CN116814768A - 一种单核细胞亚群用于诊断或治疗新冠感染后遗症的用途 - Google Patents
一种单核细胞亚群用于诊断或治疗新冠感染后遗症的用途 Download PDFInfo
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Abstract
本发明生物医学技术领域,具体涉及制备用于诊断和/或治疗新型冠状病毒感染后遗症(long COVID)的技术和产品中的用途。从受试者获得外周血样品;确定样品中细胞周期蛋白依赖性激酶抑制剂1C阳性(CDKN1C+)的非经典单核细胞比例,和比较受试者的CDKN1C+的非经典单核细胞比例与预定参考值;其中,预定参考值以健康人群体中CDKN1C+非经典单核细胞的比例的中位数为基础,且与预定参考值相比,样品中升高的CDKN1C+非经典单核细胞的比例显示受试者患有long COVID。本发明对对非经典单核细胞进行细胞功能实验和体外IL‑1β刺激实验的结果强调了对CDKN1C+非经典单核细胞以及对IL‑1β的阻断可作为long COVID治疗靶标的前景。
Description
技术领域
本发明涉及生物医学技术领域,具体涉及制备用于诊断和/或治疗新型冠状病毒感染后遗症的技术和产品中的用途。
背景技术
截止2013年4月,由严重急性呼吸综合征冠状病毒2(SARS-CoV-2)引起的2019冠状病毒病(COVID-19)已导致超过7.63亿人感染和690万人死亡(参见https://covid19.who.int/)。越来越多的患者在急性感染后逐渐康复,但部分康复者出现了长期的后遗症(long COVID,post COVID-19condition或Post-acute sequelae of COVID-19,PASC,参见World Health Organization.A clinical case definition of post COVID-19condition by a Delphi consensus,6October 2021.2021.https://apps.who.int/iris/handle/10665/345824),并已成为全球关注的问题。我国针对第一波新冠感染康复者的长期随访队列研究中,出院一年后49%的患者以及出院两年后55%的患者仍至少存在一种后遗症(参见Huang L,Yao Q,Gu X,et al.1-year outcomes in hospital survivorswith COVID-19:a longitudinal cohort study.Lancet.2021Aug 28;398(10302):747-758.和Huang L,Li X,Gu X,et al.Health outcomes in people 2years aftersurviving hospitalisation with COVID-19:a longitudinal cohort study.LancetRespir Med.2022Sep;10(9):863-876.)。此外美国国家卫生统计中心在实验性家庭脉搏调查中添加了一些问题以评估long COVID的流行程度,在最近为期两周(2023年3月29日至4月10日)的阶段调查中仍有15.5%的新冠康复者患有后遗症(参见https://www.cdc.gov/nchs/covid19/pulse/long-covid.htm)。因此亟需对新冠感染后遗症的诊断和治疗方法以减轻新冠后遗症所带来的全球性公共卫生负担。
单细胞转录组测序(scRNA-seq)是一种提供单个细胞的表达谱的技术,能够揭示细胞群中单个细胞间的关键转录信息差异。因此scRNA-seq可用于监测与疾病相关的细胞分布和基因转录模式,从而筛选出疾病诊断和预后相关的候选生物标志物和治疗靶标。例如多项研究发现高表达钙卫蛋白且低表达第Ⅱ型人类白细胞DR抗原的经典单核细胞在COVID-19重症患者中富集,可作为区分COVID-19重症患者的标志物之一以及潜在的治疗靶点(参见Schulte-Schrepping J,Reusch N,Paclik D,et al.Severe COVID-19Is Markedby aDysregulated Myeloid Cell Compartment.Cell.2020Sep 17;182(6):1419-1440.e23.和Silvin A,Chapuis N,Dunsmore G,et al.Elevated Calprotectin andAbnormal Myeloid Cell Subsets Discriminate Severe from Mild COVID-19.Cell.2020Sep 17;182(6):1401-1418.e18.)