NFAT-independent negative signaling by calcineurin and inhibitory function of CD94/NKG2A. (A) A total of 2 × 10⁵ total spleen cells from 2C TCR Tg mice were stimulated with 10μM SIYR peptide with or without calcineurin and NFAT inhibitor, FK506 and VIVIT, respectively, at the indicated concentrations (0.3-20nM for FK506 and 0.3-20μM for VIVIT). (B-C) A total of 1 × 10⁵ naive WT and IL2^−/− 2C CD8⁺ T cells were stimulated with 2 × 10⁵ irradiated T-depleted B6 spleen cells pulsed with 10μM SIYR peptide (sAPC-SIYRp) with or without 5nM FK506 or 5μM VIVIT. On day 3 after culture, cells were analyzed for expression of the indicated NK receptors (A, CD94; B, NKG2ACE; C, NKG2D) by flow cytometry. (D) Schematic diagram for signaling pathways regulating CD94/NKG2A expression. TCR ligation induces both positive and negative signaling for CD94/NKG2A expression via PI3K and MAPK and through the calcineurin pathway, respectively. Negative signaling through the calcineurin pathway is NFAT independent and is further potentiated by B7/CD28 interaction, although only with concomitant interaction with IL-2; in the absence of IL-2, CD28/B7 interaction enhances positive signaling, thereby resulting in increased CD94/NKG2A expression compared with CD3 ligation alone. Thus, under defined conditions, the NFAT-dependent classic calcineurin pathway does serve to induce positive signaling for CD94/NKG2A expression, together with the PI3K and MAPK pathways; these pathways may act as potential targets for the positive and negative regulation of the various cytokines indicated. (E-G) A total of 4 × 10⁴ NKG2ACE⁺ CD44^hi OT-I CD8⁺ T cells purified on day 3 after primary (1°) stimulation of naive OT-I cells with plate-bound anti-CD3 monoclonal antibody (5 μg/mL) in the presence or absence of cytokines IL-6, IL-10, or IL-21 (50 ng/mL) were restimulated with plate-bound anti-CD3 monoclonal antibody (2.5 μg/mL) plus either isotype control or anti-NKG2A monoclonal antibody (10 μg/mL). Proliferation (E), IFN-γ production (F), and apoptotic cell death (G) were analyzed on the indicated time points after secondary (2°) culture. The percentages of cells in the respective regions (B-C,F) are indicated. (A-C,E-G) Data are representative of 2 or 3 independent experiments (A,E,G; mean ± SD).