Actin cloud formation lowers the threshold of T-cell activation. (A) Actin cloud formation augments actin rearrangement with very low doses of anti-CD3 stimulation. Jurkat cells were stimulated with either anti-CD8 (1 μg/mL), control Ab (1 μg/mL), anti–LFA-1 (1 μg/mL) Ab, or ICAM-1/Ig (0.5 μg/mL) together with graded concentrations of anti-CD3 mAb for 10 minutes, fixed, and stained with phalloidin–Alexa 568. One hundred cells were analyzed. White arrows indicate representative actin clouds. (B) Functional costimulation by actin cloud–inducing stimulation. (Left panel) Jurkat cells expressing CD8/ADAP were stimulated with a fixed amount of anti-CD3 (10 μg/mL) together with graded amounts of anti-CD8 or anti–LFA-1, and IL-2 production was measured by ELISA. (Right panel) The CD8/ADAP-Jurkat cells were stimulated with graded amounts of anti-CD3 together with a fixed amount of anti-CD8 or control rat Ig (10 μg/mL), and the induction of the cell-surface expression of CD69 was stained with PE–anti-CD69 Ab (BD Pharmingen) and analyzed by flow cytometry. MFI indicates mean fluorescent intensity. Data represent the mean ± SD of triplicate cultures.