Translational and Clinical Research Program, Cancer Research Center (IBMCC, CSIC - University of Salamanca), and Department of Medicine, University of Salamanca, Salamanca, Spain.
Cytometry Service, NUCLEUS, University of Salamanca, Salamanca, Spain.
Front Immunol. 2024 Jul 8;15:1423689. doi: 10.3389/fimmu.2024.1423689. eCollection 2024.
Natural killer (NK) cells are traditionally identified by flow cytometry using a combination of markers (CD16/CD56/CD3), because a specific NK-cell marker is still missing. Here we investigated the utility of CD314, CD335 and NKp80, compared to CD16/CD56/CD3, for more robust identification of NK-cells in human blood, for diagnostic purposes.
A total of 156 peripheral blood (PB) samples collected from healthy donors (HD) and patients with diseases frequently associated with loss/downregulation of classical NK-cell markers were immunophenotyped following EuroFlow protocols, aimed at comparing the staining profile of total blood NK-cells for CD314, CD335 and NKp80, and the performance of distinct marker combinations for their accurate identification.
NKp80 showed a superior performance (vs. CD314 and CD335) for the identification of NK-cells in HD blood. Besides, NKp80 improved the conventional CD16/CD56/CD3-based strategy to identify PB NK-cells in HD and reactive processes, particularly when combined with CD16 for further accurate NK-cell-subsetting. Although NKp80+CD16 improved the identification of clonal/tumor NK-cells, particularly among CD56 cases (53%), aberrant downregulation of NKp80 was observed in 25% of patients, in whom CD56 was useful as a complementary NK-cell marker. As NKp80 is also expressed on T-cells, we noted increased numbers of NKp80 cytotoxic T-cells at the more advanced maturation stages, mostly in adults.
Here we propose a new robust approach for the identification of PB NK-cells, based on the combination of NKp80 plus CD16. However, in chronic lymphoproliferative disorders of NK-cells, addition of CD56 is recommended to identify clonal NK-cells, due to their frequent aberrant NKp80 phenotype.
自然杀伤 (NK) 细胞传统上通过使用标记物（CD16/CD56/CD3）的组合通过流式细胞术进行鉴定，因为仍然缺少特定的 NK 细胞标记物。在这里，我们研究了 CD314、CD335 和 NKp80 的用途，与 CD16/CD56/CD3 相比，它们在用于诊断目的的人类血液中更准确地鉴定 NK 细胞。
总共对 156 份来自健康供体 (HD) 和经常与经典 NK 细胞标记物的丢失/下调相关的疾病患者的外周血 (PB) 样本进行免疫表型分析，根据 EuroFlow 方案进行，旨在比较总血液 NK 细胞的 CD314、CD335 和 NKp80 的染色模式，以及不同标记物组合用于准确鉴定的性能。
NKp80 对 HD 血液中 NK 细胞的鉴定表现出优越的性能（与 CD314 和 CD335 相比）。此外，NKp80 改善了基于传统 CD16/CD56/CD3 的策略，用于鉴定 HD 和反应性过程中的 PB NK 细胞，特别是与 CD16 结合使用时，可进一步准确地对 NK 细胞进行亚群分析。尽管 NKp80+CD16 改善了克隆/肿瘤 NK 细胞的鉴定，特别是在 CD56 病例中（53%），但在 25%的患者中观察到 NKp80 的异常下调，在这些患者中，CD56 可作为补充 NK 细胞标记物。由于 NKp80 也在 T 细胞上表达，我们注意到 NKp80 细胞毒性 T 细胞在更成熟的成熟阶段数量增加，主要在成人中。
在这里，我们提出了一种新的基于 NKp80 加 CD16 组合的 PB NK 细胞鉴定的稳健方法。然而，在 NK 细胞慢性淋巴增殖性疾病中，由于其频繁出现异常的 NKp80 表型，建议添加 CD56 以识别克隆 NK 细胞。
