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首页> 外文期刊>Journal of Translational Medicine >Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70
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Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70

机译：产生具有生物活性IL-12p70能力的临床级树突状细胞的生成

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Background For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4+ T helper 1 cells, which then support the development of CD8+ cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development. Methods After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I:C). Results Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I:C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I:C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I:C) were unable to express proteins following RNA transfer by electroporation. Conclusion Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.
机译：背景技术为了获得最佳的T细胞活化作用，希望树突状细胞（DC）将MHC分子内的肽显示为信号1，将共刺激分子显示为信号2，并另外产生IL-12p70作为信号3。IL-12p70会使T细胞的反应极化为CD4 + T辅助细胞1，然后支持CD8 + 细胞毒性T淋巴细胞的发育。因此，我们开发了新的成熟鸡尾酒，使DC可以生产具有生物活性的IL-12p70，用于大规模的癌症疫苗开发。方法在封闭袋系统中淘洗白细胞分离产物后，将富集的单核细胞与GM-CSF和IL-4培养六天，以生成不成熟的DC，然后将其与含有细胞因子，干扰素-γ，前列腺素E2和a Toll样受体8的配体，带有或不带有聚（I：C）。结果成熟的DCs表达了适当的成熟标志物和淋巴结归巢趋化因子受体CCR7。培养两天后，它们保持完全的成熟，没有成熟的鸡尾酒和冷冻保存。 TLR配体刺激诱导的DC能够在原代培养物中和与表达CD40L的成纤维细胞共培养一天后分泌IL-12p70，从而模拟与T细胞的相遇。 DCs用我们的新的含TLR8配体的鸡尾酒（具有或不具有聚（I：C））成熟，可在肽脉冲后诱导同种异体反应并刺激病毒特异性T细胞。在含有TLR8配体而没有聚（I：C）的鸡尾酒中成熟的DC也可以装载RNA作为抗原源，而在含有聚（I：C）的鸡尾酒中成熟的DC在通过电穿孔转移RNA后无法表达蛋白质。结论我们的新的成熟鸡尾酒可以使DC易于收获，稳定的成熟，并能在冷冻保存后大量回收成熟的DC。我们产生DC的程序很容易适应GMP的要求，并产生分泌IL-12p70的DC，适合使用肽或RNA作为免疫抗原来源开发癌症疫苗。

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