Clínica Universitaria de Navarra, Universidad de Navarra, Av. Pio XII 36, 31008 Pamplona, Spain.
Centre for Applied Medical Research, Universidad de Navarra, Av. Pio XII 55, 31008 Pamplona, Spain.
Front Biosci (Elite Ed). 2022 Jan 20;14(1):2. doi: 10.31083/j.fbe1401002.
Stimulation of dendritic cells (DC) is considered critical in cancer immunotherapy. BATF-3-dependent subsets, that express in humans CD141 (BDCA-3), promote CD8 T-cell cross-priming against tumor antigens. Here, we evaluate two clinical-grade stimuli for peripheral blood CD141+ myeloid dendritic cells (mDCs), a rare DC subset that is currently being explored for use in immunotherapy. In contrast to routine evaluation methods, which focus on predefined maturation markers on the surface or factors released from the activated cells, we applied an unbiased transcriptome-based method using both RNA-sequencing (RNA-seq) and microarrays. Specifically, we analyzed the mRNA of CD141+ mDCs from five human donors upon activation with two clinical-grade adjuvants, Hiltonol (poly-ICLC, a TLR3 ligand) and protamine RNA (pRNA, a TLR7/8 ligand), and compared these samples to unstimulated counterparts. Both methods, RNA-seq, and microarray showed that Hiltonol and pRNA lead to almost identical changes in the transcriptome of CD141+ mDCs. A gene ontology (GO) term analysis suggested that these changes were mainly related to activation and maturation pathways, including induction of type I IFN and IL-12 transcription, while pathways related to adverse effects or cell damage were not strongly affected. The combination of both reagents in the DC cultures gave a very similar result as compared to either stimulus alone, suggesting no synergistic effect. Furthermore, our analysis demonstrates that microarray and RNA-seq analysis gave similar conclusions about the activation status of these cells. Importantly, microarray analyses instead of the advantages of RNA sequencing may still be suitable for studying the activation of rare cell types that are minimally represented or in very low frequency in the organism. Together, our results indicate that both stimuli are potent clinical grade adjuvants with comparable effects to mature CD141+ mDCs in short-term cultures to be used in immunotherapy.
树突状细胞（DC）的刺激被认为是癌症免疫治疗的关键。在人类中表达 CD141（BDCA-3）的 BATF-3 依赖性亚群促进了针对肿瘤抗原的 CD8 T 细胞交叉呈递。在这里，我们评估了两种临床级别的刺激物，用于外周血 CD141+髓样树突状细胞（mDC），这是一种目前正在探索用于免疫治疗的罕见 DC 亚群。与专注于表面上预先定义的成熟标志物或激活细胞释放的因子的常规评估方法相反，我们应用了一种基于无偏转录组的方法，同时使用 RNA 测序（RNA-seq）和微阵列。具体而言，我们分析了来自五个供体的 CD141+mDC 在两种临床级别的佐剂（Hiltonol（聚 ICLC，TLR3 配体）和鱼精蛋白 RNA（pRNA，TLR7/8 配体））激活后的 mRNA，将这些样本与未刺激的对照进行比较。两种方法，RNA-seq 和微阵列均显示，Hiltonol 和 pRNA 导致 CD141+mDC 转录组几乎相同的变化。GO 术语分析表明，这些变化主要与激活和成熟途径有关，包括诱导 I 型 IFN 和 IL-12 转录，而与不良反应或细胞损伤相关的途径则没有受到强烈影响。与单独的刺激物相比，两种试剂在 DC 培养物中的组合产生了非常相似的结果，表明没有协同作用。此外，我们的分析表明，微阵列和 RNA-seq 分析对这些细胞的激活状态得出了相似的结论。重要的是，微阵列分析而不是 RNA 测序的优势可能仍然适合研究在生物体中代表性最小或频率非常低的稀有细胞类型的激活。总之，我们的结果表明，这两种刺激物都是有效的临床级佐剂，可在短期培养中诱导 CD141+mDC 成熟，并可用于免疫治疗。
