该研究在西奈山卫生系统（MSHS）（IRB-18-00638/ studing-18-00627和IRB-20-03276），波士顿儿童医院（04-09-113R），国家过敏和感染疾病（NIAID，NIAID，NIHIH）（NIAID，NIH）（05-I-I-I-02213）（JACA和ROCEFELERERERERER）（JACA FILLERARERERER）（JACA FILLERARERENS（JACA）（JACA FILLERIAD）（JACA FILLESING），批准了这项研究。XFK-0815），法国伦理委员会COMITéDeProtection Des vensnes，法国国家医学和健康产品安全机构以及国家De laSanté等人研究所（CONTOALITS C10-13和C10-14）。亲自或通过NIH的DS-Connect国家注册表（https://dsconnect.nih.gov）亲自与患者联系。所有患者数据均已识别。本研究中所有个人的书面知情同意书是根据机构审查委员会协议提供的。在门诊常规访问的背景下，从未感染的患者中抽取了所有样本，并且患者没有感染的迹象（发烧，流鼻涕，咳嗽，喉咙痛）。在扩展数据表1和2中概述了患有DS的个体的人口统计学和特征。对于图3中的六名SLE和扩展数据，图4，四个患有严重疾病，患有肾脏介入，其中一个患有中度疾病而无需肾脏介入，其中一名患有轻度疾病，肾脏涉及轻度疾病，并在采样时被采样时解决了肾脏介入。一半的患者患有活性疾病，其他三名捐助者在治疗后正在缓解。从每个供体中，将血液抽入用肝素钠（BD真空吸蛋白）中的细胞制备管中。从Ficoll分离中分离血浆，并在-80°C下储存直至使用。全血是使用蛋白质组学稳定剂Prot1（SmartTube）直接固定的，并在-80°C下冷冻，或者通过将270 µL血液直接添加到含有冻干抗体面板的MDIPA管中，将新鲜的全血样品染色，混合并在室温下孵化30分钟 然后用Prot1稳定并冷冻。 　　患者的法定监护人或转诊医师填写了临床问卷。为每种自身免疫性表现或感染性发作施用一个点。如果将发作（例如肺炎）表征为经常性或报告三倍以上的情况，我们将此类别的分数限制在三点。例如，如果患者患有甲状腺功能减退症和12种耳朵感染，则该患者的总免疫临床评分为4分。 　　先前描述了质量细胞仪的详细方案51。根据制造商的推荐方案将冷冻的稳定血液样品解冻，然后用条形码通透性缓冲液（Fluidigm）洗涤。使用细胞-ID 20-plex PD条形码试剂盒（流体配置）将样品唯一的条形码化，并合并在一起。对于以前未染色的样品，将细胞与抗体鸡尾酒孵育，以鉴定主要的免疫群体，其次是甲醇通透性，肝素阻滞和染色，并用针对细胞内靶标的抗体鸡尾酒，包括磷酸化和信号传导的标记。以下所有抗体均购自Fluidigm：CD111-Granzyme B，CD112-IGA，In113-CD57，In115-CD11C，CD116-IGD，I127-127I，CE140-140CE，PR141-KI67，PR141-KI67，ND142-CD19，ND143434343434343434,4,43434,4,4343434,4,4 dra，Nd145-CD4, Nd146-CD8, Sm147-pSTAT5, 150Nd-pSTAT5, Nd148-CD16, Sm149-CD127, Sm149-pSTAT6, Nd150-CD1c, Eu151-CD123, Sm152-CD66b, Eu153-pSTAT1, Sm154-ICOS,GD155-CD27，GD156-P38，158GD-PSTAT3，TB159-PMAPKAP2，GD160-CD14，DY161-CD56，DY162-TCRGD，DY162-CD169Er166-CD25, Er167-pERK1/2, Er168-CD3, Tm169-CD71, Tm169-STAT1, Er170-CD38, Yb171-CD95, Yb171-CD141, Yb172-CD39, Yb173-Tbet, Yb174-HLADR, Lu175-pS6,Yb176-CD54, Pr141-IFNγ, Nd144-CD141, 171Yb-CD141, Sm147-IL-1B, Sm149-IL-1RA, Eu153-TNF, Gd156-IL-6, Gd158-IL-2, Tb159-GM-CSF, Dy164-IL-17A, Ho165-CCL4,ER166-IL-10，TM169-IFNα2B，YB173-IL-8，LU175-IL-29和YB176-CXCL10。洗涤后，将细胞在新鲜稀释的2.4％甲醛中孵育，其中含有125 nm ir Intercarator（流体化），0.02％皂苷和30 nm OSO4（Acros Organics）在室温下30分钟。然后将样品立即洗涤并采集。 　　为了获取，将样品用PBS+0.2％BSA，PBS和CAS缓冲液（流体）洗涤。CAS缓冲液中的最终溶液由每毫升100万个细胞和1/20 EQ珠（Fluidigm）组成。在常规仪器优化后，以以下速度采集样品 <300 events per second on a Helios mass cytometer (Fluidigm) with a modified wide-bore injector (Fluidigm). 　　