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/hiFceRI+ cells in blood have previously been shown to represent a mast cell precursor.  Methods: We enumerated FceRI-, FceRI+ and FceRIhi 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+FceRI+ blood cells than controls. Lineage-CD34+CD117+FceRI+ 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+FceRI+ 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 FceRI- and FceRI+ myeloid progenitors.  Conclusion: Increased blood CD34+CD117+FceRI+ 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.