Influence of CD28 costimulation on CD94/NKG2A expression. (A-F) A total of 0.5 to 1 × 10⁵ unlabeled (A-C,E-F) or CFSE-labeled (D) purified naive 2C CD8⁺ T cells were stimulated with either (A-C) plate-bound anti-CD3 monoclonal antibody (A,C; 2.5 μg/mL; B, 1-10 μg/mL) with or without anti-CD28 monoclonal antibody (A-B, 10 μg/mL; C, 0-10 μg/mL). (D-E) A total of 1 to 2 × 10⁵ transfected fly cells as APCs (flyL^d.ICAM, flyL^d.B7 and flyL^d.B7.ICAM) pulsed with 10μM QL9 peptide (E; with or without 10 μg/mL anti–ICAM-1 or anti-B7–1 monoclonal antibody), or (F) 5 × 10⁵ irradiated T-depleted B10.D2 (H-2^d) spleen cells (B10.D2 sAPCs) pulsed with 10μM p2Ca or QL9 peptide with or without 10 μg/mL CTLA4Ig or anti–IL-2 monoclonal antibody; note that fly APCs die within day 1 when cultured at 37 and were therefore added either once (at day 0; E) or daily for 3 days (at days 0-2; D). On day 3 after culture, cells were analyzed for the indicated NK receptors by flow cytometry. The percentages of cells in the respective regions (A,D) are indicated. (A-F) Data are representative of at least 4 independent experiments (B-C,E-F; mean ± SD). *P < .05. **P < .005.