。尽管目前已有针对long COVID中部分后遗症症状的诊断工具,例如用于诊断后遗症中体位性直立性心动过速综合征的抬头倾斜台试验和用于检测心血管损伤的磁共振成像扫描,但目前尚无普遍性筛查且灵敏度高的longCOVID的诊断工具。同样地,虽然针对部分症状的治疗方法对特定患者有效,但仍缺乏针对long COVID的广泛且有效的治疗方法,且大多仍处于开发阶段。因此,筛选更多特异性强、灵敏度高的外周血生物标志物,对long COVID的临床诊断和治疗均具有重要的应用价值。
发明内容
为实现上述目的,本发明提供了针对细胞周期蛋白依赖性激酶抑制剂1C阳性(CDKN1C+)的非经典单核细胞亚群的检测试剂在制备诊断和/或治疗long COVID的产品中的用途。优选地,上述检测试剂包含流式细胞术和质谱流式细胞技术所使用的抗体、单细胞RNA检测的探针和扩增测序所使用的引物。
一方面,本发明提供确定外周血样品中CDKN1C+的非经典单核细胞比例的试剂在制备诊断产品中的用途。
所述产品用于诊断long COVID,与预定参考值相比较,外周血样品中升高的CDKN1C+非经典单核细胞的比例显示受试者患有long COVID;其中,预定参考值以健康人群体中CDKN1C+非经典单核细胞的比例的中位数为基础。
进一步的,CDKN1C+非经典单核细胞为蛋白质水平上分化簇3阴性(CD3-)、分化簇19阴性(CD19-)、分化簇56阴性(CD56-)、分化簇16阳性(CD16+)、补体成分1q阴性(C1Q-)且CDKN1C+表达的单核细胞;转录水平上Fcγ受体IIIa阳性(FCGR3A+)、C1Q-、CDKN1C+且高表达肌酸激酶B(CKB)、细胞间粘附分子4(ICAM4)、G蛋白亚基Gamma 2(GNG2)、细胞间粘附分子3(ICAM3)、突触结合蛋白样蛋白1(SYTL1)、细胞色素P450家族4亚家族F成员22(CYP4F22)、心肌营养素样细胞因子因子1(CLCF1)、干扰素诱导的跨膜蛋白2(IFITM2)、动脉粥样硬化和炎症性肠巨噬细胞调节中富含斑块的LncRNA(SMIM25)、跨膜4结构域亚家族成员7(MS4A7)、钙素(TESC)、卵黄膜外层1同系物(VMO1)和血小板内皮细胞粘附分子1(PECAM1)的单核细胞。
进一步的,long COVID符合世界卫生组织通过德尔菲共识形成的对COVID-19长期影响的临床定义,其症状包括疲劳、气促、认知障碍、嗅觉/味觉改变、抑郁、焦虑、胸痛、咳嗽、头晕、胃肠道问题、头痛、关节痛、肌肉疼痛/痉挛、运动后不适、睡眠障碍和心动过速/心悸。
进一步的,所述产品用于诊断long COVID患者的恢复情况,或评价long COVID治疗方案的有效性。
更进一步的,当所述产品用于诊断long COVID患者的恢复情况,或用于评价longCOVID治疗方案的有效性时,其中预定参考值以患有long COVID的受试者外周血样品中CDKN1C+的非经典单核细胞比例为基础,样品中降低的CDKN1C+的非经典单核细胞比例显示恢复良好或治疗方案有效。
进一步的,适用于本发明的鉴定方法包括通过scRNA-seq、流式细胞术和其他可获得特定细胞类型比例的方法在样品及参照中确定CDKN1C+的非经典单核细胞的比例。
另一方面,本发明提供所述CDKN1C+的非经典单核细胞在制备治疗long COVID的药物中的用途。
进一步的,所述药物包含对调控CDKN1C+非经典单核细胞分化的配体IL-1β的阻断剂。
优选地,上述阻断剂为抗IL-1β抗体、利洛西普(rilonacept)(IL1 Trap)、IL-1受体拮抗剂(IL-1Ra)及相关制品阿那白滞素(anakinra)、IL-1的可溶性受体(利洛西普)和IL-1β的人单克隆抗体(卡那单抗(canakinumab)和Xoma 052)。
本发明具有如下有益效果:本发明通过scRNA-seq和流式细胞术在发现队列和验证队列中首次证实CDKN1C+的非经典单核细胞可作为诊断long COVID的新生物标志物。此外,我们对非经典单核细胞进行细胞功能实验和体外IL-1β刺激实验的结果强调了对CDKN1C+非经典单核细胞以及对IL-1β的阻断可作为long COVID治疗靶标的前景。
附图说明
图1为实施例1中流式细胞术验证所使用的分类策略。PBMC:外周血单个核细胞,FSC-A:前散射面积,SSC-A:侧散射面积,FSC-H:前散射高度。
