Jurkat cells and primary human CD4+ T cells were stimulated with either anti-CD3 (UCHT-1) monoclonal antibody, anti-CD226 monoclonal antibody (NewE1, Millipore), or a combination of both. Cells were incubated with indicated antibodies for 30 min in ice, then washed with ice-cold free medium, resuspended in warm free medium (40 × 106 cells/ml), and left for 5 min at 37°C. Antibodies were cross-linked using goat anti-mouse Fab2 secondary antibody (20 μg/ml; Jackson ImmunoResearch) for the indicated times. Stimulation was stopped with addition of 2× lysis buffer (2% Triton X-100, phosphatase inhibitors). Activation of ERK1/2 and AKT was assessed by immunoblots with phospho-specific antibodies to ERK1/2 phosphorylated at Thr202 and Tyr204, and to AKT phosphorylated at Ser473 (Cell Signaling Technology). CD226 was immunoblotted using DX11 antibody (BD Biosciences). Signals were detected with a ChemiDoc XRS system (Bio-Rad) and analyzed using the manufacturer’s software.

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