本研究的目的在于证明槟榔碱能够诱导线粒体（mt）DNA D环和程序性细胞死亡配体1（PD-L1）在细胞外囊泡（EVs）中的分泌，并通过上调调节性T细胞（Treg）的数量来削弱T细胞免疫力的假设。然而，这种免疫抑制可以通过全葡聚糖颗粒（WGP）β-葡聚糖在口腔鳞状细胞癌（OSCC）患者中被逆转。利用OSCC细胞系分析槟榔碱诱导的活性氧产物（ROS）的产生和细胞质mtDNA D环。对60例OSCC患者的手术标本和血清进行了mtDNA D环、PD-L1、干扰素γ（IFN-γ）和Treg细胞的检测。我们证明，更高的mtDNA D环、PD-L1和Treg细胞数与肿瘤大小、淋巴结转移、临床分期和槟榔嚼食有显著相关性。此外，多变量分析证实，更高的mtDNA D环水平和Treg细胞数量是不利的独立因素。槟榔碱显著诱导细胞质mtDNA D环泄漏和PD-L1表达，这些通过EVs封装以促进免疫抑制的Treg细胞数量。然而，WGP β-葡聚糖可以提高CD4+和CD8+ T细胞数量，减轻Treg细胞的数量，并促进口腔癌细胞凋亡。总之，槟榔碱诱导EVs产生携带mtDNA D环和PD-L1，并进一步引发免疫抑制。然而，WGPβ-葡聚糖对T细胞免疫和细胞凋亡具有潜在的双重效应，我们强烈推荐将其与针对OSCC的靶向和免疫疗法相结合。© 2023 The Authors. 由John Wiley & Sons Australia, Ltd代表日本癌症协会出版的《癌症科学》杂志发表。

The suppressive regulatory T cells (Treg) are frequently upregulated in cancer patients. This study aims to demonstrate the hypothesis that arecoline could induce the secretion of mitochondrial (mt) DNA D-loop and programmed cell death-ligand 1 (PD-L1) in extracellular vesicles (EVs), and attenuate T-cell immunity by upregulated Treg cell numbers. However, the immunosuppression could be reversed by whole glucan particle (WGP) β-glucan in oral squamous cell (OSCC) patients. Arecoline-induced reactive oxygen specimen (ROS) production and cytosolic mtDNA D-loop were analyzed in OSCC cell lines. mtDNA D-loop, PD-L1, IFN-γ, and Treg cells were also identified for the surgical specimens and sera of 60 OSCC patients. We demonstrated that higher mtDNA D-loop, PD-L1, and Treg cell numbers were significantly correlated with larger tumor size, nodal metastasis, advanced clinical stage, and areca quid chewing. Furthermore, multivariate analysis confirmed that higher mtDNA D-loop levels and Treg cell numbers were unfavorable independent factors for survival. Arecoline significantly induced cytosolic mtDNA D-loop leakage and PD-L1 expression, which were packaged by EVs to promote immunosuppressive Treg cell numbers. However, WGP β-glucan could elevate CD4+ and CD8+ T-cell numbers, mitigate Treg cell numbers, and promote oral cancer cell apoptosis. To sum up, arecoline induces EV production carrying mtDNA D-loop and PD-L1, and in turn elicits immune suppression. However, WGP β-glucan potentially enhances dual effects on T-cell immunity and cell apoptosis and we highly recommend its integration with targeted and immune therapies against OSCC.© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.