Characterization of mAb CD69.2.2. (A) (Top) The specific binding of CD69.2.2 mAb to CD69 in thymocytes from wt (gray shaded curve) and CD69^-/- mice (dotted line) was determined by flow cytometry analysis. Isotype control (IgG1) is indicated by solid line. (Bottom) Immunoprecipitation of cell lysates from murine thymocytes with mAb CD69.2.2. As a negative control a nonrelated mAb (IgG1) was used. The immunoprecipitated samples were separated in an SDS-PAGE gel in reducing conditions (R) and nonreducing conditions (NR). Molecular weight (MW) markers are indicated in kDa. (B-D) MAb CD69.2.2 does not fix complement or induce ADCC. (B) Results of complement-mediated cytotoxicity assays using different anti–murine CD69 mAbs. The mAbs CD69.2.1 (IgG2b) and CD69.2.3 (IgG2a) were used as positive controls. RMA-S cells served as the target. (C) mAb binding to Fc receptors in CD69^-/- macrophages was determined by flow cytometry analysis. The CD69.2.2 (IgG1) mAb is represented by the gray shaded curve, while the positive control mAb (CD69.2.3, IgG2a) is represented by the black line. The negative control is represented by the broken line. (D) ADCC activity mediated by mAb CD69.2.2 (IgG1) and CD69.2.3 (IgG2a) assessed using IL-2–activated NK cells as effectors cells and RMA-S as target cells. E/T ratio indicates effector-target ratio. Error bars represent standard deviation.