Generation of an Optimized Polyvalent Monocyte-Derived Dendritic Cell Vaccine by Transfecting Defined RNAs after Rather Than before Maturation

Abstract

Transfection with RNA is an attractive method of Ag delivery to dendritic cells (DCs), but has not yet been standardized. We describe in this study the methods to efficiently generate an optimized mature monocyte-derived DC vaccine at clinical scale based on the electroporation of several RNAs either into immature DC followed by maturation or, alternatively, directly into mature DCs, which has not been possible so far with such high efficiency. Electroporation of DCs resulted in high yield, high transfection efficiency (>90%), and high migration capacity. Intracellular staining allowed the study of the expression kinetics of Ags encoded by the transfected RNAs (MelanA, MAGE-3, and survivin) and a validation of the vaccine (≥90% transfection efficiency). Expression of all three Ags peaked 3–4 h after electroporation in DC transfected either before or after maturation, but decreased differently. The DC vaccine can also be cryopreserved and nevertheless retains its viability, stimulatory capacity as well as migratory activity. In addition, we uncover that DC transfected after rather than before maturation appear to be preferable vaccines not only from a production point of view but also because they appear to be immunologically superior for CTL induction in sharp contrast to common belief. DCs transfected after maturation not only more effectively generate and present the Mage-3.A1 and MelanA.A2.1 epitopes to T cell clones, but they even are superior in priming to the standard proteasome-dependent MelanA.A2.1 wild-type prototype tumor epitope, both in terms of T cell expansion and effector function on a per cell basis.

Dendritic cell preactivation impairs MHC class II presentation of vaccines and endogenous viral antigens

“…In the latter case, the TLR ligand may reach DCs that have not yet encountered the antigen, inducing their maturation and thus preventing their participation in antigen presentation. Finally, it is important to note that immunotherapeutic approaches (18) consisting of inoculation of DCs that are first induced to mature in vitro, and then transfected with nucleic acids encoding tumor antigens (43), might result in DCs presenting the antigens via MHC I but not via MHC II (44), although evidence to the contrary has been reported as well (45,46). Lack of induction of helper T cells might lead to poor induction of protective cytotoxic T lymphocyte immune responses (47) or even induce tolerance (48), so caution must be exerted when employing this strategy.…”

Efficient large volume electroporation of dendritic cells through micrometer scale manipulation of flow in a disposable polymer chip

“…Other groups use various pulse lengths depending on their setup, but there seems to be consensus on pulse lengths around 0.25-2 ms being optimal when using square waveforms. (Hosoi et al 2008;Met et al 2008;Saeboe-Larssen et al 2002;Schaft et al 2005) We also found that two of those pulses cause a high degree of cell death probably due to irreversible electroporation of the cell membrane.…”

“…Therefore it is not acceptable to expose individual cells to different field strengths. Both exponentially decaying (Ponsaerts et al 2002;Schuurhuis et al 2009;Tuyaerts et al 2003) and square (Hosoi et al 2008;Met et al 2008;Saeboe-Larssen et al 2002;Schaft et al 2005) waveforms are commonly used for electroporation. Square waveforms have the advantage of being well-defined and short, ensuring that the vast majority of the cells are exposed to the same electric conditions.…”

“…As the flow approaches the first mesh (slice c, distance 250 μm) the parabolic flow profile disappears. The flow through the openings in the mesh is evenly distributed between all openings (slice d) and the flow velocity in the electroporation zone between the meshes is almost uniform across the width of the channel (slice e) Met et al 2008;Saeboe-Larssen et al 2002;Schaft et al 2005) (Markovic et al 2006) Therefore we chose to test voltages that in our system would produce corresponding field strengths. The optimal voltage resulting in the highest yield was found to be 52 V corresponding to an electric field of 1.3 kV/cm (data not shown) This value is in good agreement with other reports where similar pulse lengths are used.…”

Generation of CD8+T cells expressing two additional T-cell receptors (TETARs) for personalised melanoma therapy

“…Peripheral blood mononuclear cells (PBMC) were purified by density centrifugation as described before. 31 PBMC were incubated for 1 h at standard cell culture conditions (i.e., 37 C; 95% (v/v) humidity; 5% (v/v) CO 2 ) in DC medium, consisting of RPMI 1640 (Lonza, Order Nr. BE12-167F), 1% plasma from leukapheresis products (heat-inactivated, 30 minutes, 56 C), 2 mM L-glutamine (Lonza, Order Nr.…”