Reduced expression of cytotoxic components in <i>PI3Kδ−/−</i> CTLs.

<p>WT and <i>PI3Kδ−/−</i> splenocytes were activated for 3 days with aCD3ε and cultured in T cell medium. A. mRNA expression of <i>grzmA</i> (WT: 0.097±0.036; versus <i>PI3Kδ−/−</i>: 0.014±0.01, n = 6, <i>p = 0.0003</i>), g<i>rzmB</i> (WT: 1±0.076; versus <i>PI3Kδ−/−</i>: 0.419±0.075, n = 6, <i>p<0.0001</i>) and <i>prf1</i> (WT: 0.0433±0.004; versus <i>PI3Kδ−/−</i>: 0.013±0.003, n = 6, <i>p<0.0001</i>) was quantified by qRT-PCR and normalized to the house-keeping gene g<i>apdh</i>. B. Similarly, under standard culturing conditions mRNA levels of <i>trail</i> (WT: 0.0049±0.0012; versus <i>PI3Kδ−/−</i>: 0.0026±0.0007, n = 4, <i>p = 0.0178</i>) and <i>fasl</i> (WT: 0.0138±0.0015; versus <i>PI3Kδ−/−</i>: 0.0039±0.0001, n = 4, <i>p<0.0001</i>) were measured. C. <i>Ifng</i> mRNA was quantified by qRT-PCR under standard culturing conditions (WT: 0.019±0.0005; versus <i>PI3Kδ−/−</i>: 0.004±0.001, n = 6, <i>p<0.0001</i>) and after stimulation with 5 ng/ml IL-12 for 4 h (WT: 0.241±0.06; versus <i>PI3Kδ−/−</i>: 0.087±0.016, n = 6, <i>p = 0.0002</i>). D. To quantify IFN-γ protein levels, WT and <i>PI3Kδ−/−</i> splenocytes were stimulated with ConA. After 48 h supernatants were harvested and IFN-γ release was measured by ELISA (WT: 6515±2061 pg/ml; versus <i>PI3Kδ−/−</i>: 1359±1147 pg/ml, n = 3, <i>p = 0.0193</i>). IFN-γ release of unstimulated controls of WT and <i>PI3Kδ−/−</i> splenocytes was below detection limit of the assay (<10 pg/ml). Statistics were calculated with an unpaired Student’s <i>t</i>-test, and values represent mean±SD. One out of two independently performed experiments with comparable results is shown.