Activation of NK cell (human and mouse) by immunostimulatory DNA sequence.

Abstract

A purified nucleic acid fraction extracted from Mycobacterium bovis BCG and designated MY-1, which was composed of 70.0% DNA and 28.0% RNA, 1.3% protein, 0.27% hexose and 0.1% lipid with no detectable amounts of cell wall components possesses strong antitumor activity against various syngeneic mouse and guinea pig tumors. This fraction showed no direct cytotoxicity in vitro against these tumors. MY-1 after digestion with RNase contained 97.0% single-stranded DNA with a guanine-cytosine content of 69.8%, and showed stronger antitumor activity than undigested MY-1, while MY-1 digested with DNase contained 97.0% RNA, and had reduced activity [15, 19]. Peritoneal cells from BALB/c mice injected intraperitoneally with MY-1 (100 μg) 3 h, 1 day, 2 days, 4 days or 7 days before, were tested for cytotoxic activity. An intraperitoneal injection of MY-1 1 day earlier rendered mouse peritoneal cells strongly cytotoxic to YAC-1 cells in vitro, but those obtained 3 h and 4 days after MY-1 showed only weak cytotoxicity. Those obtained 2 days after MY-1 injection induced moderate cytotoxicity. The effector cells’ were non-adherent to plastic dishes. No cytotoxicity of the adherent cell fraction was observed. Nonadherent cells activated by MY-1 were not cytotoxic against P815 cells even in a 16-h 51Cr release assay. The activity was destroyed by treatment with anti-asialo-GMl antiserum plus complement or carrageenan in vitro, but not with carbonyl-iron or anti-Thy 1.2 antiserum, suggesting that the cells are NK cel. In vivo augmentation of NK activity was dependent on MY-1 dose, and reached a peak 1 day after MY-1 injection. Since NK activity in lipopolysaccharide (LPS)-nonresponder mice could be augmented by MY-1, the possibility that LPS contaminated the MY-1 augmented-NK cell activity was excluded. MY-1 digested preliminarily with DNase lost its NK-inducing activity suggesting that the DNA entity of MY-1 was essential for the activity. When mice were pretreated with anti-asialo-GMl or carrageenan, MY-1 could not render the peritoneal cells cytotoxic. Antitumor activity of MY-1 was also abolished if the animals were pretreated with asialo GM1 antiserum or carrageenan suggesting that the activity can be ascribed mainly to activated NK cells [16].