Harnessing RNA sequencing for global, unbiased evaluation of two new adjuvants for dendritic-cell immunotherapy. Homo sapiens

Effective stimulation of immune cells is crucial for the success of cancer immunotherapies. Current approaches to evaluate the efficiency of stimuli activating immune cells are mainly defined by known flow cytometry-based cell activation or cell maturation markers. This method however does not give a complete overview of the achieved activation state and may leave important side effects unnoticed. Here, we used an unbiased RNA sequencing (RNA-seq)-based approach to compare the capacity of four clinical-grade dendritic cell (DC) activation stimuli used to prepare DC-vaccines composed of various types of DC subsets; the already clinically applied GM-CSF and Frühsommer meningoencephalitis (FSME) prophylactic vaccine and the novel clinical grade adjuvants protamine-RNA (pRNA) complexes and CpG-P. We found that GM-CSF and pRNA have similar effects on their target cells, whereas pRNA and CpG-P induce stronger tType I interferon (IFN) expression than FSME. In general, the pathways most affected by all stimuli were related to immune activity and cell migration. GM-CSF stimulation, however, also induced a significant increase of genes related to nonsense-mediated decay, indicating a possible deleterious effect of this stimulus. Taken together, all novel stimuli appear to be promising alternatives. Our study demonstrates how RNA-seq based investigation of changes in a large number of genes and gene groups can be exploited for fast and unbiased, global evaluation of clinical-grade stimuli, as opposed to the general limited evaluation of a pre-specified set of genes, by which one might miss important biological effects detrimental for vaccine efficacy. Overall design: mRNA profiles of CD1c+ mDCs and pDCs, MACS isolated from 3 healthy volunteers and stimulated with 4 clinical grade stimuli, were generated by deep RNA sequencing using HiSeq 2000 System (TruSeq SBS KIT-HS V3，Illumina)

有效刺激免疫细胞，是癌症免疫治疗取得成功的关键所在。当前用于评估免疫细胞激活刺激物效能的方法，主要基于已确立的基于流式细胞术的细胞激活或细胞成熟标志物进行定义。然而，此类方法无法全面呈现细胞已达到的激活状态，还可能遗漏重要的未被察觉的副作用。本研究采用无偏倚的基于RNA测序（RNA sequencing, RNA-seq）的方法，对比了四种用于制备包含多种树突状细胞（dendritic cell, DC）亚群的DC疫苗的临床级DC激活刺激物的效能；这四种刺激物分别为已临床应用的粒细胞-巨噬细胞集落刺激因子（granulocyte-macrophage colony-stimulating factor, GM-CSF）、春夏脑膜脑炎（Frühsommer meningoencephalitis, FSME）预防性疫苗，以及新型临床级佐剂鱼精蛋白-RNA（protamine-RNA, pRNA）复合物与CpG-P。本研究发现，GM-CSF与pRNA对靶细胞的作用效果相似；而pRNA与CpG-P诱导的I型干扰素（interferon, IFN）表达水平显著高于FSME。总体而言，所有刺激物影响最为显著的通路均与免疫活性及细胞迁移相关。然而，GM-CSF刺激还会显著上调与无义介导的mRNA降解（nonsense-mediated decay）相关的基因表达，提示该刺激物可能存在有害效应。综上，所有新型刺激物均展现出作为替代方案的良好前景。本研究证实，相较于仅对预先选定的基因集进行有限评估（此类方法可能会遗漏对疫苗效力有害的重要生物学效应），基于RNA-seq对大量基因及基因家族的表达变化进行检测，可实现对临床级刺激物的快速、无偏倚的全局评估。实验设计：从3名健康志愿者体内经磁珠激活细胞分选（magnetic activated cell sorting, MACS）分离得到的CD1c阳性髓系树突状细胞（CD1c+ mDCs）与浆细胞样树突状细胞（pDCs），分别用4种临床级刺激物进行刺激，随后采用Illumina HiSeq 2000测序系统（TruSeq SBS KIT-HS V3）进行深度RNA测序，以获取其mRNA表达谱。