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Attenuated Expression of A20 Markedly Increases the Efficacy of Double-Stranded RNA-Activated Dendritic Cells As an Anti-Cancer Vaccine¹

Karine Breckpot^2,3,*, Cindy Aerts-Toegaert^2,*, Carlo Heirman^*, Uschi Peeters^*, Rudi Beyaert^,, Joeri L. Aerts^* and Kris Thielemans^*

^* Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the Vrije Universiteit Brussel, Brussels, Belgium; Unit of Molecular Signal Transduction in Inflammation, Department for Molecular Biomedical Research, Vlaams Instituut voor Biotechnologie, Ghent, Belgium; and Department for Molecular Biology, Ghent University, Ghent, Belgium

A20 is a zinc finger protein with ubiquitin-modifying activity. A20 has been described as negatively regulating signaling induced by the TNF receptor and TLR family in a number of cell types, including mouse bone marrow-derived dendritic cells (DCs). However, the expression and effect of A20 in activated human monocyte-derived DCs have not been previously evaluated. We report that DCs activated with the TLR3 ligand poly(I:C) up-regulate A20. Down-regulating A20 demonstrated its role in the functional activation of DCs. A20 down-regulated DCs showed higher activation of the transcription factors NF-B and activator protein-1, which resulted in increased and sustained production of IL-6, IL-10, and IL-12p70. We additionally silenced the immunosuppressive cytokine IL-10 and demonstrated that IL-10 inhibits T cell proliferation. We further demonstrated that A20 down-regulated DCs skew naive CD4⁺ T cells toward IFN- producing Th1 cells, a process which is dependent on IL-12p70 and which is unaffected by IL-10. Furthermore, A20 and/or IL-10 down-regulated DCs had an enhanced capacity to prime Melan-A/MART-1 specific CD8⁺ T cells. Finally, we demonstrated that potent T cell stimulatory DCs are generated by the simultaneous delivery of poly(I:C₁₂U), A20, or A20/IL-10 small interfering RNA and Ag-encoding mRNA, introducing a one step approach to improve DC-based vaccines. Together these findings demonstrate that A20 negatively regulates NF-B and activator protein-1 in DCs and that down-regulation of A20 results in DCs with enhanced T cell stimulatory capacity.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by grants to K.T. from the Belgian Science Policy’s Interuniversity Attraction Poles Program, from the Foundation against Cancer, from an Integrated Project and Network of Excellence sponsored by the European Union, and from the Fund of Scientific Research Flanders (FWO-Vlaanderen). C.A.-T., J.L.A., and K.B. are funded by the FWO-Vlaanderen.

² K.B. and C.A.-T. contributed equally to this work.

³ Address correspondence and reprint requests to: Dr. Karine Breckpot, Laboratory of Molecular and Cellular Therapy, Department of Physiology-Immunology, Medical School of the ‘Vrije Universiteit Brussel’, Laarbeeklaan 103/E, 1090 Brussels, Belgium. E-mail address: karine.breckpot{at}vub.ac.be

⁴ Abbreviations used in this paper: DC, dendritic cell; AP-1, activator protein-1; GUS, β-glucuronidase; siRNA, small interfering RNA; TAA, tumor-associated antigen; Treg, regulatory T cell.