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Tumor Apoptotic Bodies Inhibit CTL Responses and Antitumor Immunity via Membrane-Bound Transforming Growth Factor-β1 Inducing CD8⁺ T-Cell Anergy and CD4⁺ Tr1 Cell Responses

¹ Research Unit, Saskatchewan Cancer Agency, Departments of Oncology, ² Pathology, and ³ Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Requests for reprints: Jim Xiang, Research Unit, Saskatchewan Cancer Agency, Department of Oncology, University of Saskatchewan, 20 Campus Drive, Saskatoon, Saskatchewan S7N 4H4, Canada. Phone: 306-6552917; Fax: 306-6552635; E-mail: jim.xiang{at}saskcancer.ca.

Key Words: Apoptosis • apoptotic bodies • TGF-β1 • antitumor immunity

Tumor cell apoptosis induced by radiation therapy results in apoptotic tumor cells and apparition of membrane blebs termed apoptotic bodies (APB). The immune responses induced by apoptotic tumor cells have been extensively studied. However, the role of APB in modulation of tumor immune responses is elusive. In this study, we induced apoptosis in 90% ovabumin-expressing EG7 tumor cells by in vitro irradiation (9,000 rad) of tumor cells with a subsequent cell culture for 9 hours. APB purified from irradiation-induced apoptotic EG7 cell culture supernatant by differential ultracentrifugation were vesicles with 50 to 90 nm in diameter and expressed apoptosis-specific Annexin V, 14-3-3, and Histone H3. We then investigated its potential modulation in DC_OVA–induced T-cell responses and antitumor immunity. We found that EG7-derived APB were tolerogenic and capable of suppressing DC_OVA-stimulated CD8⁺ CTL responses and antitumor immunity via its induction of CD8⁺ T-cell anergy and type 1 regulatory CD4⁺ T-cell responses. Analysis of apoptotic tumor cells and APB revealed the expression of membrane-bound transforming growth factor (TGF)-β1 associated with irradiation-induced apoptosis formation, which is a result from activation of transcriptional factor NF-AT specific for TGF-β1 promoters. Our data further elucidate that it is the membrane-bound TGF-β1 expression on APB that contributes to its in vitro antiproliferative effect as shown by using neutralizing TGF-β1–specific antibody. Administration of anti–TGF-β1 antibody in vivo also blocked APB-mediated immune suppression of CD8⁺ CTL responses and antitumor immunity. Therefore, our study may have great impact in designing a combined radiation therapy with immunotherapy of cancer. [Cancer Res 2009;69(19):7756–66]