Anti cd3 clone 2c11

Naive CD4 T cells (CD4⁺Foxp3GFP⁻CD44^low) were sorted from lymph nodes of Foxp3^GFP mice. For polyclonal iTreg generation, naive CD4 T cells were cultured with immobilized anti-CD3 (clone 2C11, 2 μg/ml), soluble anti-CD28 (clone 37.51, 2 μg/ml), rhIL-2 (100 U/ml) (obtained from the NCI Preclinical Repository Biological Resources Branch) and rhTGFβ1 (5 ng/ml, R&D system, Minneapolis, MN) for 3 days. For allogeneic iTreg generation, the sorted naive CD4 T cells were co-cultured with irradiated T cell-depleted splenocytes from BALB/c mice (2:1 ratio), rhIL-2 (100 U/ml), rhTGFβ1 (5 ng/ml), anti-CD3 (0.1 μg/ml) for 5 days (33 (link)). Tregs were sorted based on GFP expression and restimulated with immobilized anti-CD3 (2 μg/ml), anti-CD28 (2 μg/ml), rhIL-2 (100 U/ml) in the presence or absence of rIL-27 (10 ng/ml) for 24 h unless stated otherwise.

CD8 T cells (4 × 10⁶/ml) were washed and resuspended in HEPES buffer (20 mM HEPES, 140 mM NaCl, 2 g/l glucose) with 0.1% BSA. Where required, 96-well flat-bottom flexiwell plates were coated with 10 μg/ml anti-CD3 (clone 2C11; R&D Systems) at 4°C overnight. T cell aliquots of 50 μl (2 × 10⁵) were added to flexiwell plates (Dynatech, West Sussex, U.K.) with or without 5 mM Mg²⁺ and 1 mM EGTA. Soluble murine ICAM-1–Fc (10 μg/ml) was added to the cells in addition to 50 ng/ml PdBu, 100 μM N4, or 2 μg/ml anti-CD28 (R&D Systems). After 30 min incubation at 37°C, T cells were washed twice in ice-cold assay buffer with 0.1% BSA and incubated with 0.1 μg/ml Fc-specific FITC-conjugated goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 20 min on ice. Cells were washed twice in ice-cold assay buffer with 0.1% BSA to remove excess unbound mAb and fluorescence was detected with a MACSQuant (Miltenyi Biotec) flow cytometer.