Kinetic analysis of lung-infiltrating lymphocytes. (A) Experimental design. (B) Representative dot plots of lymphocytes isolated from metastatic target organs at day 10 or 21 after tumor challenge. Cells were isolated from tumor-bearing lungs as described in “Methods” and subjected to an in vitro restimulation with tumor peptide. Plots are gated on live, CD8⁺ cells. Numbers in the upper right-hand quadrant reflect the frequency of CD8⁺ T cells that are positive for both IFNγ and the activation marker CD44. Data are representative of 3 independent experiments with 4 mice per group in each experiment. (C,D) Quantification of lung infiltrates at either 10 (C) or 21 (D) days after tumor challenge. Data are plotted as means (± SEM) and represent pooled data from either 2 (C, n = 8 mice/group) or 3 (D, n = 12 mice/group) independent experiments, with 4 mice/group in each experiment. (E) Effector phenotype of CD8⁺ T cells isolated from lungs of mice vaccinated with tumor antigen plus αCD40/TLR7* at either 10 or 21 days following tumor inoculation. The dot plots are first gated on live CD8⁺ cells and then further gated on IFNγ⁺CD44⁺ populations. Data are representative of at least 2 independent experiments, with 4 mice/group in each experiment.