drugs are used to treat ITP,approximately 50%of patients fail to respond to therapy or relapse in a short term.Therefore,it is necessary to further explore the risk factors for ITP to identify novel potential therapeutic targets.The histone deacetylase(HDAC)superfamily comprises 11 HD AC isoforms encoded by the mammalian genome.We previously reported that low-dose chidamide,a selective HDAC inhibitor,restores immune tolerance in patients with ITP.In adult mammals,HDAC3 uniquely regulates environmental challenges,circadian,nutrient,metabolic pathways,and limits autoimmunity,among other homeostatic functions.HDAC3 modulates the functions of immune cells and influences cell homeostasis through systemic immune responses,thereby regulating autoimmunity and inflammation.For example,HDAC3 is an attractive protein target to mediate tolerance induction and reactivity in inflammatory macrophages,and depletion of HDAC3 in macrophages displays an anti-inflammatory histone acetylation gene expression pattern,with a protective effect against inflammation.HDAC3 deletion of T cells impaired their transition to the double-positive stage and displayed downregulation of genes for T cell function and cell cycle.Moreover,HDAC3 deficiency in Tregs perturbed the HDAC3-dependent function of immunosuppressive Tregs,which led to aggravated inflammatory bowel disease(IBD)and other lethal autoimmune outcomes in animals.Therefore,HDAC3 acts as a dynamic chromatin regulator to regulate autoimmunity,but its function in ITP remains to be elucidated.Genetic studies have revealed some genetic variations,especially single nucleotide polymorphisms(SNPs),are related to autoimmune diseases.HDAC3 rs2530223 has been implicated in diabetes mellitus and tumor immunity.Considering the vital immunomodulatory function of HDAC3,we hypothesized that HDAC3 polymorphism may play an important role in ITP pathogenesis.Objective:To investigate the association between HDA C3 rs2530223 polymorphism and the susceptibility or severity or therapeutic response of ITP in the Chinese Han population.Methods:(1)Clinical sample collection:Clinical data and peripheral venous blood samples from ITP patients were collected and stratified according to platelet counts,corticosteroid sensitivity,and refractoriness.Peripheral venous blood samples were collected from age-and sex-matched healthy donors as controls.All participants belonged to the Han population.(2)Determination of HDAC3 gene polymorphism:Peripheral blood mononuclear cells(PBMCs)were isolated from peripheral venous blood samples of ITP patients and healthy donors and genomic DNA was extracted from the PBMCs.Genotyping of the HDAC3 rs2530223 polymorphism was performed using MassARRAY platform.(3)Statistical analysis:The Hardy-Weinberg equilibrium(HWE)of HDAC3 rs2530223 genotypes was calculated in the control group.The chi-squared(χ2)test,Fisher’s exact test,and univariate binary logistic regression analyses were used in SPSS software(version 26.0)for statistical analyses.Results:(1)Study cohortNo significant difference was found in age or sex between ITP patients(n=209)and healthy donors(n=210)(p=0.25 and 0.25,respectively).75.60%of ITP patients had platelet counts<30×109/L(n=158),6.22%were refractory(n=13),and 80.86%received corticosteroid treatment(n=169),of which 86 were corticosteroid-resistant.The HDAC3 SNP rs2530223 conformed to HWE in the healthy control group(p=0.41).(2)Relationship between HDAC3 gene polymorphism and ITP susceptibilityUnder the recessive,dominant,codominant,and allelic models,the genotypic and allelic frequencies of HDAC3 rs2530223 were significantly associated with ITP susceptibility(p=0.036,0.033,0.035,and 0.009,respectively).Individuals with T allele of HDAC3 rs2530223 exhibited a 1.472-fold increased risk of ITP susceptibility(OR 1.472;95%CI 1.100-1.969;p=0.009),while ones with the TT genotype under the codominant and recessive models,and the TC/TT genotypes under the dominant model all revealed increased risk of ITP susceptibility(Dominant:OR 1.965;95%CI 1.046-3.656;p=0.036;Codominant:OR 2.264;95%CI 1.1754.360;p=0.015;Recessive:OR 1.512;95%CI 1.028-2.224;p=0.036).(3)Relationship between HDAC3 gene polymorphism and platelet countWe divided ITP patients into platelet counts<30×109/L(n=158)and platelet counts≥30×109/L(n=51)groups to evaluate the relationship between HDA C3 rs2530223 and platelet count in patients with ITP.Under dominant and codominant models,and statistical analysis revealed different genotypic frequency distributions of HDAC3 rs2530223 between the two groups,suggesting a significant relation with platelet count in patients with ITP(p=0.008 and 0.030,respectively).However,there was no significant difference in the recessive and allele models of HDAC3 rs2530223(p=0.448 and 0.068,respectively).Under the codominant and dominant models,the TC/TT genotypes of HDAC3 rs2530223 are relevant to the in increased risk of P platelet counts<30×109/L(OR 3.932;95%CI 1.426-10.842;p=0.008).(4)Relationship between HDA C3 gene polymorphism and corticosteroid sensitivityWe stratified the patients with corticosteroid therapy into a corticosteroid-sensitive group and a corticosteroid-resistant group(n=83 and 86,respectively),to explore the association between HDAC3 rs2530223 and the susceptibility to corticosteroid treatment.However,there was no significant difference in the allelic or genotypic frequencies of HDAC3 rs2530223 between the corticosteroid-sensitive group and the corticosteroid-resistant group(recessive,p=0.494;dominant,p=0.169;codominant,p=0.110;allele,p=0.968).(5)Relationship between HDAC3 gene polymorphism and ITP refractorinessWe further assigned those patients who responded to medical treatment and splenectomy to the non-refractory group,and the others were assigned to the refractory group(n=196 and 13,respectively).Neither allelic nor genotypic frequencies of HDAC3 rs2530223 were significantly different between the two groups(recessive,p=0.871;dominant,p=0.606;codominant,p=0.753;allele,p=0.547).Conclusion:The results revealed that HDAC3 single-nucleotide polymorphism rs2530223 was associated with increased susceptibility and severity of ITP,but not with refractoriness or corticosteroid sensitivity.HDAC3 may be involved in the molecular pathogenesis of ITP.更多还原