Abstract

BackgroundmRNA electroporation of dendritic cells (DCs) facilitates processing and presentation of multiple peptides derived from whole antigen, tailored to different HLA molecules. Clinical responses to electroporated moDC vaccines, however, have been suboptimal. Human Langerhans-type DCs (LCs) are the most potent conventional DC subtype for inducing CD8+ cytotoxic T lymphocytes (CTLs) in vitro. We recently demonstrated that Wilms’ tumor 1 (WT1) mRNA-electroporated LCs are superior to moDCs as stimulators of tumor antigen-specific CD8+ CTLs, even though they are comparable stimulators of allogeneic T cell proliferative responses. A detailed comparative evaluation of the effects of mRNA electroporation on LCs versus moDCs, however, is needed.MethodsImmature and partially-matured human moDCs and LCs electroporated with mRNA were compared for transfection efficiency, phenotypic changes, viability, retention of transgene expression after cryopreservation, and immunogenicity. Student t test was used for each pairwise comparison. One-way analysis of variance was used for multiple group comparisons.ResultsTransfection efficiency after electroporation with enhanced green fluorescent protein (eGFP) mRNA was higher for immature than for partially-matured moDCs. In contrast, transfection efficiency was higher for partially-matured than for immature LCs, with the additional benefit that electroporation itself increased maturation and activation of CD83+HLA-DRbright LCs but not moDCs. Electroporation did not impair final maturation and activation of either DC subtype, after which both mRNA-electroporated LCs and moDCs were functionally similar in stimulating allogeneic T cell proliferation, a standard assay of DC immunogenicity.ConclusionsThese findings support mRNA electroporation of DCs, and in particular LCs, as an effective non-viral method to stimulate specific, potent CD8+ CTL responses. The differences between LCs and moDCs regarding this form of antigen-loading have important implications for DC-based immunotherapies.

Highlights

  • Effective therapeutic cancer vaccination requires the optimization of tumor antigen presentation by antigenpresenting cells (APCs) to induce strong antigen-specific T cell responses, especially CD8+ cytolytic T lymphocytes (CTLs) with a sufficiently broad repertoire and immunologic memory [1]

  • The transfection efficiency of mRNA electroporation varies with the maturation status of Monocyte-derived dendritic cells (DCs) (moDC) and Langerhans-type DC (LC) Immature and 24-hour, partially-matured moDCs and LCs were electroporated with enhanced green fluorescent protein (eGFP) mRNA

  • Optimal electroporation parameters for immature moDCs and partially-matured LCs were determined by varying set voltage, number of electroporation pulses, and amount of mRNA to maximize transfection efficiency while minimizing cell loss

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Summary

Introduction

Effective therapeutic cancer vaccination requires the optimization of tumor antigen presentation by antigenpresenting cells (APCs) to induce strong antigen-specific T cell responses, especially CD8+ cytolytic T lymphocytes (CTLs) with a sufficiently broad repertoire and immunologic memory [1]. Electroporation of DCs with mRNA encoding specific tumor-associated antigens is an effective non-viral method to stimulate T cell responses in vitro and in vivo [4,5,6,7,8,9,10,11]. This method of antigen loading, which facilitates processing and presentation of multiple class I and II MHC-restricted epitopes from the translated protein [12], is more efficient than peptide pulsing and less problematic than retroviral transgenes, which carry the risk of genome integration [13]. A detailed comparative evaluation of the effects of mRNA electroporation on LCs versus moDCs, is needed

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