FCS files of acquired events were normalized and concatenated with Fluidigm acquisition software, deconvoluted using a MATLAB-based debarcoding application and the resulting files were analysed using Cytobank. Cell events were identified as Ir191/193-positive and Ce140-negative events. Doublets were excluded on the basis of Mahalanobis distance and barcode separation and with the Gaussian parameters calculated using the Helios CyTOF software. Downstream data analysis was performed on Cytobank, by biaxial gating of immune populations according to a previously published gating scheme51 outlined in Supplementary Fig. 1. All cell frequency analyses were restricted to adult HCs and adults with DS (aged at least 18 years) unless otherwise indicated. 　　Cryopreserved PBMCs were thawed and allowed to rest briefly in complete RPMI medium supplemented with 10% FBS, 1% penicillin–streptomycin and 1% GlutaMax. Cells were immunostained with antibodies in 0.5% BSA in PBS for 1 h, washed 3 times in 0.5% BSA in PBS for 1 h and acquired immediately. The following antibodies were used: CD19 APC-Cy7 (SJ25C1), CD27 FITC (M-T271), CD38 APC (HIT2), CD38 PE-Cy7 (HIT2), CD11c PE (B LY6), IgD BV421 (IA6), CD21 APC (Bu32) and anti-human 9G4 IgG APC (provided by J. Farmer). 　　The IgG fraction was isolated from HC and DS plasma using the Protein G HP SpinTrap (Sigma-Aldrich). THP-1 cells were incubated with the IgG fraction diluted 1:3 in complete RPMI with 10% FBS for 20 min or recombinant anti-IFNGR2 antibodies (2 μg ml−1, Thermo Fisher Scientific, PA5-47938) at 37 °C for 20 min followed by stimulation with IFNγ (10 ng ml−1) for 20 min at 37 °C. Cells were stained with Zombie Aqua Live-Dead (BioLegend) for 30 min on ice, then fixed/permeabilized in 90% ice-cold methanol and stained with anti-phospho-STAT1-PE (1:25, BD) for 1 h on ice. Flow cytometry was acquired on the BD LSR Fortessa II system, and data were analysed with FlowJo. 　　Whole blood was incubated for 4 h with tofacitinib (500 nM, ApexBio) or tocilizumab (50 μg ml−1, Selleckchem) or vehicle controls (DMSO and human IgG control, 50 μg ml−1, BioLegend, respectively). For IFN-I blocking, whole blood was incubated with a combination of anti-IFNAR2 (2.5 μg ml−1 PBL Assay Science), anti-IFNα (0.2 μg ml−1, PBL 31110–1) and IFNβ (0.2 μg ml−1, PBL 31401-1) antibodies or vehicle control for 4 h. For IL-10 blocking, whole blood was incubated with a combination of anti-IL-10 (5 μg ml−1, BioLegend) and anti-IL-10R (5 μg ml−1, BioLegend) for 4 h. After blocking, whole blood was fixed using Proteomic Stabilizer PROT1 (1.4× blood volume, SmartTube) for 10 min at room temperature and frozen at −80 °C. 　　