Antigen priming with IL-21 directs CD8⁺ T cells to express L-selectin during secondary stimulation. (A) Wild-type CD8⁺ T cells were anti-CD3/anti-CD28 stimulated with 10 ng/mL of the indicated cytokine for 3 days. Sell expression relative to β-actin expression was determined 4 hours after restimulation in cytokine-neutral conditions. (B) Four days after antigen priming with the indicated cytokine, pmel-1 Thy1.1 CD8⁺ T cells were restimulated with cognate antigen and 10 ng/mL IL-2 (regardless of the initial priming cytokine). Flow cytometry was performed 3 days after initiating the secondary response and histograms of L-selectin expression are displayed. The indicated cytokines and concentrations refer to the initial priming conditions. (C) Dot plots showing CFSE dilution and L-selectin expression by pmel-1 Thy1.1 CD8⁺ T cells that were antigen-primed with 100 ng/mL of the indicated cytokine for 4 days, labeled with CFSE, and restimulated with antigen and 10 ng/mL IL-2 (regardless of the cytokine present during initial priming). (D) Graphs of the mean fluorescence intensity of CFSE and L-selectin during restimulation. (E) Secondary expansion of cells antigen-primed with 10 ng/mL of the indicated cytokine for 4 days then restimulated with antigen and 10 ng/mL IL-2. Error bars indicate the standard error of the mean. (F) Tumor regression in response to adoptive transfer of 10⁶ pmel-1 CD8⁺ T cells primed in 10 ng/mL of the indicated cytokine for 4 days, restimulated with antigen and IL-2 for 6 days, and then adoptively transferred. Vaccine and IL-2 were administered to mice in all except the “No treatment” group. Error bars reflect the standard error of the mean.