Proteomic data (FCS files, flow cytometry) for expanded regulatory T cells in autoimmune polyendocrine syndrome type 1
Description
The deposit contains flow cytometric characterization and Treg suppression assay 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. 1. Flow cytometry characterization of expanded Tregs Tregs were isolated from whole blood from 17 APS-1 patients and 14 healthy controls and expanded in vitro for 14 days and then frozen. These were then characterized by flow cytometry. Please see the publication for details. 2. Tregs suppression assay Thawed PBMCs from 15 APS-1 patients and 15 healthy controls were used to obtain responder T cells (Tresp). Cells were then cultured to a concentration of 1x106 cells/ml and rested overnight. Next day, Tresp cells were stained with the CellTrace Violet Cell Proliferation Kit. To assess the suppressive capacity, recovered expanded Tregs were activated and co-cultured at different ratios for 5 days at 37℃ and 5% CO2. Cells were harvested and stained for flow cytometry.
Files
Steps to reproduce
1. Flow cytometry characterization of Tregs Expanded Tregs from 17 APS-I patients and 14 healthy blood donors were stained with a modified protocol according to Santegoets et al (Table S1A, publication for details). Fixation and permeabilization was achieved using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to instructions from the manufacturer. Cells were analysed using the BD LSRFortessa Cell Analyser and the BD FACSDiva Software. FlowJo v10.2 and v10.8 CL (BD) was used to analyse flow cytometric data. 2. Tregs suppression assay Thawed PBMCs from 15 APS-1 patients and 15 healthy controls were run through the Pan T Cell Isolation Kit Human (Miltenyi Biotec) and were used to obtain responder T cells (Tresp), according to the manufacturer’s protocol. Cells were then cultured in TexMACS Medium supplemented with 5% AB serum or FBS and 50 U/ml rIL2, to a concentration of 1x106 cells/ml. Cells were rested overnight at 37℃ and 5% CO2. Next day, Tresp cells were stained with the CellTrace Violet Cell Proliferation Kit (Invitrogen) for flow cytometry according to instructions from the manufacturer. Cells were dissolved to a concentration of 5x105 cells/ml in TexMACS Medium supplemented with 5% AB serum or FBS, 1% penicillin-streptomycin and 50 U/ml rIL2, further referred to as Treg suppression medium. To assess the suppressive capacity, recovered expanded Tregs were activated with 3 μL/mL cells Immunocult Human CD3/CD28 T Cell Activator (Stemcell Technologies) and co-cultured at different ratios (Tresp:Tregs 1:1, 2:1, 4:1 and 8:1) in Treg suppression medium for 5 days at 37℃ and 5% CO2. Expanded Tregs from patients were added to autologous patient responder cells and expanded Tregs from healthy controls were added to autologous responder cells from healthy controls. Cells were harvested and stained with Live/Dead Fixable Yellow Dead Cell Stain Kit (Invitrogen), and directly conjugated mouse anti-human antibodies against CD3, CD4, CD8 and CD25 (Table S1, please see publication for details). Cells were fixed using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) according to instructions from the manufacturer. Suppressive capacity was assessed using flow cytometry (BD LSRFortessa Cell Analyser and the BD FACSDiva Software). Percentage of Treg suppression was calculated as % Treg suppression=((〖Tresp〗_alone-〖Tresp〗_(treated with Tregs))/〖Tresp〗_alone )∙100% FlowJo v10.2 and v10.8 CL (BD) was used to analyse flow cytometric data.