FIGURE 3.

Intrinsic Notch1 signaling directly regulates perforin and granzyme B expression in CD8⁺ T cells. A, Purified CD8⁺ T cells from un-stimulated splenocytes were pretreated in vitro with GSI, washed two times, mixed with CD8-depleted cells, and stimulated with anti-CD3ε plus anti-CD28. As a control, similar to parallel samples analyzed by real-time quantitative PCR and by intracellular staining as in Fig. 1D, stimulated bulk splenocytes in the absence or presence of GSI in vitro were compared (top panels). Using intracellular staining and flow cytometry, the percentage of CD8⁺ T cells expressing perforin and granzyme B after 2 days was determined. All perforin- or granzyme B-positive plots are gated on CD8⁺. B, CD8⁺ T cells were isolated from splenocytes, pretreated in vitro with GSI then stimulated with anti-CD3ε plus anti-CD28 for 2 days. We verified N1ICD inhibition by immunoblot (top band); heat shock protein HSP70 was used as a loading control (bottom band). C, CD8⁺ T cells prepared as in A were stimulated for 1–3 days, supernatants were harvested, and IFN-γ determined by ELISA. Data represent the mean ± SD of three individual replicates. All experiments were repeated three times.