Ueno Hideki, Tcherepanova Irina, Reygrobellet Olivier, Laughner Erik, Ventura Claire, Palucka A Karolina, Banchereau Jacques
Baylor Institute for Immunology Research, 3434 Live Oak, Dallas, TX 75204, USA.
J Immunol Methods. 2004 Feb 15;285(2):171-80. doi: 10.1016/j.jim.2003.11.012.
Human dendritic cells (DCs) generated in culture from either monocytes or CD34+ hematopoietic progenitor cells (CD34-HPCs) have been used in cancer immunotherapy protocols with encouraging results. Yet an optimal strategy for the delivery of antigen(s) to DCs still remains to be established. Recent studies demonstrated the feasibility of mRNA transfection to load monocyte-derived DCs. It is not known, however, whether DCs derived by culturing CD34-HPC with GM-CSF and TNF-alpha for 9 days (CD34-DCs) can be efficiently transduced with mRNA. Here we show that clinical-grade CD34-DCs generated after 8 days of culture can be transfected with mRNA without significant alteration of cell viability. About 90% of cells transfected with GFP-RNA express GFP 24 h post-transfection. Remarkably, transfected CD34-DCs retain high levels of GFP expression for at least 14 days. CD34-DCs transfected with Flu-MP RNA were highly efficient in inducing the proliferation of Flu-MP-specific CD8+ T cells as measured by tetramer staining. Furthermore, the stimulated CD8+ T cells produced IFN-gamma upon antigenic stimulation and were able to kill targets pulsed with Flu-MP peptide. Both DC subsets in CD34-DCs, CD1a+-DC (Langerhans cells) and CD14+-DC (interstitial DC), were equally transfected with GFP-RNA, and yielded Flu-specific cytotoxic T cells upon transfection with Flu-MP RNA. Thus, RNA can be used to deliver antigens to two distinct myeloid DC subsets in CD34-DC cultures.
从单核细胞或CD34⁺造血祖细胞（CD34⁺-HPCs）体外培养产生的人树突状细胞（DCs）已被用于癌症免疫治疗方案，并取得了令人鼓舞的结果。然而，将抗原递送至DCs的最佳策略仍有待确立。最近的研究证明了mRNA转染加载单核细胞来源DCs的可行性。然而，尚不清楚用GM-CSF和TNF-α培养9天产生的DCs（CD34-DCs）是否能被mRNA有效转导。在这里，我们表明培养8天后产生的临床级CD34-DCs可以用mRNA转染，而细胞活力无明显改变。转染GFP-RNA的细胞中约90%在转染后24小时表达GFP。值得注意的是，转染的CD34-DCs至少14天保持高水平的GFP表达。用Flu-MP RNA转染的CD34-DCs在通过四聚体染色测量时，在诱导Flu-MP特异性CD8⁺ T细胞增殖方面非常高效。此外，受刺激的CD8⁺ T细胞在抗原刺激后产生IFN-γ，并能够杀死用Flu-MP肽脉冲处理的靶细胞。CD34-DCs中的两个DC亚群，CD1a⁺-DC（朗格汉斯细胞）和CD14⁺-DC（间质DC），均被GFP-RNA同等转染，并在用Flu-MP RNA转染后产生Flu特异性细胞毒性T细胞。因此，RNA可用于将抗原递送至CD34-DC培养物中的两个不同髓样DC亚群。
