Proleukin

After 4 days of stimulation of CD25-depleted LN cells with D665 beads CD4 + T cells were purified and the beads depleted by magnetic separation (Miltenyi) as described in the previous section. The CD4 + T cells were then cultured for 3 days at 1 × 10 6 /mL in the presence of 10 -7 M recombinant human IL-2 (Proleukin; Novartis). The precultured cells or freshly isolated CD4 + CD25 - T cells for comparison were then seeded at a density of 2 × 10 4 cells/well of a 96-well round bottom plate (Greiner) together with 5 × 10 4 Dynabeads Pan Mouse IgG (Invitrogen) coated either with 10 μg/mL normal mouse Ig (Sigma) or 10 μg/mL anti-CD3 mAb (clone 145-2C11; Biolegend) and 1 μg/mL anti-CD28 mAb (clone E18 [45] ) according to the manufacturer's instructions. Where indicated Proleukin, recombinant mouse IL-7 (R&D Systems) and recombinant mouse IL-15 (Biolegend) were added at a final concentration of 10 -6 M each. The total volume per well was 50 μL. All cultures were set up in triplicates. 3 H-thymidine was added for the last 16 h of the 3-day culturing period.