Icos bv605

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.).

To quantify CD4⁺ICOS⁺CD38⁺ and CD8⁺ICOS⁺CD38⁺ T cells, PBMCs were first resuspended with Human TruStain Fcx (Biolegend) for 10 min at room temperature and then stained with the following antibodies in FACS buffer (PBS + 2% fetal bovine serum): CD8a e450 (Invitrogen 48-0086-42, 1:200) ICOS BV605 (Biolegend 313538, 1:50), CCR7 PE (Biolegend 353204, 1:200), CD38 PerCP (Biolegend 303520, 1:100), CD4 PECy7 (Biolegend 357410, 1:100), and CD3 APCCy7 (Biolegend 300318, 1:200). Tfh markers consisted of CXCR5 APC (Biolegend 356907, 1:200) and PD-1 PE (Biolegend 135205, 1:100). CD71 APC (Biolegend 334108, 1:100) was also measured on sorted cells. Cells were analyzed on a Miltenyi MACSQuant16 Analyzer with single-stain control PBMC samples used for compensation conducted in FlowJo v10.6.2.