Anti cd3 clone 2c 11

CD25⁺CD4⁺ and CD25⁻CD4⁺ T cells were isolated from erythrocyte-depleted cell suspensions of spleens and lymph nodes using the CD4⁺ T cell isolation kit II followed by CD25-PE and anti-PE MicroBeads (all Miltenyi Biotec) according to the manufacturer’s instructions. Sorted CD25⁻CD4⁺ T cells were labeled with 2.5 μM CFSE (Molecular Probes) for 4 min at 37 °C; labeling was stopped by the addition of FCS. T cell-depleted splenocytes (using CD4 and CD8a MicroBeads; Miltenyi Biotec) treated for 45 min with 50 μg/ml mitomycin C (AppliChem) were used, after extensive washing, as antigen-presenting cells. To induce proliferation, 0.5 μg/ml of anti-CD3 (clone 2C-11; BioLegend) was added. 1 × 10⁵ CFSE-labeled CD25⁻CD4⁺ responder T cells were cultured with 1 × 10⁵ APCs in 96-well U-bottom tissue culture plates (Falcon). CD25⁺CD4⁺ T cells were added at the ratios 1 + 1, 1 + 4 and 1 + 9. On day 3 of co-culture, proliferation (based on CFSE dilution) was analyzed by flow cytometry; 7-AAD was added to exclude dead cells from the analysis.