RNA Transfer by Electroporation into Mature Dendritic Cells Leading to Reactivation of Effector-Memory Cytotoxic T Lymphocytes: A Quantitative Analysis

Abstract

Previous studies have analyzed transfer of RNA-encoded tumor-associated antigens (TAAs) into immature dendritic cells (DCs) because of their exceptional ability to internalize antigens. Concerns have been raised regarding the use of immature DCs in clinical studies because of their capacity to tolerize T cells. Therefore, we focused on optimizing RNA transfer into mature DCs using the method of electroporation and obtained high protein expression in 90% of mature DCs. Particular emphasis was placed on quantifying RNA transfer. Reconstitution of peptide-MHC (pMHC) ligands on RNA-pulsed DCs was measured with the help of effector-memory cytotoxic T lymphocytes (CTLs) specific for the melanoma-associated antigens tyrosinase and mutated cyclin-dependent kinase 4. In contrast to single-species RNA, transfer of native tumor-derived RNA or amplified tumor RNA into DCs resulted in very low or no capacity to reactivate these CTLs. A correlation was found between TAA message levels in tumor-derived RNA and pMHC ligand reconstitution on DCs. These results demonstrate that even TAAs with highly immunogenic mutated epitopes are not necessarily transferred when tumor-derived RNA is transfected into the DCs, thereby resulting in a lack of DC capacity to reactivate some preexisting effector-memory CTLs.

High-Level Antigen Expression and Sustained Antigen Presentation in Dendritic Cells Nucleofected with Wild-Type Viral mRNA but Not DNA

“…Prior or concurrent maturation of human monocyte-derived DC with CD40L did not alter nucleofection efficiency with either DNA or mRNA in our studies, similar to results in previous reports (29,34), although in other studies, electroporation of mRNA into mature DC led to a modest increase in transgene expression (22). Larregina et al reported that simultaneous transfection and CD40-mediated maturation of murine bone marrow-derived DC significantly enhanced DNA transfection efficiency (28), suggesting that maturation may have a greater effect on gene expression in the murine system.…”

“…This discrepancy between our results and those of others using electroporation might be explained by the use of different electroporation devices leading to differences in the delivery of electric current to cells. In our studies, the efficiency of nucleofection was such that as little as 5 g mRNA resulted in near-total transfection of DC, yet at least 50 g mRNA can be safely delivered to DC without apparent toxicity (22). This provides the advantage of being able to introduce relatively small amounts of mRNA encoding multiple genes into DC simultaneously, such as different patient-derived viral genes for immunotherapy of HIV infection.…”

“…Immature DC are specialized in antigen uptake, and as such, transfection of DC with either DNA or mRNA is traditionally done at the immature stage of differentiation. However, electroporation of mature human DC with mRNA is reported to be efficient (22,31), and in the murine system, DNA transfection may be enhanced in mature DC (28). To determine the influence of DC maturation on transfection efficiency, we nucleofected human monocyte-derived DC with DNA and mRNA encoding gfp with and without prior treatment of cells with CD40L for 24 h to induce maturation.…”

“…Other comparative studies have shown that human DC expressing mRNAencoded influenza virus matrix protein by electroporation were far superior in their capacity to stimulate M1-specific cytotoxic T cells than DC expressing DNA-encoded antigen (41). The durable antigen presentation in mRNA-nucleofected DC is likely to be a function of sustained protein expression and presentation rather than the persistence of intracellular mRNA, as introduced mRNA is rapidly degraded in cells (13,22).…”

High immunogenic potential of p53 mRNA-transfected dendritic cells in patients with primary breast cancer

“…Subsequent studies have highlighted that DCs electroporated after maturation were at least as efficient in antigen presentation and had even greater potency to invoke immune response than DCs electroporated before maturation [12,45,46]. We further expand on Numerous studies have demonstrated a correlation between antigen persistence in the DC and magnitude of the immune response [47][48][49].…”