Figueiredo Constança, Seltsam Axel, Blasczyk Rainer
Institute for Transfusion Medicine, Hannover Medical School, Carl-Neuberg-Str. 1, 30625, Hannover, Germany.
J Mol Med (Berl). 2009 Feb;87(2):199-210. doi: 10.1007/s00109-008-0417-0. Epub 2008 Nov 11.
Natural killer (NK) and T-cell cytotoxicity is significantly reduced by signaling via CD94/NKG2A receptors. High levels of NKG2A on NK cells have been shown to compromise the graft-versus-leukemia effect in hematopoietic stem cell transplantation. We therefore evaluated the functional relevance of NKG2A silencing for the cytotoxic potential of genetically engineered NK and T cells. Lentiviral vectors containing short hairpin RNA (shRNA) sequences targeting NKG2A transcripts were used to transduce NKG2A(+) primary NK and T cells. NKG2A expression levels were measured by flow cytometry and real-time PCR. The effect of NKG2A silencing on the cytolytic potential of NK and T cells was evaluated in cytotoxicity assays using K562 and B lymphoblastoid cells as targets. Granzyme B mRNA transcript levels were detected by real-time PCR. The transduction of inducible RNAi cassettes containing the sequences for shRNAs targeting NKG2A reduced protein expression in NK and T cells by up to 95%. The cytotoxicity assays demonstrated that NKG2A silencing effectively enhanced NK and CD8+ T-cell lysis by up to 40% and 15%, respectively. However, lysis of K562 cells which lack human leukocyte antigen-E, the ligand of NKG2A, was associated with an upregulation of the natural cytotoxicity receptor NKp30 in NKG2A-silenced NK cells. Our data suggest that RNAi-mediated silencing of NKG2A in effector cells could improve the efficacy of cell-based immunotherapies but also show that indirect effects of NKG2A knockdown exist that have to be considered when designing therapeutic protocols with genetically engineered NK or T cells.
通过CD94/NKG2A受体发出的信号会显著降低自然杀伤（NK）细胞和T细胞的细胞毒性。研究表明，NK细胞上高水平的NKG2A会损害造血干细胞移植中的移植物抗白血病效应。因此，我们评估了NKG2A沉默对于基因工程改造的NK细胞和T细胞细胞毒性潜力的功能相关性。使用含有靶向NKG2A转录本的短发夹RNA（shRNA）序列的慢病毒载体转导NKG2A(+)原代NK细胞和T细胞。通过流式细胞术和实时PCR测量NKG2A的表达水平。在以K562细胞和B淋巴母细胞为靶标的细胞毒性试验中，评估了NKG2A沉默对NK细胞和T细胞溶细胞潜力的影响。通过实时PCR检测颗粒酶B mRNA转录水平。转导含有靶向NKG2A的shRNA序列的可诱导RNAi盒，可使NK细胞和T细胞中的蛋白质表达降低多达95%。细胞毒性试验表明，NKG2A沉默分别有效地增强了NK细胞和CD8+ T细胞的裂解作用，增强幅度高达40%和15%。然而，缺乏NKG2A配体人类白细胞抗原-E的K562细胞的裂解与NKG2A沉默的NK细胞中自然细胞毒性受体NKp30的上调有关。我们的数据表明，效应细胞中RNAi介导的NKG2A沉默可以提高基于细胞的免疫疗法的疗效，但也表明存在NKG knockdown的间接效应设计基因工程改造的NK细胞或T细胞治疗方案时必须予以考虑。
