Experiment Overview
| Repository ID: | FR-FCM-Z7ZS | Experiment name: | Single cell transcriptomic and proteomic signatures of paradoxical eczema in psoriasis patients treated with biologics | MIFlowCyt score: | 77.75% |
| Primary researcher: | Ali Al-Janabi | PI/manager: | Ali Al-Janabi | Uploaded by: | Ali Al-Janabi |
| Experiment dates: | 2023-06-01 - | Dataset uploaded: | Oct 2023 | Last updated: | Nov 2023 |
| Keywords: | [human PBMCs] [CyTOF] [atopic dermatitis] [psoriasis] | Manuscripts: | [40157420] | ||
| Organizations: |
University of Manchester, Division of Musculoskeletal and Dermatological Sciences, Manchester, (United Kingdom)
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| Purpose: | Biologics targeting the tumour necrosis factor (TNF) and interleukin (IL)-17/23 axis are highly effective treatments for psoriasis but can result in cutaneous adverse events. The pathogenesis of paradoxical eczema, the occurrence of an atopic dermatitis phenotype after biologic initiation in people with psoriasis, is unknown. In combination with single cell RNA-seq data, we explored the systemic disease signature of paradoxical eczema using Cytometry by Time of Flight (CytTOF) on stimulated and unstimulated peripheral blood mononuclear cells from patients with paradoxical eczema, matched psoriasis controls and healthy volunteers. | ||||
| Conclusion: | Using single cell RNA-seq and mass cytometry, we found increased expression of TNF, interferon (IFN)-γ and IFN-α and their signalling pathways in paradoxical eczema case cell clusters compared with matched psoriasis controls. | ||||
| Comments: | The experiment was undertaken in four batches, with anti-CD45 barcodes applied to individual donor samples before pooling. The included data has undergone debarcoding, manual gating (FlowJo v10.9) to isolate live singlets and normalisation using CytoNorm. The experimental protocol is summarised below (wash and centrifugation steps excluded to improve readability). Cells were thawed, washed, and rested overnight. Half of the cells from each donor were stimulated for 4 hours in PMA and ionomycin in the presence of Brefeldin A (BD GolgiPlug) at 37°C. Each sample (stimulated and unstimulated from each donor) was barcoded separately using combinations of 110Cd, 111Cd, 195Pt, 195Pt or 198Pt-tagged anti-CD45 antibody. Samples were then pooled, and 12 million cells total taken forward for FcR blocking (TruStain FcX) and surface staining using antibodies from four Maxpar Direct Immune Profiling Assay (MDIPA) tubes (Standard Biotools). Cells were fixed in 2% formaldehyde (FA) solution for 10 minutes, permeabilised and stained with the following intracellular antibodies: 114Cd-TNFa, 116Cd-IFNg, 142Nd-IL-4, 159Tb-GM-CSF, 162Dy-IL-17A, 165Ho-IL-10,169Tm-IL-13 and 175Lu-IFNa. The cells were then fixed again in 2% FA and 31.25nM Cell-ID Intercalator-Ir for 1 hour before freezing at -20°C overnight. The following morning, cells were thawed, washed, suspended in Maxpar Cell Acquisition Solution containing 0.2X EQ Four Element Calibration Beads and acquired on a Helios system. Due to the number of samples, this workflow was undertaken in four batches with stimulated cells from one psoriasis control donor being included in each run to enable downstream batch normalisation. | ||||
| Funding: | This work was funded by the Medical Research Council (Award Ref. MR/N025989/1). The CyTOF experiments were funded by UCB. | ||||
| Quality control: | Identification of live singlets and de-barcoding was undertaken manually for each batch using FlowJo v10.9. | ||||