For plasmablast differentiation of CD11c+ cells (Fig. 4a), 2–5 × 104 naive (CD19+CD38−CD27−CD11c−) and CD11c+ (CD19+CD11c+) B cells were sort-purified from the PBMCs of healthy donors using the IMI5L sorter (BD) and rested overnight in complete RPMI. They were then stimulated with IL-21 (50 ng ml−1; BioLegend), BAFF (100 ng ml−1, BioLegend), IL-10 (250 ng ml−1, BioLegend), IL-2 (5 ng ml−1) and R848 (1 μg ml−1; Invivogen) with or without IFNγ diluted in complete RPMI. Cells were collected at day 4 and stained for flow cytometry. 　　For the cytokine-induced plasmablast differentiation of B cells (Fig. 4d,e and Extended Data Fig. 5a–f), 2–5 × 104 sorted naive or total B cells were sorted from healthy donor PBMCs using fluorescence-activated cell sorting, the Pan B Cell Isolation Kit II or the Naive B Cell Isolation Kit II (Miltenyi Biotech), as indicated. They were cultured for 3–6 days in complete RPMI supplemented with IL-21 (50 ng ml−1; BioLegend), BAFF (10 ng ml−1, BioLegend), IL-2 (50 ng ml−1, BioLegend), R848 (1 μg ml−1; Invivogen) with or without IL-10 (250 ng ml−1, BioLegend) and/or anti-human IgM (5 μg ml−1, Southern Biotech) as indicated, in addition to either (1) plasma from HC individuals or individuals with DS that was depleted from IgG using the Protein G HP SpinTrap (Sigma-Aldrich) at a final dilution of 1:4 or (2) exogenous addition of IL-6 (100 ng ml−1, BioLegend), IFNα2b (100 U ml−1, Merck), IL-4 (20 ng ml−1, BioLegend) and IFNγ (20 ng ml−1, BioLegend). At least two healthy donors and four individuals with DS were tested for each experiment. Cells were collected at indicated days and stained for flow cytometry. 　　For cytokine-blocking experiments, sorted naive B cells were cultured as described above in the presence of the following blocking antibodies: anti-IFNGR2 (2 μg ml−1, Thermo Fisher Scientific, PA5-47938), anti-IFNAR2 (2.5 μg ml−1 PBL Assay Science), anti-IFNα (0.2 μg ml−1, PBL 31110–1) and IFNβ (0.2 μg ml−1, PBL 31401-1), tocilizumab (50 μg ml−1, Selleckchem), adalimumab (2 μg ml−1, Selleckchem) or a combination. Cells were collected at indicated days and stained for flow cytometry. 　　For co-cultures of in vitro polarized T cells (Fig. 4f–h and Extended Data Fig. 5g–i). CD4 T cells were purified using MACS (Miltenyi Biotec) from healthy donor PBMCs and cultured in the presence of plate-bound anti-CD3 (OKT3, 10 μg ml−1) and anti-CD28 (CD28.2, 5 μg ml−1) antibodies in complete RPMI alone (unstimulated control) or with (1) IL-2 (50 ng ml−1), IL-12 (5 ng ml−1) and anti-IL-4 (1 μg ml−1, R&D MAB204-SP) (TH1 conditions); (2) IL-6 (50 ng ml−1) with or without IL-2 (50 ng ml−1); or (3) plasma from HC individuals or individuals with DS (1:2 ratio). After 