Lin-CD117+CD34+FceRI+ progenitor cells are increased in chronic spontaneous urticaria and predict clinical responsiveness to anti-IgE therapy. Lin-CD117+CD34+FceRI+ progenitor cells are increased in chronic spontaneous urticaria and predict clinical responsiveness to anti-IgE therapy

Background: Chronic spontaneous urticaria (CSU) is a common, debilitating skin disorder characterised by recurring episodes of raised, itchy and sometimes painful wheals lasting longer than 6 weeks. CSU is mediated by mast cells which are absent from peripheral blood. However, lineage-CD34hiCD117int/hiFcεRI+ cells in blood have previously been shown to represent a mast cell precursor. Methods: We enumerated FcεRI-, FcεRI+ and FcεRIhi lineage-CD34+CD117+ cells using flow cytometry in blood of patients with CSU (n=55), including 12 patients receiving omalizumab and 43 not receiving omalizumab (n=43). Twenty-two control samples were studied. Disease control and patient response to omalizumab was evaluated using the Urticaria Control Test. We performed single cell RNA sequencing (scRNA-seq) on lineage-CD34hiCD117hi blood cells from a subset of patients with CSU (n=8) and healthy controls (n=4). Results: CSU patients had more Lineage-CD34+CD117+FcεRI+ blood cells than controls. Lineage-CD34+CD117+FcεRI+ cells were significantly higher in patients with CSU who had an objective clinical response to omalizumab when compared to patients who had poor disease control 90 days after initiation of omalizumab. scRNA-seq revealed that lineage-CD34+CD117+FcεRI+ cells contained both lymphoid and myeloid progenitor lineages, with omalizumab responsive patients having proportionally more myeloid progenitors. The myeloid progenitor lineage contained small numbers of true mast cell precursors along with more immature FcεRI- and FcεRI+ myeloid progenitors. Conclusion: Increased blood CD34+CD117+FcεRI+ cells may reflect enhanced bone marrow egress in the setting of CSU. High expression of these cells strongly predicts better clinical responses to the anti-IgE therapy, omalizumab. Overall design: PBMC were isolated from controls and paitents with CSU prior to treatment with omalizumab. PBMC were labelled with fluorescent antibodies and propidium iodide (PI) and purified using the FACSAria Fusion cell sorter (BD Biosciences). The lineage (Lin) channel included antibodies targeting the markers CD4, CD8, CD14 and CD19, all conjugated to V500. Cells were sorted at low pressure (100 µM nozzle at 20 psi) into PBS. For each sample, two populations of single, live (PI-) cells were sorted; (i) CD34+CD117+Lin- and (ii) Lin+, we recombined these populations at a ratio of 1:1. Data is from a single BD Rhapsody run, containing 12 samples multiplexed using the human multiplexing kit. Fastq files contain all three library types, WTA (RNA), AbSeq (CITE-Seq) and SMK (sample tag). Fastq files should be processed via the BD Rhapsody pipeline. Supplementary data contains outputs of this pipeline (revision 11), including UMI gene counts table (DBEC_MolsPerCell), sample tag reads and calls (specifying sample information for each barcode). We also include minimal sample information (Sample_info) to identify samples and their original clinical groupings.

背景：慢性自发性荨麻疹（Chronic spontaneous urticaria, CSU）是一种常见的致残性皮肤疾病，以反复发作的隆起、瘙痒且有时伴疼痛的风团持续超过6周为特征。CSU由肥大细胞介导，但外周血中并无肥大细胞。不过此前研究表明，血液中谱系阴性CD34hiCD117int/hiFcεRI+细胞可作为肥大细胞前体。
方法：我们通过流式细胞术对55例CSU患者的血液样本中FcεRI-、FcεRI+及FcεRIhi谱系阴性CD34+CD117+细胞进行计数，其中12例患者接受奥马珠单抗（omalizumab）治疗，43例未接受该治疗；同时纳入22例健康对照样本。采用荨麻疹控制测试评估疾病控制情况及患者对奥马珠单抗的应答。我们对8例CSU患者及4例健康对照的血液中谱系阴性CD34hiCD117hi细胞进行了单细胞RNA测序（single cell RNA sequencing, scRNA-seq）。
结果：CSU患者血液中的谱系阴性CD34+CD117+FcεRI+细胞数量高于健康对照。与奥马珠单抗治疗90天后疾病控制不佳的患者相比，对奥马珠单抗有客观临床应答的CSU患者，其谱系阴性CD34+CD117+FcεRI+细胞水平显著更高。单细胞RNA测序结果显示，谱系阴性CD34+CD117+FcεRI+细胞同时包含淋巴系及髓系祖细胞谱系，对奥马珠单抗应答的患者体内髓系祖细胞比例更高。髓系祖细胞谱系中包含少量真正的肥大细胞前体，以及更多未成熟的FcεRI-和FcεRI+髓系祖细胞。
结论：血液中CD34+CD117+FcεRI+细胞数量增加可能反映了CSU状态下骨髓向外周血的迁出增强。这类细胞的高表达可有效预测抗IgE治疗药物奥马珠单抗的临床应答效果更佳。
整体实验设计：在接受奥马珠单抗治疗前，从健康对照及CSU患者体内分离外周血单个核细胞（peripheral blood mononuclear cell, PBMC）。将PBMC用荧光抗体及碘化丙啶（propidium iodide, PI）标记，通过FACSAria Fusion细胞分选仪（BD Biosciences）进行纯化。谱系（Lin）通道使用结合V500荧光素的CD4、CD8、CD14及CD19靶向抗体。以低压（20 psi压力下使用100 μM喷嘴）将细胞分选至磷酸盐缓冲液（PBS）中。对每个样本，分选两类单活性（PI-）细胞群：(i) CD34+CD117+Lin-细胞，以及(ii) Lin+细胞，并以1:1的比例将这两类细胞群进行重组。本次数据来自单次BD Rhapsody测序运行，包含12个使用人类多重试剂盒进行多重标记的样本。Fastq文件包含三种文库类型：WTA（RNA）、AbSeq（CITE-Seq）及SMK（样本标签）。Fastq文件需通过BD Rhapsody分析流程进行处理。补充数据包含该分析流程（版本11）的输出结果，包括UMI基因计数表格（DBEC_MolsPerCell）、样本标签读取结果及标记调用结果（用于为每个条形码标注样本信息）。我们同时提供了最小化样本信息表（Sample_info），用于识别样本及其原始临床分组。