Involvement of Toll-Like Receptor 4 Signaling in Interferon- Production and Antitumor Effect by Streptococcal Agent OK-432

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Abstract
Background: The streptococcal agent OK-432 has been used for immunotherapy of head and neck cancer, among other malignancies, but its mechanism of action is unknown. Because the Toll-like receptor 4 (TLR4)/MD-2 complex is important in enabling the mammalian immune system to recognize bacterial components, we investigated whether expression of the TLR4 and MD-2 genes is associated with OK-432-induced anticancer immunity. Methods: Peripheral blood mononuclear cells (PBMCs) from 28 patients with head and neck cancer were analyzed for TLR4 and MD-2 mRNA expression by reverse transcription–polymerase chain reaction (RT–PCR) analysis. PBMCs were treated in vitro with OK-432 or with OK-PSA (a lipoteichoic-acid-related molecule that is an active component of OK-432), and interferon-gamma (IFN-γ) mRNA expression, an immune response measure, was analyzed by RT–PCR. Patient sera collected 24 hours after OK-432 administration were examined for IFN-γ protein using an enzyme-linked immunosorbent assay. Lewis lung carcinoma-bearing wild-type C57BL/6 and TLR4-deficient mice (four mice per group) received intraperitoneal injections of OK-432, and tumor volumes and sera IFN-γ levels were measured over time. All statistical tests were two-sided. Results: Twenty patients expressed both TLR4 and MD-2. Expression of TLR4 and MD-2 genes was associated with the in vivo IFN-γ induction in 19 patients administered OK-432 (Fisher's exact test P<.001). Although both OK-432 and OK-PSA induced IFN-γ expression from PBMCs in vitro, expression of TLR4 and MD-2 was associated only with IFN-γ expression induced by OK-PSA (P<.001). In vivo intraperitoneal administration of OK-432 resulted in an increase of IFN-γ in sera from wild-type mice but not in sera from TLR4-deficient mice. Tumors in wild-type mice treated with OK-432 were statistically significantly smaller than those in mice treated with saline (P = .007). By contrast, in TLR4-deficient mice, there was no difference in tumor volume between the two treatment groups. Conclusions: TLR4 and MD-2 may mediate OK-432-induced anticancer immunity.

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