Gene expression profiling upon knockdown of JAK1 in IM9 cells. Homo sapiens

Natural Killer (NK) cells are primary effectors of innate immunity directed against transformed cells. In response, tumor cells have developed mechanisms to evade NK cell-mediated lysis but the molecular basis for target cell resistance is not well understood. In the present study, we used a lentiviral shRNA library targeting more than 1000 human genes to identify 83 genes that promote target cell resistance to human NK cells. Many of the genes identified in this genetic screen belong to common signaling pathways, however, none of these genes have previously been known to modulate susceptibility of human tumor cells to immunologic destruction. In particular, gene silencing of two members of the JAK family (JAK1 and JAK2) in a variety of tumor cell targets increased their susceptibility to NK-mediated lysis and induced increased secretion of interferon gamma (IFN-gamma by NK cells. Treatment of tumor cells with JAK inhibitors also induced increased susceptibility to NK cell activity. These findings may have important clinical implications and suggest that small molecule inhibitors of tyrosine kinases being developed as therapeutic anti-tumor agents may also have significant immunologic effects in vivo. Overall design: IM9 cells were transduced with shRNA-encoding vectors and selected with Puromycin. Two vectors were specifically targeting JAK1 (JAK1-1 and JAK1-3) and one vector encoded an irrelevant control shRNA (CTRL-2). Total RNA was obtained from the parental IM9 cell line, the control-shRNA expressing IM9 cells, the JAK1-1-shRNA and JAK1-3-shRNA expressing IM9 cells in 2 separate experiments (Exp1 and Exp2).

自然杀伤（NK）细胞是固有免疫系统中靶向杀伤转化细胞的主要效应细胞。作为应对，肿瘤细胞进化出了逃避NK细胞介导的细胞裂解的机制，但靶细胞产生抗性的分子基础尚未被充分阐明。本研究使用靶向超过1000个人类基因的慢病毒短发夹RNA（shRNA）文库，筛选得到83个可增强靶细胞对人NK细胞抗性的基因。本次遗传筛选得到的基因中，许多属于常见信号通路，但此前尚无研究表明这些基因可调控人类肿瘤细胞对免疫杀伤的易感性。具体而言，在多种肿瘤靶细胞中沉默JAK家族的两个成员（JAK1与JAK2），可增强这些细胞对NK细胞介导裂解的易感性，并促进NK细胞分泌干扰素γ（IFN-γ）。使用JAK抑制剂处理肿瘤细胞，同样可提升其对NK细胞活性的敏感性。本研究结果具有重要的临床意义，提示当前作为抗肿瘤治疗药物开发的酪氨酸激酶小分子抑制剂，在体内可能同时具备显著的免疫调控效应。整体实验设计：将携带shRNA编码序列的载体转导至IM9细胞，并用嘌呤霉素（Puromycin）进行筛选。其中两个载体特异性靶向JAK1（JAK1-1与JAK1-3），另有一个载体编码无关对照shRNA（CTRL-2）。在两次独立实验（实验1与实验2）中，分别从亲本IM9细胞系、表达对照shRNA的IM9细胞、表达JAK1-1-shRNA与JAK1-3-shRNA的IM9细胞中提取总RNA。