Gehring Stephan, Gregory Stephen H, Wintermeyer Philip, San Martin Maryann, Aloman Costica, Wands Jack R
The Liver Research Center, Providence, Rhode Island 02903, USA; The Warren Alpert Medical School of Brown University, Providence, Rhode Island 02903, USA.
J Immunol Methods. 2008 Mar 20;332(1-2):18-30. doi: 10.1016/j.jim.2007.12.007. Epub 2008 Jan 14.
Dendritic cells (DCs) capture, internalize and process antigens leading to the induction of antigen-specific immune responses. The aim of this study was to develop, implement and characterize an efficient approach for DC-based immunization. Dendritic cells were expanded in vivo by hydrodynamic delivery of a human flt3 ligand expression plasmid. Splenic DCs were isolated and purified with magnetic beads linked to hepatitis C virus (HCV) nonstructural protein-5 (NS5), anti-CD40 and/or LPS. The DCs that contained beads were purified by passage over a magnetic column and subsequently phenotyped. Enrichment resulted in a population consisting of 80% CD11c(+) cells. Uptake of uncoated microparticles promoted DC maturation and the expression of CD80, CD86, and MHC-II molecules; beads coated with LPS and anti-CD40 further increased the expression of these co-stimulatory molecules, as well as the secretion of IL-12. Mice immunized subcutaneously with DCs containing beads coated with HCV NS5 protein, anti-CD40 and LPS exhibited significant antigen-specific, increases in IFN-gamma-producing CD4(+) T cells and CTL activity. This approach combines three critical elements necessary for efficient DC-based immunization that include DC enrichment, maturation and antigen targeting.
树突状细胞（DCs）捕获、内化并处理抗原，从而诱导抗原特异性免疫反应。本研究的目的是开发、实施并表征一种基于DC的免疫接种有效方法。通过水动力递送人flt3配体表达质粒在体内扩增树突状细胞。用与丙型肝炎病毒（HCV）非结构蛋白-5（NS5）、抗CD40和/或脂多糖（LPS）相连的磁珠分离并纯化脾DC。含有磁珠的DC通过在磁柱上通过进行纯化，随后进行表型分析。富集产生了一个由80% CD11c(+)细胞组成的群体。摄取未包被的微粒促进DC成熟以及CD80、CD86和MHC-II分子的表达；包被有LPS和抗CD40的磁珠进一步增加了这些共刺激分子的表达以及IL-12的分泌。用含有包被有HCV NS5蛋白、抗CD40和LPS的磁珠的DC皮下免疫的小鼠表现出显著的抗原特异性，产生IFN-γ的CD4(+) T细胞增加以及CTL活性增强。该方法结合了基于DC的有效免疫接种所需的三个关键要素，包括DC富集、成熟和抗原靶向。