4 days, T cells were washed and irradiated (5,000 rad) and co-cultured with freshly MACS-sorted naive B cells from the same donor. Cells were co-cultured for 3–6 days in complete RPMI supplemented with IL-21 (50 ng ml−1), IL-2 (50 ng ml−1) and anti-human IgM (5 μg ml−1, Southern Biotech) and collected for flow cytometry. The supernatants were collected for IFNγ ELISA quantification (BioLegend) according to the manufacturer’s instructions. 　　For T cell–B cell co-cultures from HC donors and donors with DS (Fig. 4i,j), CD4 T cells were MACS-purified and co-incubated with sort-purified CD19+CD38−CD27−CD11c− B cells on the same day. B and T cells were co-cultured for 3 days in complete RPMI supplemented with IL-21, IL-2 and anti-human IgM as above and collected for flow cytometry. 　　Donor PBMCs were thawed in complete RPMI and rested overnight. Naive (CD19+CD27−CD38lowCD11c−), CD11c+ (CD19+CD27−CD38lowCD11c−) and memory (CD19+CD27+CD38−) B cells were sort-purified using an IMI5L sorter (BD) and genomic DNA was isolated with QIAamp DNA Blood Mini Kit (Qiagen). 　　Sample data were generated using the immunoSEQ Assay (Adaptive Biotechnologies). The somatically rearranged Homo sapiens BCR IgH locus CDR3 was amplified from genomic DNA using a two-step, amplification-bias-controlled multiplex PCR approach52,53. The first PCR consists of forward and reverse amplification primers specific for every V and J gene segment and amplifies the hypervariable complementarity-determining region 3 (CDR3) of the immune receptor locus. Furthermore, primers are included to amplify B cell precursor DJ rearrangements. The second PCR adds a proprietary barcode sequence and Illumina adapter sequences54. CDR3 libraries were sequenced on the Illumina instrument according to the manufacturer’s instructions. 　　Raw sequencing reads were demultiplexed according to Adaptive’s proprietary barcode sequences. Demultiplexed reads were then processed to remove adapter and primer sequences, identify and remove primer dimer, germline and other contaminant sequences. The filtered data are clustered using the relative frequency ratio between similar clones and a modified nearest-neighbour algorithm to merge closely related sequences to correct technical errors introduced through PCR and sequencing while allowing for somatic hypermutation in the V segment. The resulting sequences were sufficient to allow annotation of the V, D and J genes, and the N1 and N2 regions constituting each unique CDR3 and the translation of the encoded CDR3 amino acid sequence. Gene definitions were based on annotation in accordance with the IMGT database (www.imgt.org). The set of observed biological BCR IgH CDR3 sequences was normalized to correct for residual multiplex PCR amplification bias and quantified against a set of synthetic BCR IgH CDR3 sequence analogues53. Data were analysed using the immunoSEQ Analyzer toolset. 　　