图2为实施例1中发现的新的C1Q-CDKN1C+非经典单核细胞亚群及其特征性差异表达基因。(A)包含所有单核细胞亚群的的UMAP图。(B)以CDKN1C+非经典单核细胞(c34)标记基因的平均表达量着色的单核细胞UMAP图。(C)比较特定基因在CDKN1C+非经典单核细胞(c34)和C1Q+非经典单核细胞(c35)之间表达百分比的箱型图(双侧Wilcoxon秩和检验)。(D)火山图显示c34和c35细胞之间的差异表达基因(双侧Mann-Whitney U检验,P值通过Benjamini-Hochberg进行多重检验校正)。UMAP:统一流形逼近与投影,padj:Benjamini-Hochberg法校正后的P值。
图3为实施例1中CDKN1C+非经典单核细胞和新型冠状病毒感染后遗症之间的关联。(A和B)基于发现队列的scRNA-seq结果;(C-E)基于验证队列的流式细胞术结果。(A)在六个月随访时(F1),9名患者的c34细胞比例与CT评分之间的关联。(B)所有随访患者中c34细胞比例与关节痛之间的关联(单侧Mann-Whitney U检验)。(C)验证队列中10名患者在六个月随访时通过流式细胞术获得的CDKN1C+CD16+单核细胞比例与其CT评分之间的关联。(D)在验证队列中通过流式细胞术测得的CDKN1C+CD16+单核细胞比例与关节痛之间的关联(双侧Mann-Whitney U检验)。(E)在验证队列中通过流式细胞术测得的CDKN1C+CD16+单核细胞比例与F1时PASC之间的关联(双侧Mann-Whitney U检验)。CT,计算机断层扫描,+:患有,-:未患,PASC:新型冠状病毒感染后遗症,HC:健康对照。
图4为实施例2中细胞功能验证中所使用的队列示意图和流式细胞术分析使用的门控策略。其中12名患者在F1时无PASC。PBMC:外周血单个核细胞,FSC-A:前散射面积,SSC-A:侧散射面积,FSC-H:前散射高度,TNF-a:肿瘤坏死因子-α,IP-10:干扰素伽玛诱导的10千道尔顿蛋白,G-CSF:粒细胞集落刺激因子,F1:第一次随访(发病后约半年),F2:第二次随访(发病后约一年),PASC:新型冠状病毒感染后遗症。
图5为实施例2中CDKN1C+非经典单核细胞的促炎特性及其调控配体。(A)与其他细胞亚群比较,CDKN1C+非经典单核细胞中不同细胞因子/趋化因子编码基因的相对表达情况(双侧Wilcoxon秩和检验,P值通过Benjamini-Hochberg进行多重检验校正)。(B)发现队列中所有患者的PBMC中血浆细胞因子水平与CDKN1C+非经典单核细胞间的相关性(Pearson相关性)。(C-E)CDKN1C+和C1Q+非经典单核细胞亚群中G-CSF+(C)、TNF-α+(D)和IP-10+(E)细胞的比例(双侧Mann-Whitney U检验)。(F)NicheNet分析表明调控CDKN1C+非经典单核细胞(左)和C1Q+非经典单核细胞(右)之间差异基因表达的潜在配体。(G)分离的非经典单核细胞接种IL-1β后,使用流式细胞术检测CD16+单核细胞中CDKN1C+、G-CSF+、C1Q+表达单核细胞的比例(双侧Mann-Whitney U检验)。c34:c34_Mono_FCGR3A_CDKN1C(CDKN1C+非经典单核细胞),c35:c35_Mono_FCGR3A_C1QA(C1Q+非经典单核细胞),IL:白细胞介素,FGF basic:碱性成纤维细胞生长因子,G-CSF:粒细胞集落刺激因子,GM-CSF:粒细胞-巨噬细胞集落刺激因子,IFN:干扰素,IP:干扰素γ诱导蛋白,MCP:单核细胞趋化蛋白,MIP:巨噬细胞炎症蛋白,PDGF:血小板衍生生长因子,TNF:肿瘤坏死因子,VEGF:血管内皮生长因子,padj:Benjamini-Hochberg法校正后的P值。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不应理解为本发明的限制。如未特殊说明,下述实施例中所使用材料试剂等均可从商业途径获得,所使用的技术手段均为本领域技术人员熟知的常规操作方法或商业试剂盒使用说明书中所使用的。
实施例1:CDKN1C+的非经典单核细胞的诊断用途
1.材料和方法
1.1研究人口和样本采集
本实施例建立了两个COVID-19患者的长期随访队列,包括基于scRNA-seq的发现队列和基于流式细胞术的验证队列。两个队列的所有患者均招募于中国武汉市金银潭医院。纳入标准如下:(1)年龄>18岁;(2)实验室检测曾确证为SARS-CoV-2感染;(3)入院时出现发热、呼吸道感染症状和异常的胸片检查结果;(4)无SARS-CoV-2二次感染;(5)无SARS-CoV-2疫苗接种史;(6)居住地址为武汉市;(7)签署了知情同意书。本实施例中的队列排除了以下患者:(1)在随访前死亡;(2)因精神障碍、痴呆或因基础疾病再次入院而难以随访;(3)因患骨关节疾病或因中风或肺栓塞等疾病而无法自由活动;(4)拒绝参加此研究;(5)无法获得联系;(6)居住在武汉市以外或养老院、福利院。该研究经金银潭医院伦理审查委员会(KY-2020-02.01,KY-2020-80.01)批准。
