Proteomic data (FCS files, mass cytometry) for expanded regulatory T cells in autoimmune polyendocrine syndrome type 1
Description
The deposit contains mass cytometric (CyTOF) characterization data for the paper "Single cell characterization of blood and expanded regulatory T cells in autoimmune polyendocrine syndrome type 1", published in iScience in 2024. The dataset includes thawed expanded Tregs from 17 APS-1 patients and 17 healthy controls. Please see more details in the publication.
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The dataset includes expanded Tregs from 17 APS-I patients and 17 age- and gender-matched healthy controls. CD4+CD25+CD127low cells (from here-on referred to as “expanded Tregs”) were isolated directly from EDTA-blood using the MACSxpress Whole blood Treg Isolation Kit Human (Miltenyi Biotec, cat. 130-109-557). The purity of up-concentrated Tregs was not investigated because we needed all cells for the downstream protocol but has been evaluated to contain >65% FOXP3+ Tregs in similar experiments. Cells were then expanded in TexMACS Medium (Miltenyi Biotec, cat. 130-097-196) supplemented with 500 U/mL recombinant (r) - IL2 (Miltenyi Biotec, cat. 130-097-744) and 5% FBS or human AB serum for 14 days at 37℃ and 5% CO2. At day 0, 2x107 CD3/CD28 MACSiBead Particles/mL were added, according to the Treg Expansion Kit Human protocol (Miltenyi Biotec, cat. 130-095-345). Cells were given new medium containing 500 U/mL rIL2 every 2-3 days and split when necessary. At day 14 cells were harvested, counted and frozen in AB serum or FBS supplemented with 10% DMSO. Thawed expanded Tregs from the patients and healthy controls were barcoded according to the Cell-ID 20-Plex Pd Barcoding Kit User Guide. Before antibody labelling, cells were incubated for 10 minutes at room temperature with 1 μL Fc-block solution and 1 μL heparin (10U). A cocktail of 27 metal-conjugated antibodies (Table S1B, see publication for details) was added and incubated at room temperature for 30 minutes. After washing, an intercalation solution (700 μL PBS + 250 μL fresh 16% PFA + 100 μL 10X BioLegend Intracellular Staining Perm Wash Buffer + 0.25 (125nM) μL 500 μM Iridium Intercalator) was added and incubated at 4℃ overnight. Cells were washed, pelleted and frozen in CRYO#20 (Cytodelics) at -80℃ until acquisition on the CyTOF XT instrument (Fluidigm). Samples were debarcoded using the Fluidigm Debarcoding Software (7.0.8493.0) with a 20-plex-debarcoding key (Fluidigm). Raw CyTOF XT FCS files were bulk normalized in the fluidic CyTOF Software v8.0 using EQ four-element calibration bead (bead normalization passport) to normalize the data sets. Randomization was selected automatically on the uniform negative distribution (UND) in linear value, compatible with FlowJo v10.2 (BD), and on the default time interval normalization. The normalized FCS files were exported to RStudio for concatenation and debarcoding using CATALYST. Please see publication for details.