Virogenetics Laboratory of Virology, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
Flow Cytometry Core Facility, Malopolska Centre of Biotechnology, Jagiellonian University, Krakow, Poland.
Front Immunol. 2023 May 23;14:1123155. doi: 10.3389/fimmu.2023.1123155. eCollection 2023.
Natural killer (NK) cells plays a pivotal role in the control of viral infections, and their function depend on the balance between their activating and inhibitory receptors. The immune dysregulation observed in COVID-19 patients was previously associated with downregulation of NK cell numbers and function, yet the mechanism of inhibition of NK cell functions and the interplay between infected cells and NK cells remain largely unknown.
In this study we show that SARS-CoV-2 infection of airway epithelial cells can directly influence NK cell phenotype and functions in the infection microenvironment. NK cells were co-cultured with SARS-CoV-2 infected epithelial cells, in a direct contact with A549 cell line or in a microenvironment of the infection in a 3D ex vivo human airway epithelium (HAE) model and NK cell surface expression of a set of most important receptors (CD16, NKG2D, NKp46, DNAM-1, NKG2C, CD161, NKG2A, TIM-3, TIGIT, and PD-1) was analyzed.
We observed a selective, in both utilized experimental models, significant downregulation the proportion of CD161 (NKR-P1A or KLRB1) expressing NK cells, and its expression level, which was followed by a significant impairment of NK cells cytotoxicity level against K562 cells. What is more, we confirmed that SARS-CoV-2 infection upregulates the expression of the ligand for CD161 receptor, lectin-like transcript 1 (LLT1, CLEC2D or OCIL), on infected epithelial cells. LLT1 protein can be also detected not only in supernatants of SARS-CoV-2 infected A549 cells and HAE basolateral medium, but also in serum of COVID-19 patients. Finally, we proved that soluble LLT1 protein treatment of NK cells significantly reduces the proportion of CD161+ NK cells, the ability of NK cells to control SARS-CoV-2 infection in A549 cells and the production of granzyme B by NK cells and their cytotoxicity capacity, yet not degranulation level.
We propose a novel mechanism of SARS-CoV-2 inhibition of NK cell functions via activation of the LLT1-CD161 axis.
自然杀伤 (NK) 细胞在控制病毒感染方面发挥着关键作用，其功能取决于其激活和抑制受体之间的平衡。先前，COVID-19 患者的免疫失调与 NK 细胞数量和功能的下调有关，但 NK 细胞功能抑制的机制以及受感染细胞与 NK 细胞之间的相互作用在很大程度上仍然未知。
在这项研究中，我们表明 SARS-CoV-2 感染气道上皮细胞可以直接影响感染微环境中的 NK 细胞表型和功能。NK 细胞与 SARS-CoV-2 感染的上皮细胞共培养，与 A549 细胞系直接接触，或在 3D 离体人气道上皮 (HAE) 模型中的感染微环境中培养，并分析一组最重要的受体（CD16、NKG2D、NKp46、DNAM-1、NKG2C、CD161、NKG2A、TIM-3、TIGIT 和 PD-1）在 NK 细胞表面的表达。
我们观察到，在两种使用的实验模型中，表达 CD161（NKR-P1A 或 KLRB1）的 NK 细胞的比例及其表达水平均显著下调，随后 NK 细胞对 K562 细胞的细胞毒性水平显著受损，这是一种选择性下调。此外，我们证实 SARS-CoV-2 感染可上调感染上皮细胞上 CD161 受体配体凝集素样转录物 1 (LLT1、CLEC2D 或 OCIL) 的表达。LLT1 蛋白不仅可以在 SARS-CoV-2 感染的 A549 细胞上清液和 HAE 基底外侧培养基中检测到，还可以在 COVID-19 患者的血清中检测到。最后，我们证明了可溶性 LLT1 蛋白处理 NK 细胞可显著降低 CD161+NK 细胞的比例、NK 细胞控制 A549 细胞中 SARS-CoV-2 感染的能力以及 NK 细胞产生颗粒酶 B 的能力及其细胞毒性能力，但不降低脱颗粒水平。
我们提出了一种新型的 SARS-CoV-2 通过激活 LLT1-CD161 轴抑制 NK 细胞功能的机制。
