Figure 3.

Reductions in CD4⁺, CD25⁺ and Foxp3⁺ T regulatory cell numbers in the vaccine with NYT diet group are more apparent compared with those in the vaccine with control diet group. Irradiated (20,000 rad) CT26 cells (1×10⁵) were s.c. injected into BALB/c mice. Vaccinated mice were fed the control diet or NYT diet from the vaccination until four weeks following the tumor challenge. Three weeks following the vaccination, 1×10⁶ live CT26 cells were s.c. injected into these mice. Twenty-eight days following the tumor challenge, (A) tumor-draining lymph nodes and (B) spleens were recovered and stained with anti-CD3, anti-CD4, anti-CD25 and anti-Foxp3. The proportions of CD3⁺, CD4⁺, CD25⁺ and Foxp3⁺ cells were determined using flow cytometry. Four to six female mice were used for each group. Results were pooled from two independent experiments. Each symbol represents one data point; the bars are the median. All P-values were calculated using the Kruskal-Wallis test with Dunn's multiple comparison test. The naïve group contained mice that received no vaccination or tumor challenge. NYT, ninjin'yoeito; CD, cluster of differentiation; foxp3, forkhead box P3; s.c., subcutaneously.