发现队列共纳入47名COVID-19患者,其中10名患者分别在住院时(HO)、症状出现后半年(2020年7月,F1)和症状出现后一年(2020年12月至2021年1月,F2)接受了三次调查采样。另外10名COVID-19康复者在F1时入组,F2时再次被调查采样。剩余的27名患者仅进行了单个时间点的调查采样,包括采样于HO的11名患者、采样于F1的15名患者和采样于F2的1名患者。此外7名无COVID-19病史和SARS-CoV-2疫苗接种史的健康对照(HC)被招募采样以进行scRNA-seq分析,同时使用了公开发表的18份健康参与者外周血单个核细胞(PBMC)的scRNA-seq数据集以进行合并分析(参见Ren X,Wen W,Fan X,et al.COVID-19immunefeatures revealed by a large-scale single-cell transcriptomeatlas.Cell.2021Apr 1;184(7):1895-1913.e19.和Zhang J Y,Wang X M,Xing X,etal.Single-cell landscape of immunological responses in patients with COVID-19.Nat Immunol.2020Sep;21(9):1107-1118.)。
验证队列共纳入71名COVID-19康复者,并获得95份外周血样本。其中包括发现队列中的34名COVID-19康复者及其43份外周血样本(F1采集25份,F2采集18份),和新入组的37名COVID-19康复者及其52份外周血样本(F1采集29份,F2采集23份)。此外18名无COVID-19病史和SARS-CoV-2疫苗接种史的HC被招募采样。
在随访期间,所有康复者接受随访检查并完成一份设计好的调查问卷以收集有关自我报告症状(包括气促、咳嗽、疲劳、头痛、肌肉疼痛、胸痛、关节痛、心悸、头晕、胃肠道问题、嗅觉/味觉改变、睡眠障碍、运动后不适、焦虑或抑郁),同时使用乙二胺四乙酸(EDTA)抗凝管采集静脉血。以上自我报告症状和随访检查结果根据WHO通过德尔菲共识形成的对COVID-19长期影响的临床定义进行参与者的long COVID判定。处于仰卧位的受试者在吸气结束时进行胸部计算机断层扫描(SIEMENS SOMATOM PERSPECTIVE 64CT扫描仪),随后使用经过临床验证的人工智能算法计算五个肺叶中每个肺叶的解剖受累程度,其定义为肺炎病灶体积与每个肺叶体积的比率,随后计算半定量CT评分以评估肺受累等级:0,无受累;1,受累小于5%;2,5–25%的受累;3,26–50%的受累;4,51–75%的受累;5,受累超过75%(参见Liu F,Zhang Q,Huang C,et al.CT quantification of pneumonia lesions in earlydays predicts progression to severe illness in a cohort of COVID-19patients.Theranostics.2020Apr 27;10(12):5613-5622.doi:10.7150/thno.45985.)。
1.2单细胞测序文库构建与测序
所有采集的静脉血通过以300×g离心10分钟以分离血浆并冷冻保存于-80℃直至试验。外周血采集12小时内根据制造商的说明使用Ficoll-Paque PLUS(GE Healthcare,芝加哥,伊利诺伊州,美国)分离新鲜的PBMC用于scRNA-seq。
使用Chromium单细胞5'端文库和凝胶珠试剂盒V1.1(10×Genomics,旧金山,加利福尼亚州,美国)进行scRNA-seq文库构建。将细胞悬液于含有0.5%牛血清白蛋白(BSA)的DPBS中稀释至1,000个细胞/μl的浓度,并使用血细胞计数仪测量浓度。将6,000个单细胞乳液凝胶珠(GEM)所需体积的单细胞悬浮液置于到单细胞5'端芯片A(10×Genomics)上的单独泳道中。最终文库使用Agilent 2100Bioanalyzer(安捷伦科技公司,圣克拉拉,加利福尼亚州,美国)进行评估,并使用定量试剂盒(天根公司,北京,中国)和QuantStudio12K Flexreal-time PCR system(Thermo Fisher Scientific,沃尔瑟姆,马萨诸塞州,美国)通过qPCR进行定量。最终将每个样品的文库稀释至4nM并使用150-bp双端策略在NovaSeq(Illumina,Inc.,San Diego,CA,USA)上机测序。
