Frequency of tumor-infiltrating lymphocytes (TILs) in ex vivo tumor specimens over the course of tumor development. Specimens were obtained prior to and at three time-points following tumor induction and stained for CD3, CD4, and CD45 expression. (A) An increase in the frequency of TILs as characterized by CD3 and CD45 expression was observed at the late time-point (0.3% vs. 2.0%, P = 0.047). (B) The percentage of CD4+ T cells among TILs was reduced at the late time point (76.5% vs. 47.5%, P = 0.0003). 13 mice were analyzed at 13 dpi (n = 4), 26 dpi (n = 4), and at 29 dpi upon exhibiting clinical tumor morbidity (n = 1) or at 40 dpi (n = 4). Differences between the means at each time point were tested using two-sided t tests with unequal variances. *, P < 0.05; **, P < 0.01, ***, P < 0.001.