Negative signaling for CD94/NKG2A expression by calcineurin pathway. (A-C) A total of 0.5 to 1 × 10⁵ unlabeled (B) or CFSE-labeled (A,C) purified naive 2C CD8⁺ T cells were stimulated either with (A-B) plate-bound anti-CD3 monoclonal antibody alone (2.5 μg/mL) or (A-C) anti-CD3 (2.5 μg/mL) plus anti-CD28 monoclonal antibodies (10 μg/mL) in the presence or absence of (A) the indicated phosphatidylinositol 3-kinase and mitogen-activated protein kinase inhibitors, 5μM LY294002, 10μM PD98059, or 10μM SB203580, or (B,C) calcineurin inhibitors, FK506 (B, 0.2nM; B,C, 5nM) or CsA (B, 50 ng/mL; B,C, 200 ng/mL). (D) A total of 1 × 10⁵ purified naive 2C CD8⁺ T cells were stimulated with plate-bound anti-CD3 monoclonal antibody alone (2.5 μg/mL) with or without anti-CD28 monoclonal antibody (10 μg/mL) in the presence or absence of FK506 (0.1 and 0.5nM for anti-CD3; 1 and 5nM for anti-CD3 plus anti-CD28). (E) A total of 1 × 10⁵ purified naive IL2^−/− 2C CD8⁺ T cells were stimulated with plate-bound anti-CD3 monoclonal antibody (2.5 μg/mL) and anti-CD28 monoclonal antibody (10 μg/mL) in the presence or absence of IL-2 (100 ng/mL), FK506 (5nM), or both (5nM for FK506; 1, 10, and 100 ng/mL for IL-2). (F-G) A total of 0.5 × 10⁵ naive WT and CnAβ^−/− CD8⁺ T cells purified from WT (from CnAβ^+/+ littermate) and CnAβ^−/− mice were stimulated with plate-bound anti-CD3 monoclonal antibody (F, 2.5 μg/mL; G, 0.1-10 μg/mL) with or without 10 μg/mL anti-CD28 monoclonal antibody. On day 3 after culture, cells were analyzed for expression of either the indicated NK receptors (A-G, CD94; C-E, NKG2D) or the activation markers (F; CD25 and CD44) by flow cytometry. The percentages of cells in the respective regions (A,C,F) are indicated. (A-G) Data are representative of more than 3 independent experiments (B,D-E,G; mean ± SD).