Ko Meng-Wei, Mei Ao, Senjor Emanuela, Nanut Milica Perišić, Gao Lucy Wanrong, Wong Paul, Chen Po-Chun, Cohn Whitaker, Whitelegge Julian P, Kos Janko, Kaur Kawaljit, Malarkannan Subramaniam, Jewett Anahid
Division of Oral Biology and Medicine, The Jane and Jerry Weintraub Center for Reconstructive Biotechnology, University of California Los Angeles School of Dentistry, Los Angeles, California, USA.
Department of Microbiology and Immunology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
BMJ Oncol. 2025 Jun 10;4(1):e000676. doi: 10.1136/bmjonc-2024-000676. eCollection 2025.
Natural killer (NK) cells are the largest innate lymphocyte subset with potent antitumour and antiviral functions. However, clinical utilisation of human NK cells is hampered due to a lack of reliable methods to augment their antitumour potential. We demonstrated technology in which human NK cells were cocultured with osteoclasts in the presence of probiotic bacteria. This approach significantly augmented the antitumour cytotoxicity and polyfunctionality of human NK cells, resulting in the generation of supercharged NK (sNK) cells.
We explored the proteomic, transcriptomic and functional characterisation of sNK cells using cell imaging, flow cytometric analysis, 51-chromium release cytotoxicity assay, ELISA, ELIspot, IsoPLexis single-cell secretome analysis, proteomic analysis, RNA analysis, western blot and enzyme kinetics.
We found that sNK cells were less susceptible to split anergy and tumour-induced exhaustion. Proteomic analyses revealed that sNK cells significantly increased their cell motility and proliferation. Single-cell transcriptomes uncovered sNK cells undertaking a unique differentiation trajectory and turning on STAT1, JUN, BHLHE40, ELF1, MAX and MYC regulons essential for augmenting antitumour effector functions and proliferation, respectively. Both proteomic and single-cell transcriptomes revealed that an increase in Cathepsin C helped to augment the quantity and function of Granzyme B.
These results support that this unique method produces potent NK cells for clinical utilisation and delineate the molecular mechanisms associated with this process.
自然杀伤（NK）细胞是最大的先天性淋巴细胞亚群，具有强大的抗肿瘤和抗病毒功能。然而，由于缺乏可靠的方法来增强其抗肿瘤潜力，人类NK细胞的临床应用受到阻碍。我们展示了一种技术，即在益生菌存在的情况下，将人类NK细胞与破骨细胞共培养。这种方法显著增强了人类NK细胞的抗肿瘤细胞毒性和多功能性，从而产生了超强化NK（sNK）细胞。
我们使用细胞成像、流式细胞术分析、51铬释放细胞毒性测定、酶联免疫吸附测定（ELISA）、酶联免疫斑点测定（ELIspot）、IsoPLexis单细胞分泌组分析、蛋白质组分析、RNA分析、蛋白质免疫印迹和酶动力学，对sNK细胞进行了蛋白质组学、转录组学和功能表征研究。
我们发现sNK细胞较不易发生分裂无能和肿瘤诱导的耗竭。蛋白质组分析表明，sNK细胞显著提高了其细胞运动性和增殖能力。单细胞转录组揭示，sNK细胞沿着独特的分化轨迹发展，并分别开启了对增强抗肿瘤效应功能和增殖至关重要的信号转导和转录激活因子1（STAT1）、原癌基因蛋白c-Jun（JUN）、转录抑制因子BHLHE40、E74样因子1（ELF1）、原癌基因蛋白MAX和原癌基因蛋白MYC调控子。蛋白质组学和单细胞转录组分析均显示，组织蛋白酶C的增加有助于增强颗粒酶B的数量和功能。
这些结果支持了这种独特的方法可产生用于临床的高效NK细胞，并阐明了与此过程相关的分子机制。
