CELLULAR IMMUNOLOGY 68, 187- 192 (1982) Induction of Interferon-y in Mouse Spleen Cells by OK-432, a Preparation of Streptococcus pyogenes MOTOO SAITO,* TAKUSABURO EBINA,* MINORU KoI,* TAKAHIRO YAMAGUCHI,~ YOSHIMI KAWADE$ AND NAKAO ISHIDA* *Department of Bacteriology and TDepartment of Radiology, Tohoku University School of Medicine, 2-1 Seiryo-machi, Sendai 980. and $lnstitute for Virus Research, Kyoro Universiry, Kyoro 606, Japan Received January 5. 1982; accepted January 28, 1982 A bacterial antitumor and immunopotentiating agent, OK-432, induced interferon in the spleen cell cultures but not in the thymus cell cultures of various inbred strains of mice. When 1 X 10’ spleen cells were cultured in the presence of 5 pg/ml of OK-432, interferon activity was detected as early as 4 hr later and reached a maximum level of about 160 to 500 units/ ml 24 hr later. OK-432-induced interferon was mainly an IFN--, of molecular weight ap- proximately 40,000, but also contained IFN-a and IFN-8. INTRODUCTION A streptococcal preparation, OK-432 (1) has been known to have potent anti- tumor activities in experimental animals and humans (2, 3), although its mecha- nisms of tumor regression remain unclear. OK-432 injected intraperitoneally into mice (0.1 mg per mouse) induced interferon (IFN) 24 hr after injection, and interferon induction was reproducible in humans after repeated clinical trials (4). These findings introduced two new means of studying the antitumor effect of OK- 432 in mice, first by characterizing the IFN it induces and second by analyzing the cells which participate in this interferon induction. In this article, the first study was conducted to witness the production of IFN in vitro using spleen lymphocytes and thymocytes obtained from normal mice, and the second to characterize the interferon species obtained under such conditions using antibodies against various types of IFN. A later article will describe the persisting memory of macrophages for IFN induction in mice which received weekly doses of OK-432. MATERIALS AND METHODS OK-432.OK-432 (Chugai Pharmaceutical Co., Tokyo) is a penicillin- and H202- killed lyophilized preparation of Su strain of Streptococcus pyogenes (1). This lyophilized preparation was easily suspendedin RPM1 1640 medium (Gibco, Grand Island, N.Y.). Mice. Eight- to ten-week-old inbred DDI, BALB/C, and C57BL/6 mice were obtained from the Institute for Experimental Animals, Tohoku University School 187 000%8749/82/050187-06$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.