Plasma collected by Ficoll isolation from heparinized whole blood was clarified by centrifugation. Magnetic Luminex assays with the MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel (MilliporeSigma, HCYTMAG-60 K-PX30) were run according to the manufacturer’s protocol. The samples were quantified on the MAGPIX xMAP Instrument (Luminex). For each sample, >每个分析物收集50个珠子。记录了这些珠的中值荧光强度，并使用5P回归算法使用Milliplex分析算软件进行分析。在没有信号的情况下，使用了每种细胞因子的检测下限。DS年龄为7个月至37岁（n = 21）和6岁至39岁（n = 10）的患者包括患者。 　　如先前所述55进行了自身抗体的血清组分析。CDI Huprot肽阵列根据制造商的说明运行，以1/500的速度避免使用低引发的交叉反应性，并使用强大的阻止缓冲液防止非特异性结合。应用患者血清后，洗涤芯片，并在制造商推荐的浓度下施用了量化IgG（CY3; 532 nm）和IgA（CY5; 635 nm）抗体同种型的二级荧光抗体。阵列上的每个斑点都使用Genepix软件对齐方式门控，然后手动控制质量，以确保适当的量化和去除可能的伪影。标记了复制点（CV> 0.5）之间的高差异，以及有关染色伪影的信息（稀有和排除在外）。然后将原始样品肽基质读取到R（V.4.0.4）统计环境中，Limma微阵列分析套件（v.3.46.0）56。在按样本和每个类型（IgG或IgA）的基础上，使用背景校正函数（方法：NormExp，NormExp.Method：RMA）减去背景强度，然后使用所有样本使用AnmorranizeBetweenween Arneween Arrays函数（方法=“ Cyclicloess”）进行归一化。接下来，将所有对照和未检测的肽从样品 - 肽基质中过滤，并以每样本样本为基础将所有其余肽在所有其余肽中平均。将单独处理的样品矩阵在柱方面加和生成一个实验矩阵，该矩阵包含所有样品中所有样品和肽在所有阵列中共有的，并使用Limma差异表达函数进行分析以获得差异肽反应性。首先，为排除低反应性肽和异常反应性，对肽进行过滤，以包括至少5个样品中中位信号强度高于中位信号强度的肽（在处理的50个总样品中，有50个）。然后使用基本R（v.4.0.4）PRCOMP函数（比例：true）进行PCA，并使用FACTOMiner/FactoExtra软件包（v.1.0.7）57使用FVIZ_PCA_IND函数可视化PCA和PCA和加载。为了确定差异丰富的自身抗体，我们首先使用Limma的LMFIT函数在所有阵列上拟合每个肽的线性模型，以模拟肽反应性的变化（反对自动抗体）。构建模型后，我们产生了一个对比矩阵，以测试针对健康对照组的疾病组。在开发人员描述的经验贝叶斯框架下进行了差异自身抗体丰度测试，并在经验贝叶斯框架下进行了调节，并针对绝对log2 [fold Change]> 1和p滤除了结果肽列表。< 0.05. Overlap analyses were conducted and visualized using the UpSetR (v.1.4.0)58 and ggvennDiagram (https://github.com/gaospecial/ggVennDiagram) (v.1.2.1) packages. Heat maps were generated using the ComplexHeatmap (v.2.7.4)59 library. 　　To assess whether autoantibody reactivity was influenced by the gene dosage effect in DS, filtered differentially abundant autoantibodies (obtained from the DS versus HC contrast; see Supplementary Data 1) were plotted across the 23 human chromosomes (hg38) in a location-dependent manner. In brief, gene names were extracted from their associated peptides and chromosomal locations were identified using the org.Hs.eg.db (v.3.12.0)60 database. Hits were further filtered against a fold-change of 1.5 for plotting purposes and the karyoplotR (v.1.16.0)61 package was then used for visualization. 　　