Activation of T cells using antibody-coated beads

Anti-Biotin MACS iBeads (Miltenyi Biotec) were labeled with 10 μg of anti-CD3–biotin (OKT3, BioLegend), 10 μg of anti-IgG (Fc)–biotin (Novex/Life Technologies), or 10 μg of anti-CD3–biotin and 10 μg of anti-IgG (Fc)–biotin. After incubation (10 min, 4°C), beads were washed twice with PBS and resuspended in T cell medium. Bead suspensions (100 μl) were plated in 96-well U-bottom plates (Thermo Fisher Scientific, Massachusetts, USA). Effector T cell preparations were added (0.2 × 10⁶ cells per well in 100 μl of T cell medium) and incubated with beads for 24 hours. No exogenous IL-2 was added to the medium in either case. After 23 hours, cells were harvested for analysis of cytokine secretion by intracellular cytokine staining. The gating strategy was identical to that in fig. S15, with the exception that “% cytokine + ve” was used as the readout.