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International Immunology Advance Access originally published online on August 26, 2009
International Immunology 2009 21(10):1145-1150; doi:10.1093/intimm/dxp080
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© The Japanese Society for Immunology. 2009. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Time-lapse observation of cellular function with fluorescent probe reveals novel CTL–target cell interactions

Michio Tomura1, Yoshiko Saito Mori1, Ryousuke Watanabe1, Masato Tanaka2, Atsushi Miyawaki3 and Osami Kanagawa1

1 Laboratory for Autoimmune Regulation
2 Laboratory for Innate Cellular Immunity, Research Center for Allergy and Immunology, RIKEN, Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama City, Kanagawa 230-0045, Japan
3 Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako City, Saitama 351-0198, Japan

Correspondence to: O. Kanagawa; E-mail: kanagawa{at}rcai.riken.jp

Fluorescent protein that detects caspase-3 activation was used for the time-lapse observation of CTL–target cell interaction. In the target cells transfected with SCAT3.1 (caspase-3-sensitive fusion protein) complementary DNA, caspase-3 activation can be detected significantly earlier than the commonly used annexin-V binding that detects membrane change in apoptotic cells. Moreover, during the cytolytic interaction between OE4 CTL and W3 tumor target cells, detachment of CTL from the target cells occurred prior to caspase-3 activation and death of the target cells, indicating very early sensing of apoptotic target cells by CTL. This early detachment of OE4 CTL from W3 target cells was inhibited by the expression of CD80 co-stimulatory molecule on the target cells. Taken together, time-lapse observation of cellular interaction with functional probe, SCAT3.1 provides new kinetic information and demonstrates that co-stimulatory molecules regulate the kinetics of CTL–target cell interaction.

Keywords: apoptosis, caspase-3, cell death, FRET, SCAT3.1

Transmitting editor: T. Watanabe

Received 26 January 2009, accepted 24 July 2009.


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