1.3单细胞测序数据处理、聚类和细胞注释
下机Fatstq数据首先通过CellRanger(v3.1.0/5.0.1,10×Genomics)使用默认参数将测序reads和人类参考基因组(GRCh38)进行比对得到基因表达矩阵。随后使用Scanpy包(版本1.8.1)对测序数据进行整合、质控、标准化、选择高变基因、降维和聚类。我们过滤掉基因数量低于150个的低质量细胞,并对标准化后的数据选择高变基因进行主成分分析(PCA)降维,使用KNN算法(bbknn,版本1.3.1)进行批间校正。使用Leiden算法将细胞进行聚类,并使用过统一流形逼近与投影(UMAP)算法进行二次降维和可视化。
根据细胞亚群的差异基因表达情况,结合参考已发表PBMC的scRNA-seq数据集和文献对细胞亚群进行注释。细胞亚群间的差异基因分析中使用Scanpy包中的Wilcoxon秩和检验并对结果P值进行Benjamini–Hochberg矫正。细胞间通讯通过NicheNet(版本0.1.0,https://github.com/saeyslab/nichenetr)使用默认参数进行分析。
1.4流式细胞术分析
将冻存的PBMC解冻、洗涤并重悬于染色缓冲液(添加了0.5%BSA和2mM EDTA的DPBS)中。PBMC用Fc受体阻断溶液(Human TruStain FcX,BioLegend,圣地亚哥,加利福尼亚州,美国)和Horizon Fixable Viability Stain 510(BD Biosciences,圣何塞,加利福尼亚州,美国)进行染色。生物素化的人源化抗C1q抗体(Abcam,剑桥,英国)和人源化抗CDKN1C抗体(CUSABIO,休斯敦,德克萨斯州,美国)分别与FITC和PE链霉亲和素-荧光团偶联物(BIOSS Antibodies,波士顿,马萨诸塞州,美国)偶联。然后使用BioLegend的人源化抗PerC-Cy5.5-CD19、PerC-Cy5.5-CD3、PerC-Cy5.5-CD56、BV421-CD14、BV711-CD16抗体孵育30分钟进行细胞表面染色。在使用固定/透化染色缓冲液(Invitrogen,卡尔斯巴德,加利福尼亚州,美国)孵育后,细胞用人源化抗PE-CDKN1C和FITC-C1q抗体染色。附图1详细描述了流式细胞术分析所使用的门控策略。
所有样本均在BD LSRFortessa(BD Biosciences)流式细胞仪上进行分析,结果使用FlowJo软件(BD Biosciences)进行分析。单染CompBeads(BD Biosciences)或单染PBMC用于荧光补偿。
2.结果
2.1COVID-19康复患者的人口统计学和临床症状
47名COVID-19患者被纳入用于scRNA-seq分析的发现队列,其年龄中位数为54.0(四分位数间距(IQR):44.0–61.5)岁,48.9%为男性。其中囊括了36名COVID-19康复患者,年龄中位数为49.5(IQR:43.5–56.0)岁,44.4%为男性。根据WHO德尔菲共识对PASC的定义(参见World Health Organization.Aclinical case definition of post COVID-19condition by a Delphi consensus,6October 2021.2021.https://apps.who.int/iris/handle/10665/345824),F1时79.4%(27/34)的COVID-19康复患者中患有PASC,包括气促(7/32,21.9%)、咳嗽(3/33,9.1%)、疲劳或肌肉无力(12/34,35.3%)、头痛(2/33,6.1%)、肌痛(2/33,6.1%)、胸痛(1/33,3.0%)、关节痛(2/33,6.1%)、心悸(2/34,5.9%)、头晕(1/34,2.9%)、嗅觉/味觉改变(5/34,14.7%)、睡眠障碍(11/34,32.4%)、疼痛或不适(12/34,35.3%)以及焦虑或抑郁(6/35,17.1%)(表1)。此外我们共招募了71名COVID-19康复患者形成验证队列,通过流式细胞术分析以确证经发现队列获得的结果,其年龄中位数为54.0(IQR:44.0–63.0)岁,49.3%为男性。36名COVID-19康复期患者在F1时患有PASC(表1)。
