Polybrene and interleukin-4: two opposing factors for retroviral transduction of bone-marrow-derived dendritic cells.
BACKGROUND
Gene transfer using retroviral transduction offers the advantage of long-term transgene expression in developing strategies that use dendritic cells (DCs) for immunotherapy. The goal of this study was to infect DCs in an immature state in order to take advantage of their proliferating and tolerogenic potential.


METHODS
Immature DCs were generated from murine bone marrow (BM) using either GM-CSF alone or GM-CSF plus IL-4. The cells were transduced directly with retroviral supernatants or by co-culture with the GP + E-86 retroviral packaging cell line in the presence of two different cationic polymers: polybrene and protamine sulfate. Phenotypic and functional characterization of the transduced cells were then performed.


RESULTS
Our results show a low efficiency of retroviral infection of DCs in the presence of polybrene. This cationic polymer was found to be directly cytotoxic to murine DCs and thus favored the growth of contaminating macrophages. This effect was not observed using protamine sulfate. Furthermore, stimulation by IL-4 early in the culture increased DC differentiation, proliferation and transduction. However, we found that DCs generated in GM-CSF plus IL-4 presented a more mature phenotype with an enhanced allogeneic stimulating activity. Finally, we showed that DCs themselves down-regulated transgene expression in the co-cultured packaging cell line in a promoter-dependent manner.


CONCLUSIONS
We have defined optimal conditions to generate and transduce murine BM-derived DCs. This included: the use of protamine sulfate during exposure to retroviral infectious supernatant and the addition of IL-4 at an early stage of the culture. Nevertheless, this cytokine also induced DC maturation. These findings have potential implications in experimental gene therapy.
Fresnay
Stéphanie
S
Etablissement Français du Sang de Bourgogne Franche-Comté, INSERM E0119, UPRES MEN2284, Université de Franche-Comté, F-25020 Besançon cedex, France.
Chalmers
David E
DE
Ferrand
Christophe
C
Colombain
Christine
C
Newton
Isobel
I
Yerly-Motta
Véronique
V
Lienard
Agnès
A
Darodes de Tailly
Patrick
P
Hervé
Patrick
P
Tiberghien
Pierre
P
Saas
Philippe
P
D000818:Animals; D001854:Bone Marrow Cells / Q000821:virology*; D002455:Cell Division / Q000187:drug effects; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000821:virology*; D006583:Hexadimethrine Bromide / Q000494:pharmacology*; D015847:Interleukin-4 / Q000494:pharmacology*; D007959:Lymphocyte Culture Test, Mixed; D051379:Mice; D008815:Mice, Inbred Strains; D012190:Retroviridae / Q000235:genetics*; D014161:Transduction, Genetic
D015847:Interleukin-4; D006583:Hexadimethrine Bromide