Tissue-protein detection values of all human proteins were extracted from the Human Protein Atlas API using the HPAanalyze (v.1.8.1)62 package. Differentially abundant autoantibodies (obtained from the DS versus HC contrast; Supplementary Data 1) were subset from the full dataset (Supplementary Data 2) and visualized using the ComplexHeatmap library. 　　GO enrichment analysis was conducted on over-abundant (log2[fold change] >1）具有Limma的Goana功能的自身抗体，生物学过程的术语为p <0.0001，并使用GGPLOT263（v.3.3.3）软件包进行了可视化。 　　一夜之间，将96孔的肋板在4°C下涂层，每孔100μl的重组IFNGR2，MSTN或ATP6V1G2蛋白（Origene）悬浮在1×PBS中的1μgml-1溶液。第二天早晨，除去涂料溶液，并用100μl的洗涤缓冲液（PBS，为0.05％（v/v）Tween 20; PBS-T）洗涤3次。接下来，在室温下将200μl的阻塞缓冲液（含牛等离子体白蛋白（无内毒素））添加到每个井中，并在室温下孵育1小时，将其添加到每个孔中。在阻塞缓冲液中将血浆样品稀释为1：100。 　　一夜之间，将96孔的肋板用4°C涂在山羊抗人IgG的2μgml-1溶液中100μl，F（AB'）2-Fragment特异性（Jackson Immunoresearch）或1：100抗性9GMAN 9GG4 IGG抗体（由J.抗体提供的稀释）（1：100稀释）。第二天早晨，除去涂料溶液，并用100μl的洗涤缓冲液（PBS（v/v）Tween-20； PBS-T）用100μl洗涤缓冲液洗涤孔。接下来，在室温下将200μl的阻塞缓冲液（含牛等离子体白蛋白（无内毒素））添加到每个井中，并在室温下孵育1小时，将其添加到每个孔中。将B细胞刺激实验的上清液稀释1:10。 　　然后将板洗涤3次，然后再添加山羊抗人IgG F（AB） - 荷里德什过氧化物酶（HRP）偶联的二抗（稀释1：3,000在PBS-T中）。将板在37°C下孵育1小时，并洗涤3次。板再次用洗涤缓冲液洗涤3次。一旦完全干燥，将100μLSigmafastOPD（O-苯基二氨基二氨基二氯化物； Sigma-Aldrich）添加到每个孔中。将此基材放在板上5分钟，然后通过添加50μl的每孔为3 m盐酸来停止反应。使用Polarstar Omega（BMG LabTech）读取器测量490 nm处的光密度。 　　如先前所述，使用陀螺仪进行抗型I IFN自身抗体。64。细胞因子，重组人IFNα2（Miltenyi Biotec，130-108-984）或重组人IFNΩ（Merck，SRP3061）首先用EZ-Link-link Sulfo-NHS-NHS-LC-Biotin（Thermo Fisher Scientific，a39257）使用EZ-link-link Sulfo-NHS-LC-Biotin（A39257）进行生物素化。1:12。检测试剂含有二抗（Alexa Fluor 647山羊抗人IgG（Thermo Fisher Scientific，A21445））在REXXIP F中稀释（Gyros蛋白技术，P0004825; 1：500稀释2 mg Ml-1 pock of Final-1 final-1 final-1 final lince final coptation final-4最终浓度均为4°1μml-1。根据制造商的说明，制备了磷酸盐缓冲盐水，0.01％Tween-20（PBS-T）和Gyros Wash缓冲液（Gyros蛋白技术，P0020087）。然后将血浆或血清样品在0.01％PBS-T中稀释1：100，并用BioAffy 1000 CD（Gyros蛋白技术，P0004253）和Gyrolab Xpand（Gyros蛋白技术，P0020520）进行测试。清洁周期在20％乙醇中进行。 　　如先前所述，抗IFNα2和抗IFNΩ自身抗体的阻塞活性是用报告基因荧光素酶活性确定的64。用含有萤火虫荧光素酶基因的质粒转染HEK293T细胞在PGL4.45骨架中的人类ISRE启动子的控制下转染，质粒组成构型表达肾荧光素酶进行归一化（PRL-SV40）。在存在X-Tremegene9转染试剂（Sigma-Aldrich，6365779001）的情况下转染细胞24小时。Dulbecco修饰的Eagle培养基（DMEM； Thermo Fisher Scientific）中补充了2％的胎牛血清和10％健康对照或患者血清/血浆（在56°C下失活20分钟后）被IFNα2刺激（Miltenyi Biotec，130-108-984）和默克（Merc），默克（Mert）（Merck）和ifncy（默克）（Merc），SR，100 pg ml -1在37°C下16小时。每种细胞因子和剂量测试了每个样品一次。最后，将细胞在室温下裂解20分钟，并根据制造商的协议，用双酸酶报道器1000分析系统（Promega，E1980）测量荧光素酶水平。使用Victor-X多标签读取器（Perkinelmer Life Sciences）测量发光强度。将萤火虫荧光素酶活性值针对Renilla荧光素酶活性值归一化。 　　有关研究设计的更多信息可在与本文有关的自然投资组合报告摘要中获得。