表1本实施例107名COVID-19康复患者的人口学和临床症状
2.2COVID-19康复患者外周血中非经典单核细胞的异质性
通过对scRNA-seq数据的聚类和细胞注释,如图2A所示,COVID-19康复患者的非经典单核细胞包含两个不同的亚群,即c34_Mono_FCGR3A_CDKN1C(CDKN1C+非经典单核细胞)和c35_Mono_FCGR3A_C1QA(C1Q+非经典单核细胞)。其中c35细胞亚群主要以高表达补体相关基因为特征,包括C1QA、C1QB和C1QC(图2B和C)。与此相反,c34细胞亚群则表现为C1Q-,但高表达CDKN1C、CKB、ICAM4、GNG2、ICAM3、SYTL1、CYP4F22和CLCF1(图2B和C)。c34细胞亚群和c35细胞亚群之间的差异基因分析表明,和c35比较,c34还高表达PECAM1、IFITM2、SMIM25、MS4A7、FAM110A、TESC和VMO1(图2D)。
2.3CDKN1C+非经典单核细胞与多种新冠后遗症相关
已有多项针对COVID-19康复患者的临床调查表明,部分COVID-19患者在感染一年后肺部CT结果仍存在异常。我们的结果表明,外周血中CDKN1C+非经典单核细胞的比例和胸部CT评分呈正相关(图3A),其Pearson相关系数可达0.781(P=0.013),Spearman相关系数可达0.831(P=5.56×10-3)。此外CDKN1C+非经典单核细胞与COVID-19康复患者在随访过程中出现的关节痛有关(图3B),关节痛也是long COVID临床研究中经常报告的一种症状。
我们通过流式细胞术分析在验证队列中进一步验证了CDKN1C+非经典单核细胞和后遗症之间的关联。与从发现队列中获得的scRNA-seq数据结果一致,验证队列中CDKN1C+非经典单核细胞数与胸部CT评分呈正相关(图3D),在随访期间患关节痛的患者中检测到CDKN1C+非经典单核细胞比例更高(图3D)。更重要的是,与没有患PASC的康复者和HC比较,CDKN1C+非经典单核细胞在患有PASC的康复患者中比例更高(图3E)。
实施例2:CDKN1C+的非经典单核细胞的治疗用途
1.材料和方法
1.1研究人口和样本采集
本实施例建立了两个COVID-19患者的长期随访队列,包括基于scRNA-seq的发现队列和基于细胞功能实验的验证队列。两个队列的所有患者均招募于中国武汉市金银潭医院。参与者的纳入排除标准、伦理批文均如实施例1中所述。
其中的发现队列为实施例1中的发现队列。为了通过流式细胞术对CDKN1C+非经典单核细胞的功能进行验证,我们纳入了24名COVID-19康复期患者组成验证队列,在F1和F2时均进行了随访调查,其中12名患者在两次随访均无异常症状,共采集了48份PBMC样本。随访期间所采集的临床调查信息和CT评分如实施例1中所述。
1.2单细胞测序文库构建、测序和下游分析
如实施例1中所述。
1.3细胞内细胞因子染色和流式细胞术分析
将冻存的PBMC解冻、洗涤并与布雷菲德菌素A(GolgiPlug,Biolegend,圣地亚哥,加利福尼亚州,美国)和莫能菌素(GolgiStop,Biolegend)一起孵育4小时,然后重悬于染色缓冲液(添加了0.5%BSA和2mM EDTA的DPBS)中。PBMC用Fc受体阻断溶液(Human TruStainFcX)和Horizon Fixable Viability Stain 510(BD Biosciences)进行染色。人源化抗C1QB抗体(CUSABIO)和人源化抗CDKN1C抗体(CUSABIO)分别与购自BIOSS Antibodies的Alexa Fluor 488和APC偶联。生物素化的人源化抗IP-10抗体(纯化的抗CXCL10抗体,BioLegend)和生物素化的人源化抗G-CSF抗体(Abcam)分别与BV605和BV785链霉亲和素-荧光团偶联物(BioLegend)偶联。然后使用购自BioLegend的人源化抗PerC-Cy5.5-CD19、PerC-Cy5.5-CD3、PerC-Cy5.5-CD56、PE/DazzleTM 594-CD14、BV711-CD16抗体孵育30分钟进行细胞表面染色。在使用固定/透化染色缓冲液(Invitrogen)孵育后,细胞用人源化抗PE-TNF-α(BioLegend)、BV605-IP10(BioLegend)、BV785-G-CSF(Abcam)、FITC-C1QA/C(CUSABIO)、Alexa Fluor 488-C1QB(CUSABIO)和APC-CDKN1C(CUSABIO)抗体染色。附图4详细描述了流式细胞术分析所使用的门控策略。
