Institute for Tumor Immunology, Clinic for Hematology, Immunology and Oncology, Philipps University of Marburg, Hans-Meerwein-Strasse 3, 35043 Marburg, Germany.
Int J Mol Sci. 2021 Feb 22;22(4):2189. doi: 10.3390/ijms22042189.
NKp30 (Natural Cytotoxicity Receptor 1, NCR1) is a powerful cytotoxicity receptor expressed on natural killer (NK) cells which is involved in tumor cell killing and the regulation of antitumor immune responses. Ligands for NKp30, including BAG6 and B7-H6, are upregulated in virus-infected and tumor cells but rarely detectable on healthy cells. These ligands are released by tumor cells as part of the cellular secretome and interfere with NK cell activity. BAG6 is secreted via the exosomal pathway, and BAG6-positive extracellular vesicles (EV-BAG6) trigger NK cell cytotoxicity and cytokine release, whereas the soluble protein diminishes NK cell activity. However, the extracellular format and activity of B7-H6 remain elusive. Here, we used HEK293 as a model cell line to produce recombinant ligands and to study their impact on NK cell activity. Using this system, we demonstrate that soluble B7-H6 (sB7-H6), like soluble BAG6 (sBAG6), inhibits NK cell-mediated target cell killing. This was associated with a diminished cell surface expression of NKG2D and NCRs (NKp30, NKp40, and NKp46). Strikingly, a reduced mRNA expression was observed exclusively in response to sBAG6. Of note, B7-H6 was marginally released in association with EVs, and EVs collected from B7-H6 expressing cells did not stimulate NK cell-mediated killing. The molecular analysis of EVs on a single EV level using nano flow cytometry (NanoFCM) revealed a similar distribution of vesicle-associated tetraspanins within EVs purified from wildtype, BAG6, or B7-H6 overexpressing cells. NKp30 is a promising therapeutic target to overcome NK cell immune evasion in cancer patients, and it is important to unravel how extracellular NKp30 ligands inhibit NK cell functions.
自然细胞毒性受体 30（Natural Cytotoxicity Receptor 30，NKp30）是一种表达在自然杀伤（Natural Killer，NK）细胞上的强大细胞毒性受体，参与肿瘤细胞杀伤和抗肿瘤免疫反应的调节。NKp30 的配体，包括 BAG6 和 B7-H6，在病毒感染和肿瘤细胞中上调，但在健康细胞中很少检测到。这些配体作为细胞分泌组的一部分由肿瘤细胞释放，并干扰 NK 细胞的活性。BAG6 通过外泌体途径分泌，BAG6 阳性细胞外囊泡（Extracellular Vesicles，EV-BAG6）触发 NK 细胞的细胞毒性和细胞因子释放，而可溶性蛋白则减弱 NK 细胞的活性。然而，B7-H6 的细胞外形式和活性仍然难以捉摸。在这里，我们使用 HEK293 作为模型细胞系来产生重组配体，并研究它们对 NK 细胞活性的影响。使用该系统，我们证明可溶性 B7-H6（sB7-H6）与可溶性 BAG6（sBAG6）一样，抑制 NK 细胞介导的靶细胞杀伤。这与 NKG2D 和 NCRs（NKp30、NKp40 和 NKp46）的细胞表面表达减少有关。引人注目的是，仅观察到 sBAG6 引起的 mRNA 表达减少。值得注意的是，B7-H6 与 EV 一起轻微释放，并且从表达 B7-H6 的细胞中收集的 EV 不会刺激 NK 细胞介导的杀伤。使用纳米流式细胞术（Nanoflow Cytometry，NanoFCM）在单个 EV 水平上对 EV 的分子分析表明，在从野生型、BAG6 或 B7-H6 过表达细胞中纯化的 EV 中，囊泡相关四跨膜蛋白的分布相似。NKp30 是克服癌症患者 NK 细胞免疫逃逸的有前途的治疗靶点，因此揭示细胞外 NKp30 配体如何抑制 NK 细胞功能非常重要。
