CyTOF_neutrophils_inflammatory_arthritis_healthy_donors

CyTOF analysis of neutrophils from blood and inflamed synovial fluid from patients with inflammatory arthritis, as well as blood from healthy donors.
Conclusion:
Mass cytometry revealed that healthy and arthritic donor blood neutrophils are largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of FcγRI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Cytometry by Time of Flight (CyTOF) CyTOF was employed in a single batch to characterize the expression of 39 surface and intracellular proteins using a custom antibody panel (Supplementary Table 2). Blood and synovial fluid samples were washed and stained with metal-conjugated monoclonal antibodies. For all stains, viability staining by cisplatin was added to plated cells for 10 min before staining. After washing and centrifugation, Fc-Block reagent was added for 10 min before adding cell-surface CyTOF antibody staining cocktails. Cells were stained for 30 minutes, then washed once in CyTOF staining buffer (CSB, calcium/magnesium-free PBS, 0.2% BSA, 0.05% sodium azide). Cells were fixed with 1.6% paraformaldehyde diluted in PBS (PFA) for 10 minutes, washed by centrifugation, then permeabilized with eBioscience FOXP3/Transcription Factor Staining Buffer (Life Technologies # 00-5523-00) for sample barcoding with palladium reagents as described [10]. After barcoding, cells were washed by centrifugation, combined into a single tube, fixed with Fix/Perm buffer, then washed once in permeabilization buffer. The intracellular CyTOF antibody staining cocktail (antibodies against PR3, Nrf2, Arginase 1, OLFM4, Elastase, Defensin 3, LL-37 and MPO) was diluted in permeabilization buffer to stain cells for 1 hour. Cells were washed in CSB by centrifugation, then fixed overnight in 1.6% PFA. Prior to acquisition, iridium-intercalator solution (MaxPar Intercalator-Ir 500 mM; Fluidigm Sciences) was added to cells in PBS. After washing by centrifugation, cells were diluted in Cell Acquisition Solution (CAS, Fluidigm Sciences) at a concentration of 7.5 x 105 cells/ml with addition of EQ calibration beads (EQ Four Element Calibration Beads; Fluidigm Sciences) to normalize metal intensity signals using the manufacturer’s protocol. Cells were analyzed on a Helios mass cytometer (Fluidigm Sciences). Analysis of CyTOF data Data analysis was conducted using FlowJo version 10.7.1 and R. During initial quality control, contaminating leukocytes were removed (Supplementary Figure 2). We retained 280,174 single neutrophils from a total of 33 samples in our dataset. These comprised 119,354 neutrophils from healthy control blood, 94,771 neutrophils from blood of patients with inflammatory arthritis, and 66,049 neutrophils from inflamed synovial fluid. We extracted mean signal intensities for each channel per sample and calculated Spearman correlation coefficients between samples as with the RNAseq data. Using these mean expression values, fold changes were calculated and log2 transformed. For marker expression between groups, multiple t-tests were performed and p-values adjusted using Holm correction. For marker-to-marker correlation analysis, intensity on the bulk level was defined as the mean intensity of each marker from all the cells in each sample and on the single cell level, each cell was considered separately. For analysis on the single-cell level, UMAP dimensionality reduction was performed with standard settings, using all antigens (Supplementary Figures 2 and 4) or all antigens minus OLFM4, CD177 and PR3 (all other Figures) as input. Neutrophils were intentionally overclustered into k = 20 clusters. Abundance of populations between groups was tested using ANOVA, followed by independent t-tests.