Identification of the networks that regulate human monocytic myeloid-derived suppressor cell differentiation into inflammatory macrophages

Background: Monocytic myeloid-derived suppressor cells (mMDSC) support immune evasion of tumors by blocking tumoricidal T and NK cell response. Efforts to reverse mMDSC-mediated immunosuppression identified that the TLR7/8 agonist R848 induces their maturation into tumoricidal macrophages Purpose: The factors that determine whether human mMDSC differentiate into MACinflam or MACsuppress are unclear. To investigate this issue, the role of cytokines and genes activated following R848 treatment was examined. Methods: After 4 hour stimulation, cells from stimulated mMDSC were stored in RNA protect (Qiagen, Frederick, MD). Total RNA was isolated using the RNeasy micro kit (Qiagen) and RNA quality was assessed using an Agilent 2200 TapeStation. mRNA libraries were generated using the Smart-Seq ultra-low input kit (Clontech) and sequenced using a HiSeq2500 sequencer using IlluminaTruSeq v4 chemistry with 125bp paired-end reads. Sequences were aligned to the human (hg19) reference genome. Genes that were differentially expressed compared to untreated samples were identified using CLC genomics workbench (version 10). Results: TNFa combined with IL-6 effectively supports the differentiation of mMDSC into tumoricidal macrophage by activating expression of conserved set of genes. Mechanistically, transcription factors NF-KB and STAT4 emerge as important regulators and potential targets to modify mMDSC behavior. Blood was received from human donors, FACS sorted for MDSC cells and then cultured with IFNg, R848 or Il6-TNFa for 4 hours. Rna was collected and mRNA libraries were generated using the Smart-Seq ultra-low input kit (Clontech) and sequenced using a HiSeq2500 sequencer using IlluminaTruSeq v4 chemistry with 125bp paired-end reads. Sequences were aligned to the human (hg19) reference genome. Genes that were differentially expressed compared to untreated samples were identified using CLC genomics workbench (version 10)

背景：单核细胞髓系来源抑制性细胞（monocytic myeloid-derived suppressor cells, mMDSC）通过抑制杀瘤性T细胞与自然杀伤（NK）细胞的抗肿瘤应答，介导肿瘤免疫逃逸。目前针对逆转mMDSC介导的免疫抑制的研究表明，Toll样受体7/8（TLR7/8）激动剂R848可诱导其分化为杀瘤性巨噬细胞。目的：目前尚不明确哪些因素决定了人类单核细胞髓系来源抑制性细胞（mMDSC）向MACinflam型或MACsuppress型巨噬细胞分化。为阐明这一科学问题，本研究考察了细胞因子以及R848处理后激活的基因所发挥的调控作用。方法：经4小时刺激后，将受刺激的mMDSC样本保存于RNA保护试剂（Qiagen公司，马里兰州弗雷德里克）中。采用RNeasy微量试剂盒（Qiagen）提取总RNA，并通过安捷伦2200 TapeStation系统评估RNA质量。使用Smart-Seq超低起始量试剂盒（Clontech）构建mRNA文库，随后采用搭载Illumina TruSeq v4建库试剂的HiSeq2500测序仪完成125bp双端测序。将测序序列比对至人类参考基因组（hg19版本），并使用CLC基因组工作台（版本10）鉴定与未处理样本相比存在差异表达的基因。结果：研究发现，肿瘤坏死因子α（TNF-α）联合白细胞介素6（IL-6）可通过激活保守基因集的表达，有效促进mMDSC向杀瘤性巨噬细胞分化。从分子机制来看，转录因子NF-κB与STAT4是调控该过程的关键因子，同时也是修饰mMDSC功能的潜在靶点。本研究从人类供体获取外周血样本，通过荧光激活细胞分选（FACS）分离MDSC细胞，随后分别用干扰素γ（IFN-γ）、R848以及IL-6与TNF-α的组合处理细胞并培养4小时。收集RNA并使用Smart-Seq超低起始量试剂盒（Clontech）构建mRNA文库，采用搭载Illumina TruSeq v4建库试剂的HiSeq2500测序仪进行125bp双端测序，将测序序列比对至人类参考基因组（hg19版本），并通过CLC基因组工作台（版本10）鉴定与未处理样本相比存在差异表达的基因。