(A) For 48 hours, 5 × 10⁴ CD11b⁺ TIDCs isolated from CTX-BCG–treated mice were incubated with 1 × 10⁴ CT26 cells. In some wells, Z-VAD-fmk (Z-VAD), blocking anti–Fas ligand (FasL), anti-TRAIL mAbs, l-NMMA, or concanamycin A (CcmA) were added. CD11b⁺ TIDC cytotoxicity against CT26 cells was determined using a crystal violet assay. (B) FACS analysis of TRAIL expression on CD11b⁺ TIDCs (CD45⁺) that infiltrated untreated CT26 tumor and tumor treated with CTX-BCG. (C) CD11b⁺ TIDCs isolated from untreated CT26 tumors or BM-DCs were either untreated or cultured overnight with BCG (8 × 10⁵ CFU/ml). TRAIL expression was determined by FACS analysis on CD11c⁺ I-A/I-E⁺ gated cells and (D) 5 × 10⁴ CD11b⁺ TIDCs or BM-DCs were incubated with 1 × 10⁴ CT26 cells for 48 hours. A blocking anti-TRAIL mAb was also added to some of the wells containing BM-DCs activated by BCG. Cytotoxic effect on the CT26 cells was determined using a crystal violet assay. (E) BM-DCs were incubated overnight in the absence or presence of ligands for TLR2 (Pamv3CSK4 [Pam3]), TLR3 [Poly(I:C)], TLR4 (LPS), TLR9 (CpG ODN 1826 [CpG 1826]), or with BCG (8 × 10⁵ CFU/ml). TRAIL expression was determined by FACS analysis on CD11c⁺ I-A/I-E⁺ gated cells. (F) Wild-type, Myd88^–/–, Tlr2^–/–, Tlr4^–/–, or wild-type mice treated with i.p. injections of TLR9 inhibitor ODN (IRS 954) every 3 days received a subcutaneous injection of 5 × 10⁵ CT26 tumor cells. In the same experiment, 2 groups of mice received either saline or the combined treatment. Data represent mean ± SEM. *P < 0.05.