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* Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan, Republic of Korea;
Department of Internal Medicine, Inje University Busan Paik Hospital, Busan, Republic of Korea;
Department of Microbiology, Inje University College of Medicine, Busan, Republic of Korea; and
Department of Anatomy and Cancer Immunology, Seoul National University College of Medicine, Seoul, Republic of Korea
B7-H1 is a newly identified member of the B7 family with important regulatory functions in cell-mediated immune responses, and it is expressed in human immune cells and several tumors. We first observed that expression of surface B7-H1 on B cells was increased during the immortalization process by EBV, which is strongly related to both inflammation and tumorigenesis. Cross-linking of B7-H1 on EBV-transformed B cells using anti-B7-H1 Ab (clone 130002) induced reactive oxygen species (ROS) generation, mitochondrial disruption, release of apoptotic proteins from mitochondria, and subsequent apoptosis. Inhibition of caspases and ROS generation recovered B7-H1-mediated apoptosis and proteolytic activities of caspase-8, -9, and -3. We observed that B7-H1 stimulation induced both transcription and translation of fasL. ZB4, an antagonistic anti-fas Ab, and NOK-1, an antagonistic anti-fasL Ab, effectively blocked apoptosis without exerting any influence on ROS generation. N-acetylcysteine (NAC) completely blocked the induction of fasL mRNA and protein. We found that B7-H1 stimulation activated the phosphorylation of JNK and c-jun and down-regulated ERK1/2 and p-Akt. NAC blocked the activation of JNK and down-regulation of ERK, but both z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and ZB4 did not inhibit JNK activation of B7-H1 stimulation. SP600125 blocked fasL induction and apoptosis but did not affect ROS generation after B7-H1 stimulation. Taken together, we concluded that B7-H1-mediated apoptosis on EBV-transformed B cells may be involved in the induction of fasL, which is evoked by ROS generation and JNK activation after cross-linking of B7-H1. These results provide a new concept for understanding reverse signaling through B7-H1 and another mechanism of tumor immunotherapy using anti-B7-H1.
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1 This work was supported by the Science Research Center/Engineering Research Center (SRC/ERC) program of the Ministry of Science and Technology (MOST)/Korea Science and Engineering Foundation (KOSEF) (Grant R11-2005-017-02002-0), the Medical Reserve Corp (MRC) program of KOSEF funded by the Korean government (MOST, Grant R13-2007-023-00000-0), the Korea Research Foundation Grant funded by the Korean government (Ministry of Education and Human Resources Development (MOEHRD), KRF-2005-042-E00055), and Inje University Research Grant.
2 Y.S.K. and G.B.P. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Dae Young Hur, Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, 633-165 Kaekum-2-dong, Jin-gu, Busan 614-735, Republic of Korea. E-mail address: dyhur{at}inje.ac.kr
4 Abbreviations used in this paper: PD-1, programmed death-1; AIF, apoptosis-inducing factor; DCF, dichlorofluorescein; DCFH-DA, 2',7'-dichlorodihydrofluorescein diacetate; 
, mitochondrial membrane potential; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; endoG, endonuclease G; MFI, mean fluorescence intensity; NAC, N-acetylcysteine; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; ROS, reactive oxygen species; z-DEVD-fmk, N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone; z-IETD-fmk, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone; z-VAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone.
5 The online version of this article contains supplemental material.
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