所有样本均在BD LSRFortessa(BD Biosciences)流式细胞仪上进行分析,结果使用FlowJo软件(BD Biosciences)进行分析。单染CompBeads(BD Biosciences)或单染PBMC用于荧光补偿。
1.4人非经典单核细胞的纯化、培养以及流式细胞术分析
首先使用无CD16去除的EsaySepTM人单核细胞富集试剂盒(STEMCELLTechnologies,温哥华,加拿大)从PBMC中分离人单核细胞,然后使用人源化抗PE-CD16抗体(BioLegend)和抗PE微珠(Miltenyi Biotec,贝尔吉施-格拉德巴赫,德国)进行CD16+单核细胞的富集。在含有RPMI 1640(CORNING,马纳萨斯,美国)和10%热灭活胎牛血清(VivaCell biosciences,上海,中国)和1%青霉素/链霉素(Cenomcell Bio,浙江,中国)的培养基中洗涤细胞并稀释至106个细胞/mL并培养0.5小时。随后将从ACRO Biosystems(北京,中国)购得的IL-1β以150ng/mL的浓度添加至培养物中,共培养24小时后的细胞用于流式细胞术测定。在收集细胞前6小时添加布雷菲德菌素A(GolgiPlug,Biolegend,圣地亚哥,加利福尼亚州,美国)和莫能菌素(GolgiStop,Biolegend)至培养物。
对于纯化和IL-1β处理培养后的非经典单核细胞,将细胞重悬于染色缓冲液(添加了0.5% BSA和2mM EDTA的DPBS)中。PBMC用Fc受体阻断溶液(Human TruStain FcX)和Horizon Fixable Viability Stain 510(BD Biosciences)进行染色。然后使用购自BioLegend的人源化抗PerC-Cy5.5-CD19、PerC-Cy5.5-CD3、PerC-Cy5.5-CD56、BV421-CD14和PE-CD16抗体孵育30分钟。在使用固定/透化染色缓冲液(Invitrogen)孵育后,细胞用人源化抗BV785-G-CSF(Abcam)、FITC-C1QA/C(CUSABIO)、Alexa Fluor 488-C1QB(CUSABIO)和APC-CDKN1C(CUSABIO)抗体染色。所有样本均在BD LSRFortessa(BD Biosciences)流式细胞仪上进行分析,结果使用FlowJo软件(BD Biosciences)进行分析。
1.5细胞因子和趋化因子测量
血浆细胞因子和趋化因子的水平,包括白细胞介素(IL)-1ra、IL-1β、IL-2、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、IL-13、IL-15、IL-17、eotaxin、干扰素(IFN)-γ诱导蛋白(IP)-10、单核细胞趋化蛋白(MCP)-1、巨噬细胞炎症蛋白(MIP)-1α、MIP-1β、RANTES、成纤维细胞生长因子、血小板衍生生长因子-BB、血管内皮生长因子、粒细胞集落刺激因子(G-CSF)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、IFN-γ和肿瘤坏死因子(TNF)-α,使用Human Cytokine Standard 27-Plex Assay panel(Bio-Rad,赫拉克勒斯,加利福尼亚州,美国)和Bio-Plex 200系统(Bio-Rad)按照制造商的说明进行测量。
2.结果
2.1COVID-19康复患者的人口统计学和临床症状
发现队列的人口统计学和临床症状如实施例1中所述。此外我们共招募了24名COVID-19康复患者形成功能验证队列,通过细胞内细胞因子和染色流式细胞术分析以确证经发现队列获得的结果,其年龄中位数为62.0(IQR:55.25-65.5)岁,45.8%为男性。12名COVID-19康复期患者在F1时患有PASC(表2)。
表2功能验证队列中24名COVID-19康复患者的人口学和临床症状
2.2CDKN1C+非经典单核细胞介导促炎程序
我们发现与PBMC中的其他免疫细胞亚群比较,IL1RN+、IL15+、CSF3+、CXCL10+、CCL2+和TNF+细胞在CDKN1C+非经典单核细胞(c34)中显著富集(图5A)。同时血浆中由CSF3编码的G-CSF、由TNF编码的TNF-α和由CXCL10编码的IP-10等细胞因子的浓度均与CDKN1C+非经典单核细胞的比例丰度呈正相关(图5B)。尽管c34表达CCL3的水平低于c35,但外周血中c34的比例丰度与血浆中CCL3编码的MIP-1a水平呈正相关(图5B)。这些结果表明,c34细胞可能通过产生高水平的细胞因子进而介导促炎程序,这可能与long COVID有关。
