Department of Urology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai, P.R. China.
Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China
In Vivo. 2024 Jan-Feb;38(1):174-183. doi: 10.21873/invivo.13423.
BACKGROUND/AIM: The natural killer (NK) cell function of patients with malignant tumours may be suppressed by deficiency, and the poor prognosis of renal cell carcinoma (RCC) patients may be due to escape from NK cell cytotoxicity, especially with respect to natural cytotoxicity receptors (NCRs) on the NK cell surface. However, the specific mechanism remains unclear. Therefore, in this study, we sought to explore the role of NCR, especially NCR3 splice variants, in the process of NK cell deficiency in RCC patients.
We used flow cytometry to analyse the phenotype of NK cells from the peripheral blood and kidney tumour tissue of RCC patients. The NKp30-mediated NK cell killing function was measured by antibody-dependent cell-mediated cytotoxicity (ADCC) in NK and RCC cell coincubation. We extracted RNA from the peripheral blood mononuclear cells (PBMCs) of RCC patients and renal carcinoma tissue and carried out real-time quantitative PCR to detect the mRNA levels of NKp30a, NKp30b and NKp30c. mRNA expression levels of cytokines (IL-6, IL-8, IL-10, IL-18 and TGF-β) based on RNA extracted from renal carcinoma tissue and adjacent normal kidney tissues were also measured by real-time quantitative PCR.
Regarding the phenotype of NK cells in RCC patients, the proportion of NK cells in tumour tissue was significantly reduced, with changes in the NK cell proportion being most obvious in NKp30 NK cells. Furthermore, the results of the ADCC function assay showed limited NKp30 NK cell-mediated cytotoxicity in RCC patients. Through real-time quantitative PCR, we found lower expression of NKp30a and NKp30b, the immunostimulatory splice variants of NCR3 encoding NKp30, in RCC patients. Moreover, expression of activating cytokines (IL-6 and IL-8) in renal cancer tissue was decreased, though inhibitory cytokine (TGF-β) expression remained unchanged, which may result in an immunosuppressive cytokine microenvironment.
Decreased expression of immunostimulatory NCR3 splice variants and the inhibitory cytokine microenvironment in RCC patients may contribute to deficient NK cell cytotoxicity and renal carcinoma cell immune escape from NK cell killing, which may provide a theoretical basis for finding new immunotherapeutic targets for RCC.
背景/目的：恶性肿瘤患者的自然杀伤 (NK) 细胞功能可能因缺乏而受到抑制，而肾细胞癌 (RCC) 患者的预后较差可能是由于逃避了 NK 细胞的细胞毒性，特别是 NK 细胞表面的自然细胞毒性受体 (NCR)。然而，具体的机制尚不清楚。因此，在这项研究中，我们试图探讨 NCR，特别是 NCR3 剪接变体，在 RCC 患者 NK 细胞缺乏过程中的作用。
我们使用流式细胞术分析了 RCC 患者外周血和肾肿瘤组织中 NK 细胞的表型。通过 NK 和 RCC 细胞共孵育中的抗体依赖性细胞介导的细胞毒性 (ADCC) 来测量 NKp30 介导的 NK 细胞杀伤功能。我们从 RCC 患者的外周血单核细胞 (PBMC) 和肾癌细胞组织中提取 RNA，并进行实时定量 PCR 检测 NKp30a、NKp30b 和 NKp30c 的 mRNA 水平。还通过实时定量 PCR 测量了基于从肾癌细胞组织和相邻正常肾组织中提取的 RNA 的细胞因子 (IL-6、IL-8、IL-10、IL-18 和 TGF-β) 的 mRNA 表达水平。
关于 RCC 患者 NK 细胞的表型，肿瘤组织中 NK 细胞的比例明显降低，NKp30 NK 细胞的 NK 细胞比例变化最为明显。此外，ADCC 功能测定的结果表明，RCC 患者的 NKp30 NK 细胞介导的细胞毒性有限。通过实时定量 PCR，我们发现 RCC 患者 NCR3 编码 NKp30 的免疫刺激剪接变体 NKp30a 和 NKp30b 的表达较低。此外，肾癌细胞组织中激活细胞因子 (IL-6 和 IL-8) 的表达减少，而抑制细胞因子 (TGF-β) 的表达保持不变，这可能导致免疫抑制细胞因子微环境。
RCC 患者免疫刺激 NCR3 剪接变体表达降低和抑制性细胞因子微环境可能导致 NK 细胞毒性不足和肾癌细胞逃避 NK 细胞杀伤，这可能为寻找 RCC 的新免疫治疗靶点提供理论依据。
