Notch signaling is induced in established effector CD8⁺ T cells. A Mart-1–specific CD8⁺ T cell clone was incubated with nonpulsed (medium control) or Mart-1 peptide–pulsed moDCs, and 24 hours later, (A) surface expression of Notch1 and Notch2 and (B) intracellular expression levels of NICD1 were measured by flow cytometry and (C) Hes1 mRNA expression by qRT-PCR. Data are representative of 3 independent experiments. GSI treatment during activation prevented accumulation of (B) NICD intracellular but did not affect (D) surface expression of Notch1, Notch2, and CD25 nor did it affect cell viability, as measured by (E) annexin V/7AAD staining. Shown is a representative example of 3 independent experiments.