Abstract
The cell wall skeleton of Mycobacterium bovis BCG has been investigated as an immunopotentiating adjuvant for immuno-therapy of malignant tumors via Toll-like receptor (TLR) 2 and TLR4. However, due to its high molecular weight, highly complicated lipoglycan structure, and complicated purification and isolation procedure, its exact structure-activity relationship has not been well established. We have newly isolated the cell wall skeleton from M. bovis BCG Tokyo (SMP-105) and examined the binding of SMP-105 with TLR. It was revealed that highly purified SMP-105 activates the nuclear factor-kB promoter in a TLR2-dependent manner, not a TLR4-dependent manner, using a reporter gene assay system. Peritoneal exudated cells of TLR2 and MyD88 knockout mice severely reduced the induction of tumor necrosis factor-alpha and interleukin-6 in the presence of SMP-105, whereas cells from TLR4 knockout mice produced similar levels of cytokines to wild-type mice. Dendritic cells and macrophages accumulated in the draining lymph nodes of treated mice. When mice were administered both SMP-105 and mitomycin C-inactivated Lewis lung carcinoma cells simultaneously, interferon-gamma-producing cells reacting to the tumor were increased distinctly in draining lymph nodes. When C57BL/6 mice, into which splenocytes from OT-I transgenic mice had been transferred, were administered with both SMP-105 and E.G7-OVA, OVA-specific cytotoxic T lymphocytes (CTL) increased markedly. Mice treated with SMP-105 and inactivated Lewis lung carcinoma cells suppressed the growth of implanted tumors. These results suggest that the activation of TLR2 by SMP-105 sufficiently enhanced immune responses, such as the number of interferon-gamma-producing cells and CTL, and prevented the growth of tumors without the contribution of TLR4.