Ctla 4 bv421

Phenotypic markers expressed by CD4⁺ T cells isolated at day 0 and from day 14 were analysed by flow cytometry. Cells were fixed using Human FoxP3 Buffer Set (BD Biosciences, 560098), and stained with anti-human: CD4 BV510, LAG-3 Pe/Cy7, TIM-3 PE, TIGIT PeDazzle594, CTLA-4 BV421 (all BioLegend, 317444, 369310, 345006, 372716, 369606), CD3 FITC, and PD-1 APC (both ThermoFisher, 11-00390-42, 17-2799-42 respectively). Cells were then analysed using a cyAn ADP flow cytometer (Beckman Coulter).

Stimulation of cell culture was carried out with 1 μg/ml protein A (SpA, isolated from S. aureus SAC, GE Healthcare, Uppsala, Sweden), 1 µg/ml recombinant SpA (Sigma-Aldrich, Munich, Germany), 100 ng/ml synthetic lipopeptide P3C (Pam3CSK4, EMC, Tübingen, Germany) or anti-CD3/CD28 microbeads (Miltenyi Biotech, Bergisch-Gladbach, Germany).
Microbeads used for cell isolation via AutoMACS and recombinant cytokines (IL-4 and GM-CSF) were obtained from Miltenyi Biotech (Bergisch-Gladbach, Germany).
Antibodies used for flow cytometry, purity determination and cell sorting were purchased from BD Biosciences, Heidelberg, Germany, if not indicated otherwise: CD4 PErCP, CD4 PE, CD3 BV605 (BioLegend, U.S.), CD3 AF700 (BioLegend, U.S.), CD25 APC (BioLegend, U.S.), CD25 FITC, CD127 BV421, CD127 AF647, FoxP3 BV421 (BioLegend, U.S.), CCR4 PE-Cy7 (BioLegend, U.S.), ICOS BV605 (BioLegend, U.S.), CTLA4 BV421 (BioLegend, U.S.), PD-1 PE (BioLegend, U.S.) and CD14 V450. Viability staining was performed with the LIVE/DEAD Fixable dead Cell stain Kit (Thermo Fisher Scientific, U.K.).