Three-day dendritic cells for vaccine development: Antigen uptake, processing and presentation

Abstract

BackgroundAntigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.MethodsIn this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.ResultsAlthough they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivt)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivtRNA.ConclusionsThis new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivtRNA as sources of specific antigen.

Dendritic Cell–Based Immunotherapy: State of the Art and Beyond

“…The extensive culture period (7-9 days) of ex vivogenerated DCs and compounds required to differentiate them into DCs might negatively affect DC function, for example, by reducing proinflammatory cytokine production. Therefore, shorter culture protocols have been developed (11). Furthermore, natural DCs do not require extensive culture, and recently, it has been proven feasible to obtain more than 10 million pDCs and even higher numbers of BDCA-1 þ mDCs after a single leukapheresis despite their low frequency in blood (12)(13)(14).…”

Optimization of a Density Gradient Centrifugation Protocol for Isolation of Peripheral Blood Mononuclear Cells

“…Given their involvement in virtually any immune process, PBMC isolation is a key step in many immunological experiments. Once isolated, PBMCs can be used for a wide range of downstream applications such as studying autoimmune diseases [1][2][3], cancer research [4][5][6][7][8][9][10], developing new vaccines [11][12][13] and immunotherapies [6][7][8][9][10], drug discovery and testing [14][15][16] etc.…”

Fast dendritic cells matured with Poly (I:C) may acquire tolerogenic properties

“…Although the majority of studies concluded that fDCs are as good as their conventional counterparts [12,14e18], there is no strong evidence confirming that the fast DC protocol is efficient enough to undergo clinical trials. Namely, it has been reported that fDCs could have a weaker antigen uptake [15,16], antigen presentation and costimulation [14,16] and higher IL-10 production on stimulation with a TLR7/8 agonist [17]. Similarly, we showed previously that DCs generated by the fDC protocol mature poorly in the presence of proinflammatory cocktail, unlike their conventional counterparts [18].…”

“…The majority of studies comparing the conventional and fast protocol suggested that fast DCs (fDCs) are as good as their conventional counterparts [12,14e17]. However, different data showing that fDCs have a weaker antigen uptake [15,16], weaker antigen presentation and co-stimulation [14,16] and a higher IL-10 production on stimulation [17] point to their weaker potential in a clinical setting for antitumor therapy. In line with this, our previous data [18] suggest that the conventional protocol for the generation of DCs is more reliable than the fast DC protocol because the monocytes of all examined donors cultivated by standard methods had good potential to differentiate into mature, IL12eproducing DCs.…”