Functional Manipulation of Dendritic Cells by Photoswitchable Generation of Intracellular Reactive Oxygen Species Taek-Chin Cheong, †,⧫,■ Eon Pil Shin, ⊥,⧫ Eun-Kyung Kwon, † Ji-Hye Choi, † Kang-Kyun Wang, ⊥ Prashant Sharma, †,‡ Kyong Hoon Choi, ⊥ Jin-Muk Lim, # Hong-Gee Kim, # Keunhee Oh, ‡ Ju-Hong Jeon, §,‡ Insuk So, §,‡ In-Gyu Kim, ∥,‡ Myung-Sik Choi, † Young Keun Kim, ▽ Seung-Yong Seong, †,‡,⬡ Yong-Rok Kim,* ,⊥ and Nam-Hyuk Cho* ,†,‡,⬡,○ † Department of Microbiology and Immunology, ‡ Department of Biomedical Science, § Department of Physiology, ∥ Department of Biochemistry, Seoul National University College of Medicine, Seoul, Republic of Korea ⊥ Department of Chemistry, Yonsei University, Seoul, Republic of Korea # Biomedical Knowledge Engineering Laboratory, Dental Research Institute and Institute of Human-Environment Interface Biology, Seoul National University, Seoul, Republic of Korea ▽ Department of Materials Science and Engineering, Korea University, Seoul, Republic of Korea ⬡ Wide River Institute of Immunology and ○ Institute of Endemic Disease, Seoul National University Medical Research Center and Bundang Hospital, Seoul, Republic of Korea * S Supporting Information ABSTRACT: Reactive oxygen species (ROS) play an important role in cellular signaling as second messengers. However, studying the role of ROS in physiological redox signaling has been hampered by technical difficulties in controlling their generation within cells. Here, we utilize two inert components, a photosensitizer and light, to finely manipulate the generation of intracellular ROS and examine their specific role in activating dendritic cells (DCs). Photoswitchable generation of intracellular ROS rapidly induced cytosolic mobilization of Ca 2+ , differential activation of mitogen-activated protein kinases, and nuclear translocation of NF-κB. Moreover, a transient intracellular ROS surge could activate immature DCs to mature and potently enhance migration in vitro and in vivo. Finally, we observed that intracellular ROS- stimulated DCs enhanced antigen specific T-cell responses in vitro and in vivo, which led to delayed tumor growth and prolonged survival of tumor-bearing mice when immunized with a specific tumor antigen. Therefore, a transient intracellular ROS surge alone, if properly manipulated, can cause immature DCs to differentiate into a motile state and mature forms that are sufficient to initiate adaptive T cell responses in vivo. R eactive oxygen species (ROS), which include highly reactive free oxygen radicals (e.g., O 2 • and OH − ) and nonradical oxidants (e.g., H 2 O 2 ), are generated during mitochondrial respiration and cellular responses to diverse stimulation such as growth factors and pathogen infection. 1,2 Although excess ROS causes oxidative stress resulting in macromolecular damage and various disease states including cancer and aging, increasing evidence indicates that ROS also serve as critical signaling molecules in cell proliferation, differentiation, and survival. 1−3 In particular, ROS is directly involved in the activation of various cellular signaling pathways, 2 such as MAP kinase 4 and tyrosine kinase 5 signaling cascades via oxidation of redox-sensitive cysteine residues of target proteins. Transcription factors, including AP-1 and NF- κB, are also subject to redox regulation and lead to many biological changes, ranging from responding to growth factors to inflammatory responses. 3 Thus, it is now widely accepted that ROS function as important second messengers of intracellular signaling pathways. Signaling ROS are generated at the cell surface or within intracellular compartments by multiple NADPH oxidases in response to diverse stimuli and then enter the cytoplasm. 1,6 Recent evidence suggests that ROS might preferentially enter the cell through speci fic plasma membrane aquaporin channels. 7 Additionally, generation of mitochondrial ROS has been shown to be tightly regulated and participates in physiological cell signaling associated with various stresses. 8 Within the cytoplasm, intracellular ROS potentially modifies cysteine residues of over 500 proteins, as revealed by large scale Received: August 14, 2014 Accepted: December 2, 2014 Published: December 2, 2014 Articles pubs.acs.org/acschemicalbiology © 2014 American Chemical Society 757 dx.doi.org/10.1021/cb5009124 | ACS Chem. Biol. 2015, 10, 757−765