Fresnay Stéphanie, Chalmers David E, Ferrand Christophe, Colombain Christine, Newton Isobel, Yerly-Motta Véronique, Lienard Agnès, Darodes de Tailly Patrick, Hervé Patrick, Tiberghien Pierre, Saas Philippe
Etablissement Français du Sang de Bourgogne Franche-Comté, INSERM E0119, UPRES MEN2284, Université de Franche-Comté, F-25020 Besançon cedex, France.
J Gene Med. 2002 Nov-Dec;4(6):601-12. doi: 10.1002/jgm.311.
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.
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.
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.
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.
在利用树突状细胞（DCs）进行免疫治疗的策略中，使用逆转录病毒转导进行基因转移具有长期转基因表达的优势。本研究的目的是感染未成熟状态的DCs，以利用其增殖和致耐受性潜能。
使用单独的GM-CSF或GM-CSF加IL-4从鼠骨髓（BM）生成未成熟DCs。细胞直接用逆转录病毒上清液转导，或在两种不同阳离子聚合物（聚凝胺和硫酸鱼精蛋白）存在下与GP + E-86逆转录病毒包装细胞系共培养。然后对转导细胞进行表型和功能表征。
我们的结果表明，在聚凝胺存在下，DCs的逆转录病毒感染效率较低。发现这种阳离子聚合物对鼠DCs具有直接细胞毒性，因此有利于污染巨噬细胞的生长。使用硫酸鱼精蛋白未观察到这种效应。此外，培养早期IL-4刺激增加了DC分化、增殖和转导。然而，我们发现GM-CSF加IL-4生成的DCs呈现出更成熟的表型，同种异体刺激活性增强。最后，我们表明DCs自身以启动子依赖的方式下调共培养包装细胞系中的转基因表达。
我们已经确定了生成和转导鼠BM来源DCs的最佳条件。这包括：在暴露于逆转录病毒感染性上清液期间使用硫酸鱼精蛋白，以及在培养早期添加IL-4。然而，这种细胞因子也诱导DC成熟。这些发现对实验性基因治疗具有潜在意义。
