Clone 15e8

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.

PBMCs were isolated according to standard Ficoll density gradient procedures from healthy donor buffy coats. CD4+ CD25− T cells were then isolated by negative magnetic isolation using human CD4 T cell isolation kit and CD25 microbeads (Miltenyi Biotec). 12 Mio CD4+ CD25− T cells from individual donors were resuspended in 100 μl of Nucleofection® buffer solution for human primary T cells (Nucleofector™ Kits for Human T Cells, Lonza) containing 2 µM of ON-TARGETplus SH3YL1 siRNA pool or ON-TARGETplus non-targeting control pool (Dharmacon, GE). The cells were transfected using program U-014 of the Nucleofector™ 2b device using manufacturer’s recommendations. Following nucleofection, the cells were transferred to pre-warmed X-VIVO 15 medium (Lonza) and incubated for 4.5 days. The medium was changed following 5 hours of incubation meanwhile. The cells were equally distributed for 3 time points; resting, 6 hours and 24 hours. The cells for the later time points were stimulated with antibody against CD3 (0.2 µg/ml, clone OKT3; Biolegend, LEAF grade; cat. no. 317315), antibody against CD28 (2 µg/ml, clone 15E8, Miltenyi Biotec, functional grade, cat no 130-093-375), and goat anti-mouse Ig antibody as a cross-linker (2 µg/ml, SouthernBiotech, cat no. 1010-01) mimicking TCR and co-stimulation for the afore mentioned time periods at 37 °C and 5% CO₂.