Engineering lentiviral vectors for modulation of dendritic cell apoptotic pathways

James Cm Wang,Gregory A Dekaban,Bryan Cy Au,Jeffrey A Medin,Tânia C Felizardo,Daniel H Fowler

doi:10.1186/1743-422x-10-240

Abstract

BackgroundDendritic cells (DCs) are promising mediators of anti-tumor immune responses due to their potent antigen-presentation capacity. Unfortunately, cancer cells can often disarm differentiated DCs by rendering them incapable of maturation or by promoting their apoptosis. DC vaccine regimens attempt to generate functional DCs and preload them with Tumor-Associated Antigens (TAAs) to target various malignancies. Despite these efforts, the efficacy of DC vaccines in clinical trials is still rather disappointing to date. In addition to undergoing cancer-induced apoptosis, it is well established that DCs are intrinsically short-lived cell types. It is likely that a significant portion of infused DCs undergo apoptosis prior to locating and activating naïve TAA-reactive T cells.MethodsIn our current study, we constructed and investigated novel bicistronic lentivectors (LVs) encoding the cDNA for the xeno-TAA, rat HER-2/neu (rHER-2), along with five candidate mouse DC survival factors (c-FLIPS, c-FLIPL, Bcl-XL, M11L, and AKT-1) that operate in both the extrinsic and intrinsic cycles of apoptosis. The murine DC cell line, DC2.4 was transduced separately with each novel LV construct. Infected cells were enriched via flow cytometric methods based on rHER-2 expression. Transduced DC2.4 cell lines were then exposed to Fetal Calf Serum (FCS) withdrawal and to specific pharmacological apoptosis-inducing agents. DC2.4 cell death was assayed based on Annexin V and PI double-positive staining via flow cytometry. The phenotype and function of transduced DC2.4 cells and primary bone marrow-derived DCs were then assessed via expression and secretion of DC markers and cytokines, respectively.ResultsDC2.4 cells transduced with LVs encoding cDNAs for c-FLIPS, c-FLIPL, Bcl-XL, and M11L were protected from apoptosis when exposed to low FCS-containing culture media. When treated with an anti-CD95 antibody, only DC2.4 cells transduced with LVs encoding c-FLIPS and c-FLIPL were protected from apoptosis. In contrast, only DC2.4 cells transduced with LVs encoding Bcl-XL and M11L were protected from effects of staurosporine (STS) treatment. Also, LV-modified DCs maintained their original phenotype and function.ConclusionsWe present evidence that by employing novel recombinant bicistronic LVs we can simultaneously load DCs with a relevant TAA and block apoptosis; thereby confirming the usage of such LVs in the modulation of DC lifespan and function.

Highlights

Dendritic cells (DCs) are promising mediators of anti-tumor immune responses due to their potent antigen-presentation capacity
In the context of DC vaccines, LVs permit high Tumor-Associated Antigen (TAA) gene transfer efficiencies and expression [17], the generation of a broad repertoire of presented peptides, the induction of more stable peptide-MHC complexes, and the ability to co-express multiple genes such as TAAs and survival factors [18]. Along these lines we previously demonstrated that LVs encoding the sequence for the xeno-TAA HER-2 efficiently transduced murine bone marrow-derived DCs [19]
We selected the potent oncogene AKT-1 as a positive control for our anti-apoptosis experiments as it has been shown that, while isoforms AKT-1 and AKT-2 are present in hematopoietic cells, AKT-1 is the predominant isoform found in DCs [20]

Summary

Introduction

Dendritic cells (DCs) are promising mediators of anti-tumor immune responses due to their potent antigen-presentation capacity. DC vaccine regimens attempt to generate functional DCs and preload them with Tumor-Associated Antigens (TAAs) to target various malignancies. Despite these efforts, the efficacy of DC vaccines in clinical trials is still rather disappointing to date. Dendritic cells (DCs) function to initiate and orchestrate the adaptive immune response to pathogens as well as to suppress self immune responses in their absence To perform these roles, DCs are regulated by both external and internal signaling factors. External factors include IL-10, which has been shown to lower DC survival by context of immunotherapy, short-lived, ex vivo-modified DC vaccines likely undergo apoptosis prior to encountering and optimally-activating TAA-reactive T cell clones. The two main isoforms of cFLIP, the long and short forms, are capable of intervening at different checkpoints of procaspase-8 processing and both may play a role in extending DC longevity [9,11]

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