CDKN1C+非经典单核细胞的促炎功能在功能验证队列中得到进一步证实。通过流式细胞术对细胞内G-CSF、IP-10和TNF-α的表达进行检测,我们发现与C1Q+非经典单核细胞比较,CDKN1C+非经典单核细胞中G-CSF、IP-10和TNF-α的表达更高(图5C-E),这与scRNA-seq数据结果一致。
2.3CDKN1C+非经典单核细胞的特征性转录受IL-1β调控
我们通过NicheNet分析进一步探究了C1Q+非经典单核细胞(c35)和CDKN1C+非经典单核细胞之间差异性转录的调控因子。NicheNet是一种生物信息学分析工具,根据scRNA-seq数据和精选的配体靶标数据库可确定潜在调节配体的优先级顺序(参见Browaeys R,Saelens W,Saeys Y.NicheNet:modeling intercellular communication by linkingligands to target genes.Nat Methods.2020Feb;17(2):159-162.)。结果表明,C1Q-CDKN1C+非经典单核细胞更易受IL1B、VEGFA、TGFB1、FASLG和CCL4等调控,而C1Q+非经典单核细胞则更易受ICAM1、NECTIN1、SELP、CLCF1等调控。我们进一步通过分离出健康志愿者外周血中的非经典单核细胞并进行体外IL-1β刺激,发现CDKN1C和G-CSF的表达均上调,但C1Q无上调(图4G),证实了NicheNet的分析结果。
Claims (11)
1.确定外周血样品中CDKN1C+的非经典单核细胞亚群的检测试剂在制备诊断产品中的用途,其特征在于,所述产品用于诊断long COVID,与预定参考值相比,样品中升高的CDKN1C+非经典单核细胞的比例显示受试者患有long COVID;
其中,预定参考值以健康人群体中CDKN1C+非经典单核细胞的比例的中位数为基础。
2.根据权利要求1所述的用途,其特征在于,所述检测试剂包含流式细胞术和质谱流式细胞技术所使用的抗体、单细胞RNA检测的探针和扩增测序所使用的引物。
3.根据权利要求1所述的用途,其特征在于,CDKN1C+非经典单核细胞为蛋白质水平上分化簇3阴性、分化簇19阴性、分化簇56阴性、分化簇16阳性、补体成分1q阴性且CDKN1C+表达的单核细胞;转录水平上Fcγ受体IIIa阳性、C1Q-、CDKN1C+且高表达肌酸激酶B、细胞间粘附分子4、G蛋白亚基Gamma 2、细胞间粘附分子3、突触结合蛋白样蛋白1、细胞色素P450家族4亚家族F成员22、心肌营养素样细胞因子因子1、干扰素诱导的跨膜蛋白2、动脉粥样硬化和炎症性肠巨噬细胞调节中富含斑块的LncRNA、跨膜4结构域亚家族成员7、钙素、卵黄膜外层1同系物和血小板内皮细胞粘附分子1的单核细胞。
4.根据权利要求1所述的用途,其特征在于,long COVID符合世界卫生组织通过德尔菲共识形成的对COVID-19长期影响的临床定义,其症状包括疲劳、气促、认知障碍、嗅觉/味觉改变、抑郁、焦虑、胸痛、咳嗽、头晕、胃肠道问题、头痛、关节痛、肌肉疼痛/痉挛、运动后不适、睡眠障碍和心动过速/心悸。
5.根据权利要求1所述的用途,其特征在于,所述产品用于诊断long COVID患者的恢复情况,或评价long COVID治疗方案的有效性。
6.根据权利要求5所述的用途,其特征在于,所述治疗方案包括向受试者施用至少一种治疗药物。
7.根据权利要求6所述的用途,其特征在于,其中预定参考值以患有long COVID的受试者外周血样品中CDKN1C+的非经典单核细胞比例为基础,样品中降低的CDKN1C+的非经典单核细胞比例显示恢复良好或治疗方案有效。
8.根据权利要求1所述的用途,其特征在于,通过包括通过scRNA-seq、流式细胞术和其他可获得特定细胞类型比例的方法在样品及参照中确定CDKN1C+的非经典单核细胞的比例。
9.权利要求1中所述CDKN1C+的非经典单核细胞在制备治疗long COVID的药物中的用途。
10.根据权利要求9所述的用途,其特征在于,所述药物包含对调控CDKN1C+非经典单核细胞分化的配体IL-1β的阻断剂。
11.根据权利要求10所述的用途,其特征在于,所述阻断剂为抗IL-1β抗体、利洛西普、IL-1受体拮抗剂及相关制品阿那白滞素、IL-1的可溶性受体和IL-1β的人单克隆抗体卡那单抗和Xoma 052。
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