Experimental Transfusion Medicine, Faculty of Medicine Carl Gustav Carus, Dresden University of Technology, Dresden, Germany.
Institute for Transfusion Medicine, German Red Cross Blood Donation Service North-East, Dresden, Germany.
J Immunother Cancer. 2021 Jan;9(1). doi: 10.1136/jitc-2020-001334.
On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors.
Here, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92.
Unmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals.
These observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance.
自然杀伤 (NK) 细胞遇到易感靶标时，通过定向脱颗粒的高度调控步骤介导细胞毒性。细胞毒性颗粒汇聚到微管组织中心，并向免疫突触 (IS) 极化，随后颗粒外排。嵌合抗原受体 (CAR) 或单克隆抗体 (mAb) 的 NK 细胞重定向代表克服肿瘤细胞耐药性的一种很有前途的策略。然而，对于这种重定向的 NK 细胞针对 NK 细胞耐药性肿瘤的溶酶体动力学知之甚少。
在这里，我们使用旋转盘共聚焦显微镜对活细胞成像，以分析表达嵌合抗原受体的 NK-92 细胞（NK-92/5.28.z）和高亲和力 FcR 转基因 NK-92 细胞加曲妥珠单抗针对 ErbB2 阳性乳腺癌细胞（MDA-MB-453）的颗粒介导的 NK 细胞细胞毒性，这些细胞对亲本 NK-92 具有耐药性。
未经修饰的 NK-92 细胞与耐药性癌细胞共培养时会形成稳定的共轭物并聚集颗粒，但未能将颗粒极化到 IS。相比之下，CAR 或 FcR+Herceptin 对 MDA-MB-453 细胞的重定向使颗粒极化到 IS，从而实现了高效的细胞毒性。我们发现，在 NK-92 中，与耐药性 MDA-MB-453 接触后，磷酸肌醇 3-激酶 (PI3K) 途径被激活，而磷脂酶 C-γ (PLCγ) 和丝裂原活化蛋白激酶 (MEK)/细胞外信号调节激酶 (ERK) 未被激活。相比之下，CAR 或抗体依赖性细胞介导的细胞毒性 (ADCC) 的重定向提供了缺失的 PLCγ 和 MEK/ERK 信号。
这些观察结果表明，NK 细胞可以与耐药性癌细胞形成共轭物，并通过颗粒聚集做出反应，但激活信号不足以诱导颗粒极化和随后的裂解酶释放。CAR 和/或 FcR/mAb (ADCC) 轴的重定向提供了必要的信号，导致颗粒极化，从而克服肿瘤细胞耐药性。
