Isolation and Activation of Human CD4+ T Cells

Human peripheral blood mononuclear cells (PBMCs) were freshly isolated using Ficoll-Paque Plus (GE Healthcare) from anonymized healthy blood donor buffy coats which were purchased from Karolinska University Hospital. “Untouched” CD4+CD25– T cells were isolated from PBMCs using the CD4+ T cell Isolation Kit, human (Miltenyi Biotec) including additional depletion of CD25+ cells with CD25-specific MACS beads (8 μl per 10⁷ cells) as described earlier (22 (link)). Purity of the isolated T cells was accessed with flow cytometry and defined as CD3+ CD4+ CD25– CD8– T cells. T cells were stimulated with antibodies against CD3 (0.2 μg/ml, clone OKT3, BioLegend, LEAF grade), CD28 (2 μg/ml, clone 15E8, Miltenyi Biotec, functional grade), and goat anti-mouse Ig as a cross-linker (2 μg/ml, Southern Biotech) mimicking TCR and co-stimulation. The cells were stimulated for either 15 min or 1 h for proteomics studies or alternatively 3 h for mRNA studies. Jurkat T cells (clone E6.1) were stimulated with either above-described TCR and co-stimulation or “P/I stimulation” with Phorbol 12-myristate 13-acetate (PMA; 10 ng/ml; Sigma Aldrich) and ionomycin (375 ng/ml; Sigma Aldrich) for 1 h for imaging studies.