Multiparameter CyTOF Analysis of PBMCs

PBMCs were stained with 37 metal-conjugated antibodies (Lymphoid panel, Supplementary Table S3) as previously described (15 (link), 17 (link)). After staining, data acquisition was done on the Helios CyTOF mass cytometer (Fluidigm). The generated files were analyzed using FlowJo v10.5.1 (FlowJo, USA) and down-sampled to equal number of live immune cell events of 12906 (smallest possible events of the 13 files) for comparison. To identify cell populations, we clustered the merged data containing 167778 single cell events using the FlowSOM (18 (link)) method. Clusters showing significant temporal frequency changes were detected using repeated measures ANOVA, where linear mixed models were used to account for missing data (19 ). Clusters were assigned to 7 major cell lineages and visualized with t-stochastic neighbor embedding (t-SNE) (20 (link)). Clustering results were validated by manual gating using FlowJo v10.5.1. Statistical analysis and data visualization were performed with R packages (21 ). In another CyTOF experiment to investigate MDSC, PBMC were stained with metal-conjugated antibodies (Myeloid panel, Supplementary Table S3) as described above. The CyTOF data were manually gated to check for the frequencies of MDSC using FlowJo.