Activation states of cells transfected with mutant ZAP-70. (A) Basal CD69 expression in P116 cells transfected with ZAP-70 constructs. Cell populations gated on ZAP-70 expressing cells (left) and their corresponding CD69 expression (right, blue, ZAP-70⁻; red, ZAP-70⁺). (B) CD69 expression in transfected cells with equal ZAP-70 expression, unstimulated (thin lines), or stimulated (bold lines) with anti-TCR antibody C305 or PMA plus ionomycin (shading). (C) Phospho-Erk expression in PBMCs from the father (open squares), healthy sister (open diamonds), and mother (open circles) of the affected patients and from four controls (Xs), stimulated with graded dilutions of anti-CD3 antibody for 15 min. Error bars, SEM. Data are representative of four independent experiments. Note that affected patients’ cells were not available due to their having received hematopoietic stem cell transplants before identification of their gene defect. (D) CD69 expression by mean fluorescence intensity (MFI) in P116 cells either untransfected (hatched bar) or transfected with indicated amounts of plasmid vectors containing no insert or ZAP-70 cDNA constructs, as shown. All transfections contained 20 µg of plasmid DNA. Black bars, WT with or without R360P; gray bars, R192W with or without R360P. White bar, R360P plus vector with no insert. *, P < 0.05, unpaired Student’s t test. Data are representative of three independent experiments.