University of Antwerp - Faculty of Medicine, Vaccine & Infectious Disease Institute (Vaxinfectio), Laboratory of Experimental Hematology, Universiteitsplein 1, B-2610 Wilrijk (Antwerp), Belgium.
J Transl Med. 2009 Dec 18;7:109. doi: 10.1186/1479-5876-7-109.
Optimization of the current dendritic cell (DC) culture protocol in order to promote the therapeutic efficacy of DC-based immunotherapy is warranted. Alternative differentiation of monocyte-derived DCs using granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-15 has been propagated as an attractive strategy in that regard. The applicability of these so-called IL-15 DCs has not yet been firmly established. We therefore developed a novel pre-clinical approach for the generation of IL-15 DCs with potent immunostimulatory properties.
Human CD14+ monocytes were differentiated with GM-CSF and IL-15 into immature DCs. Monocyte-derived DCs, conventionally differentiated in the presence of GM-CSF and IL-4, served as control. Subsequent maturation of IL-15 DCs was induced using two clinical grade maturation protocols: (i) a classic combination of pro-inflammatory cytokines (tumor necrosis factor-alpha, IL-1beta, IL-6, prostaglandin E2) and (ii) a Toll-like receptor (TLR)7/8 agonist-based cocktail (R-848, interferon-gamma, TNF-alpha and prostaglandin E2). In addition, both short-term (2-3 days) and long-term (6-7 days) DC culture protocols were compared. The different DC populations were characterized with respect to their phenotypic profile, migratory properties, cytokine production and T cell stimulation capacity.
The use of a TLR7/8 agonist-based cocktail resulted in a more optimal maturation of IL-15 DCs, as reflected by the higher phenotypic expression of CD83 and costimulatory molecules (CD70, CD80, CD86). The functional superiority of TLR7/8-activated IL-15 DCs over conventionally matured IL-15 DCs was evidenced by their (i) higher migratory potential, (ii) advantageous cytokine secretion profile (interferon-gamma, IL-12p70) and (iii) superior capacity to stimulate autologous, antigen-specific T cell responses after passive peptide pulsing. Aside from a less pronounced production of bioactive IL-12p70, short-term versus long-term culture of TLR7/8-activated IL-15 DCs resulted in a migratory profile and T cell stimulation capacity that was in favour of short-term DC culture. In addition, we demonstrate that mRNA electroporation serves as an efficient antigen loading strategy of IL-15 DCs.
Here we show that short-term cultured and TLR7/8-activated IL-15 DCs fulfill all pre-clinical prerequisites of immunostimulatory DCs. The results of the present study might pave the way for the implementation of IL-15 DCs in immunotherapy protocols.
为了提高基于树突状细胞（DC）的免疫疗法的治疗效果，有必要优化目前的 DC 培养方案。使用粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素（IL）-15 替代分化单核细胞来源的 DC 已被认为是一种有吸引力的策略。然而，这些所谓的 IL-15 DC 的适用性尚未得到充分证实。因此，我们开发了一种新的临床前方法，用于生成具有强大免疫刺激特性的 IL-15 DC。
用 GM-CSF 和 IL-15 将人 CD14+单核细胞分化为未成熟 DC。常规在 GM-CSF 和 IL-4 存在下分化的单核细胞来源的 DC 作为对照。随后，使用两种临床级别的成熟方案诱导 IL-15 DC 的成熟：（i）促炎细胞因子（肿瘤坏死因子-α、IL-1β、IL-6、前列腺素 E2）的经典组合，和（ii）基于 Toll 样受体（TLR）7/8 激动剂的鸡尾酒（R-848、干扰素-γ、TNF-α和前列腺素 E2）。此外，还比较了短期（2-3 天）和长期（6-7 天）的 DC 培养方案。通过表型特征、迁移特性、细胞因子产生和 T 细胞刺激能力对不同的 DC 群体进行了表征。
使用 TLR7/8 激动剂鸡尾酒可使 IL-15 DC 更理想地成熟，反映在 CD83 和共刺激分子（CD70、CD80、CD86）的更高表型表达上。与常规成熟的 IL-15 DC 相比，TLR7/8 激活的 IL-15 DC 的功能优势体现在：（i）更高的迁移潜力，（ii）有利的细胞因子分泌谱（干扰素-γ、IL-12p70），和（iii）在被动肽脉冲后刺激自体、抗原特异性 T 细胞反应的能力更高。除了生物活性 IL-12p70 的产生不那么明显外，与 TLR7/8 激活的 IL-15 DC 的短期与长期培养相比，短期 DC 培养的迁移谱和 T 细胞刺激能力更具优势。此外，我们证明了 mRNA 电穿孔是一种有效的 IL-15 DC 抗原加载策略。
在这里，我们表明短期培养和 TLR7/8 激活的 IL-15 DC 满足免疫刺激 DC 的所有临床前先决条件。本研究的结果可能为 IL-15 DC 在免疫治疗方案中的应用铺平道路。
