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.
Subject(s)
Antigens, Neoplasm/genetics , Dendritic Cells/immunology , Genetic Therapy/methods , Immunotherapy, Adoptive/methods , Interleukin-12/genetics , Neoplasm Proteins/genetics , RNA, Messenger/genetics , Cancer Vaccines , Cells, Cultured , Cytotoxicity Tests, Immunologic , Cytotoxicity, Immunologic , Epitopes/immunology , Humans , Immunophenotyping , Interleukin-12/immunology , Lymphocyte Activation , MART-1 Antigen , RNA, Messenger/administration & dosage , T-Lymphocytes, Cytotoxic/immunology , Transfection/methodsABSTRACT
Replicative senescence of T cells is correlated with erosion of telomere ends. Telomerase plays a key role in maintaining telomere length. Therefore, it is thought that telomerase regulates the life span of T cells. To test this hypothesis, we have over-expressed human telomerase reverse transcriptase in human CD8(+) T cells. Ectopic expression of human telomerase reverse transcriptase led to immortalization of these T cells, without altering the phenotype and without loss of specificity or functionality. As the T cells remained dependent on cytokines and Ag stimulation for their in vitro expansion, we conclude that immortalization was achieved without malignant transformation.
