Inhibitory effect of RNA pool complexity on stimulatory capacity of RNA-pulsed dendritic cells.

Tumor cells that show downregulation of their tumor-associated antigens (TAAs) may be able to escape immune-mediated elimination. Therefore, efficient vaccine strategies attempt to target multiple TAAs simultaneously. This is easily achieved in dendritic cell (DC)-based vaccines by introducing antigens in the form of RNA. Although insufficient message may hinder adequate expression of individual TAAs when using total-tumor RNA, high amounts of individual RNAs as pools yield DCs presenting high numbers of specific peptide-major histocompatibility complex ligands with epitopes derived from different TAAs. We used the transfer of RNAs encoding the well-defined melanoma TAAs tyrosinase, Melan-A, CDK4mut, gp100, SNRP116mut, and GPNMBmut to characterize DCs at the levels of transfected RNA, expressed protein and peptide-major histocompatibility complex ligand presentation. TAA-encoding RNA was rapidly degraded in the DCs, allowing only a single surge in protein expression shortly after transfection. We compared the functional capacity of DCs transfected with pools of 3 versus 6 RNAs. Whereas functional assays demonstrated a decrease in stimulatory capacity of DCs transfected with a pool of 3 RNAs by only 30% as compared with single RNAs, a 60% loss was seen with 6 RNAs. We conclude that larger RNA pools result in diminished presentation of individual epitopes and suggest that smaller pools of RNA be transfected into separate DC populations which are then pooled to create multiplex vaccines.

Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70.

BACKGROUND: For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4+ T helper 1 cells, which then support the development of CD8+ cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development. METHODS: After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I:C). RESULTS: Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I:C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I:C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I:C) were unable to express proteins following RNA transfer by electroporation. CONCLUSION: Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.

Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal.

Efficient engulfment of the intact cell corpse is a critical end point of apoptosis, required to prevent secondary necrosis and inflammation. The presentation of "eat-me" signals on the dying cell is an important part of this process of recognition and engulfment by professional phagocytes. Here, we present evidence that apoptotic cells secrete chemotactic factor(s) that stimulate the attraction of monocytic cells and primary macrophages. The activation of caspase-3 in the apoptotic cell was found to be required for the release of this chemotactic factor(s). The putative chemoattractant was identified as the phospholipid, lysophosphatidylcholine. Further analysis showed that lysophosphatidylcholine was released from apoptotic cells due to the caspase-3 mediated activation of the calcium-independent phospholipase A(2). These data suggest that in addition to eat-me signals, apoptotic cells display attraction signals to ensure the efficient removal of apoptotic cells and prevent postapoptotic necrosis.