1. ¹Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands
2. ²Laboratory of Experimental Hematology, Faculty of Medicine, University of Antwerp, Antwerp University Hospital, Antwerp, Belgium

Correspondence: Dr HJ Bontkes, Department of Pathology, Vrije Universiteit Medical Center, CCA room 2.22, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. E-mail: hj.bontkes@vumc.nl

Received 19 June 2006; Revised 17 August 2006; Accepted 4 September 2006; Published online 12 October 2006.

Abstract

Dendritic cells (DC) transfected with messenger RNA (mRNA) encoding tumor-associated antigens (TAA) are able to induce potent tumor-specific T-cell responses directed to a broad spectrum of tumor-associated epitopes. The in vitro generation of DC possessing all the features crucial for the induction of type 1 immune responses, such as mature state, migratory potential and interleukin-12 (IL-12p70) production is complicated. Particularly migratory potential is inversely correlated with IL-12p70 production after maturation with prostaglandin E2 (PGE2), which is included in maturation cocktails currently used in most vaccination trials. Here, we show that transfection of PGE2 matured DC with a single mRNA strain encoding for ubiquitin followed by a TAA which was linked to IL-12 by a self-cleaving 2A sequence, produced biological active IL-12p70 and were able to present the transfected TAA up to 72 h after transfection. Furthermore, use of the anti-reverse cap analog for in vitro transcription of the IL-12 mRNA enabled constitutive IL-12p70 production for up to 5 days. These transfected mature DC migrated efficiently towards lymph node derived chemokines. DCs constitutively expressing IL-12p70, generate TAA-specific cytotoxic T cells with an high functional avidity, independent of CD4⁺ T-cell help.