Phenotype of OT-I CD8⁺ T cells after cytokine immunotherapy versus immunization. (A) Expression of Vα2 and Vβ5.1/5.2 on peripheral blood CD8⁺ T cells from WT (left) and OT-I (right) mice. Numbers in the quadrants denote the percentages of CD8⁺ T cells expressing each marker. (B) Percentage and (C) numbers of CD44^high expressing OT-I cells 11 days after anti-CD40 and IL-2 immunotherapy or OVA vaccination. (D) Dot plots depicting BrdU incorporation gated by CD44^high expressing population in adoptively transferred OT-I cells after immunotherapy. Percentages of (E) CD25⁻ and (F) PD-1 expression on CD44^highOT-I cells from anti-CD40/IL-2 or OVA-vaccinated mice. (G) Percentage (left) and total numbers (right) of NKG2D⁺CD25⁻ of CD44^highOT-I cells on day 12 of anti-CD40 and IL-2 treatment. (H) Frequencies of NKG2D (left), PD-1 (middle), and CD25 (right) on OT-I cells after in vitro anti-CD3 and anti-CD28 (solid line) or IL-2 (dashed line) stimulations compared with media alone (shaded). Data are representative of at least 2 independent experiments (*P < .05, **P < .01, ***P < .001).