Permanent silencing of NKG2A expression for cell-based therapeutics
Permanent silencing of NKG2A expression for cell-based therapeutics
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DOI：
10.1007/s00109-008-0417-0
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发表时间：
2009-02-01
影响因子：
4.7
通讯作者：
Blasczyk, Rainer
中科院分区：
文献类型：
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作者：
Figueiredo, Constanca;Seltsam, Axel;Blasczyk, Rainer
CD94/NKG2A 受体的信号传导可显着降低自然杀伤 (NK) 和 T 细胞的细胞毒性。 NK 细胞上高水平的 NKG2A 已被证明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 沉默与基因工程 NK 和 T 细胞的细胞毒性潜力的功能相关性。使用含有靶向 NKG2A 转录物的短发夹 RNA (shRNA) 序列的慢病毒载体转导 NKG2A(+) 原代 NK 和 T 细胞。通过流式细胞术和实时PCR测量NKG2A表达水平。使用 K562 和 B 淋巴母细胞作为靶标，在细胞毒性测定中评估 NKG2A 沉默对 NK 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。含有靶向 NKG2A 的 shRNA 序列的诱导性 RNAi 盒的转导可将 NK 和 T 细胞中的蛋白质表达降低高达 95%。细胞毒性测定表明，NKG2A 沉默可有效增强 NK 和 CD8+ T 细胞的裂解，分别高达 40% 和 15%。然而，缺乏人类白细胞抗原-E（NKG2A 的配体）的 K562 细胞的裂解与 NKG2A 沉默的 NK 细胞中天然细胞毒性受体 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 NKG2A 敲低的间接影响，在设计基因工程 NK 或 T 细胞的治疗方案时必须考虑这一影响。
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.