Isolation kit 2

Briefly, mononuclear cells were thawed from liquid N2. Next, CD4⁺ T cells were separated using the Isolation Kit II (Miltenyi Biotech, Bergisch Gladbach, Germany). Purity of samples was typically >97%. The CD4⁺ T cells were split into three cultures and an unstimulated culture served as baseline control. Two cultures were stimulated with pre-coated anti-CD3/-CD28 monoclonal antibodies (0.1 μg/ml). Cells were cultured for 48 hours in media consisting of Iscove's modified Dulbecco's medium (IMDM) supplemented with 5% fetal calf serum (Sigma-Aldrich), L-glutamine (292 mg/ml; Sigma-Aldrich), sodium bicarbonate (3.024 mg/ml; Sigma-Aldrich), penicillin (50 IE/ml; Cambrex, East rutherford, New Jersey, USA), streptomycin (50 μg/ml; Cambrex), and 100× non-essential amino acids (Gibco BRL, New York, USA). One of these cultures was supplemented with Tysabri® at a final concentration of 25 μg/ml. After culturing, cells were lysed using TRI Reagent (MRC, London UK). Total RNA was extracted according to the manufacturer’s instructions. RNA quality and quantity were assessed with the NanoDrop ND-1000 (NanoDrop Technologies Wilmington, New Jersey, USA). The Agilent Sureprint G3 Human Gene Expression 8x60k was used for gene expression analysis (Agilent Technologies, Santa Clara, California, USA).

The suspensions of spleen cells obtained from mice were prepared as reported by da Silva et al. [24 (link)]. The obtained cell suspensions were used to isolate CD4⁺ and CD8⁺ T cells using the Isolation Kit II from Miltenyi Biotec (Auburn, CA, USA), according to the manufacturer’s instructions. The negatively selected cells were stained with anti-CD3 phycoerythrin (PE) and anti-CD4 fluorescein isothiocyanate (FITC) or anti-CD8 FITC antibodies (BD Biosciences, San Jose, CA, USA) and analyzed by flow cytometry (Guava^® easyCyte, Millipore, Billerica, MA, USA). Purity grades of 94–96% were achieved.
The Jurkat E6.1 human acute leukemia T cell line was routinely grown in advanced Roswell Park Memorial Institute 1640 medium (Gibco^®, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 2500 mg/L glucose, 10 mM HEPES, and antibiotics in a humidified 5% CO₂ atmosphere at 37 °C. Cell pellets were obtained by centrifugation at 300× g for 7 min at 4 °C. The cell concentration was maintained as recommended by the ATCC cell biology collection, a protocol that was successful adopted by several authors [50 (link),77 (link),80 (link),82 (link)].

For most experiments CD4⁺ T cells were purified using the MagniSort Mouse CD4 T cell Enrichment Kit (8804-6821-74) according to the manufacturer’s instructions. For the western blotting in Figure 6E and microscopy experiments Figures 6 and S6, CD4⁺ T cells were purified by magnetic separation (Miltenyi Isolation Kit II) according to the manufacturer’s instruction.