Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
Vaccine. 2012 Jul 20;30(34):5118-31. doi: 10.1016/j.vaccine.2012.05.063. Epub 2012 Jun 9.
Integrase-defective lentiviral vectors (ID-LVs) show several hallmarks of conventional lentiviral vectors such as absence of cytotoxic effects and long-term expression in non-replicating target cells. The integration rate of ID-LVs into the genome of target cells is dramatically reduced, which enhances safety and opens perspectives for their use in vaccine development. ID-LVs have been shown to be effective vaccines in mouse models, but functional studies with human cells in vitro and in vivo are lacking. Here, we evaluated whether ID-LVs expressing combinations of cytokines (GM-CSF/IL-4 or GM-CSF/IFN-α) used to transduce human monocytes would result in functional "induced dendritic cells" (iDCs). Overnight transduction of monocytes with high titer ID-LVs generated highly viable (14 days) and immunophenotypically stable iDCs expressing GM-CSF/IL-4 ("SmartDCs") or GM-CSF/IFN-α ("SmyleDCs"). SmartDCs and SmyleDCs maintained in vitro continuously secreted the transgenic cytokines and showed up-regulation of several endogenously produced inflammatory cytokines (IFN-γ, IL-2, -5, -6, and -8). Both iDC types potently stimulated T cells in mixed lymphocyte reactions at levels comparable to conventional DCs (maintained with exogenous cytokines). A single in vitro stimulation of CD8(+) T cells with autologous SmartDCs or SmyleDCs pulsed with peptide pools of pp65 (a human cytomegalovirus antigen) resulted in high expansion of central memory and effector memory CTLs reactive against different pp65 epitopes. We further evaluated the effects of SmartDCs and SmyleDCs to expand anti-pp65 CTLs in vivo using immune deficient NOD/Rag1((-/-))/IL-2rγ((-/-)) (NRG) mice. NRG mice immunized subcutaneously with SmartDCs or SmyleDCs co-expressing the full-length pp65 were subsequently infused with autologous CD8(+) T cells. Both types of iDCs effectively stimulated human CTLs reactive against different pp65 antigenic determinants in vivo. Due to the simplicity of generation, robust viability and combined capacity to stimulate homeostatic, antigenic and multivalent responses, iDCs are promising vaccines to be explored in immunization of lymphopenic patients in the post-transplantation setting.
整合缺陷型慢病毒载体 (ID-LV) 具有传统慢病毒载体的几个特征，例如无细胞毒性作用和在非复制靶细胞中的长期表达。ID-LV 整合到靶细胞基因组中的整合率显著降低，这提高了安全性，并为其在疫苗开发中的应用开辟了前景。ID-LV 在小鼠模型中已被证明是有效的疫苗，但缺乏体外和体内用人细胞进行的功能研究。在这里，我们评估了表达细胞因子（GM-CSF/IL-4 或 GM-CSF/IFN-α）组合的 ID-LV 转导人单核细胞是否会导致功能性“诱导树突状细胞”（iDC）。用高滴度 ID-LV 过夜转导单核细胞可产生高活力（14 天）且免疫表型稳定的表达 GM-CSF/IL-4 的 iDC（“SmartDCs”）或 GM-CSF/IFN-α（“SmyleDCs”）。在体外维持的 SmartDCs 和 SmyleDCs 持续分泌转基因细胞因子，并显示出几种内源性产生的炎症细胞因子（IFN-γ、IL-2、-5、-6 和 -8）的上调。两种 iDC 类型在混合淋巴细胞反应中均能以与传统 DC（用外源性细胞因子维持）相当的水平强力刺激 T 细胞。用自身肽池 pp65（人巨细胞病毒抗原）脉冲 SmartDCs 或 SmyleDCs 对 CD8(+)T 细胞进行单次体外刺激，导致针对不同 pp65 表位的中央记忆和效应记忆 CTL 的高扩增。我们进一步使用免疫缺陷型 NOD/Rag1((-/-))/IL-2rγ((-/-))（NRG）小鼠评估了 SmartDCs 和 SmyleDCs 在体内扩增抗 pp65 CTL 的效果。NRG 小鼠皮下免疫 SmartDCs 或共表达全长 pp65 的 SmyleDCs，然后输注自身 CD8(+)T 细胞。两种类型的 iDC 都能有效地刺激体内针对不同 pp65 抗原决定簇的人 CTL。由于生成简单、活力强、结合刺激同源、抗原和多价反应的能力，iDC 是一种有前途的疫苗，可在移植后移植患者的免疫接种中进行探索。
