Peripheral changes in frequency and function of CD4+FoxP3+ Treg cells. Splenocytes were stained for extracellular CD4 and intracellular FoxP3 and IL-10 expression. (A) An increase in Treg cell frequency among CD4+ T cells was observed between the early and intermediate time points (9.0% vs. 15.2%, P = 0.0007) and this frequency remained elevated at the late time point. (B) While the proportion of Tregs expressing IL-10 increased between the early and intermediate time points (3.9% vs. 6.9%, P = 0.02), the proportion of IL-10-producing Tregs at the late time point returned to levels measured at the early time point (4.2%). 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 a Mann-Whitney test. *, P < 0.05; **, P < 0.01, ***, P < 0.001.