(A) FACS analysis of CD4⁺CD25⁺ (Treg) cells in the CD3⁺CD4⁺CD127^– tumor-infiltrating T cell subsets. The percentage of Tregs and dead cells in tumors were determined by DAPI labeling 2 days after CTX injection. Numbers indicate the percentage of cells ± SD. (B) The day of the first BCG injection, 2 groups of mice were adoptively transferred with 1 × 10⁶ Tregs or 1 × 10⁶ CD4⁺CD25^– (Tconv) cells (n = 5 in each group). (C) CD11b⁺ TIDCs from untreated CT26 tumor-bearing mice were incubated overnight with BCG and with Tregs or Tconvs at a ratio 1:1. TRAIL expression was determined by FACS analysis. (D) Same experiment as in C but using mouse BM-DCs instead of CD11b⁺ TIDCs. (E) Same experiment as in D but in some wells DCs were separated from T cells by a 0.4-μm Transwell insert. TRAIL and GAPDH mRNA levels were determined by quantitative RT-PCR on CD11c cells sorted by magnetic isolation. BM-DCs incubated with IFN-γ represent a positive control of TRAIL mRNA expression. Each value is expressed as fold increase from medium control after normalization with GAPDH. (F) CD11b⁺ TIDCs from untreated CT26 tumor-bearing animals were incubated overnight with BCG and with Tregs or Tconvs at a ratio 1:1. Then these cells (5 × 10⁴ cells) were cultured with 1 × 10⁴ CT26 cells for 48 hours. TIDC cytotoxicity on CT26 cells was determined using crystal violet staining. (G) Same experiment as in F but using mouse BM-DCs instead of CD11b⁺ TIDCs. (H) Same experiment as in F but using mouse Mo-DCs instead of CD11b⁺ TIDCs and M45 melanoma cells instead of CT26 cells. Values from 1 experiment out of 2 represent mean ± SEM. *P < 0.05.