Background

Despite recent advances in understanding the complex immunologic dysfunction in the tumor microenvironment (TME), fewer than 20% of patients with head and neck squamous cell carcinoma (HNSCC) respond to immune checkpoint blockade (ICB). Thus, it is important to understand how inhibitory IC receptors maintain the suppressed dysfunctional TME, and to develop more effective combination immunotherapy. This study evaluated the immune-modulating effects of Curcumin, which has well-established anti-cancer and chemopreventive properties, and its long-term safety as a phytochemical drug.

Methods

We carried out the western blot and small interfering RNA (siRNA) transfection assay to evaluate the effects of Curcumin on IC ligands and IC ligands function in HNSCC. Through T-cell cytotoxicity assay and measurements of cytokine secretion, we assessed the effects of combination of Curcumin with programmed death-ligand 1 (PD-L1) Ab on cancer cell killing. Flow cytometry were used to analyze the effects of Curcumin on the expression of programmed cell death protein 1 (PD-1) and T-cell immunoglobulin and mucin-domain3 (TIM-3) on CD4, CD8 and Treg. Immunofluorescence, immunohistochemistry and western blot were used to detecte the cytokine (IFN-γ, Granzyme B), IC receptors (PD-1 and TIM-3) and its ligands (PD-L1, PD-L2, Galectin-9) in xenograft mouse model and 4-nitroquinoline-1-oxide (4-NQO) oral cancer model.

Results

We found that Curcumin decreased the expression of IC ligands such as PD-L1, PD-L2, and Galectin-9 in HNSCC, leading to regulation of epithelial-to-mesenchymal transition-associated tumor invasion. Curcumin also effectively restored the ability of CD8⁺ cytotoxic T cells to lyse cancer cells. To evaluate the effect of Curcumin on the TME further, the 4-NQO oral cancer model was used. Curcumin increased T-cell proliferation, tumor-infiltrating lymphocytes (TILs), and effector cytokines, and decreased the expression of PD-1, TIM-3, suppressive IC receptors and their ligands (PD-L1, PD-L2, and Galectin-9) in the TME, implying reinvigoration of the exhausted CD8⁺ T cells. In addition, Curcumin inhibited expression of CD4⁺CD25⁺FoxP3⁺ Treg cells as well as PD-1 and TIM-3.

Conclusions

These results show that Curcumin reinvigorates defective T cells via multiple (PD-1 and TIM-3) and multi-level (IC receptors and its ligands) IC axis suppression, thus providing a rationale to combine Curcumin with conventional targeted therapy or ICB as a multi-faceted approach for treating patients with HNSCC.

中文翻译：

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姜黄素对头颈癌多层次免疫检查点阻断及T细胞功能障碍的影响

背景

尽管最近在了解肿瘤微环境 (TME) 中复杂的免疫功能障碍方面取得了进展，但只有不到 20% 的头颈部鳞状细胞癌 (HNSCC) 患者对免疫检查点阻断 (ICB) 有反应。因此，了解抑制性 IC 受体如何维持功能失调的 TME 并开发更有效的联合免疫疗法非常重要。本研究评估了姜黄素的免疫调节作用，姜黄素具有公认的抗癌和化学预防特性，以及作为植物化学药物的长期安全性。

方法

我们进行了蛋白质印迹和小干扰 RNA (siRNA) 转染试验，以评估姜黄素对 HNSCC 中 IC 配体和 IC 配体功能的影响。通过 T 细胞细胞毒性测定和细胞因子分泌的测量，我们评估了姜黄素与程序性死亡配体 1 (PD-L1) Ab 组合对癌细胞杀伤的影响。流式细胞术分析姜黄素对CD4、CD8和Treg上程序性细胞死亡蛋白1（PD-1）和T细胞免疫球蛋白和粘蛋白结构域3（TIM-3）表达的影响。使用免疫荧光、免疫组织化学和蛋白质印迹检测异种移植物中的细胞因子（IFN-γ、颗粒酶 B）、IC 受体（PD-1 和 TIM-3）及其配体（PD-L1、PD-L2、Galectin-9）小鼠模型和 4-nitroquinoline-1-oxide (4-NQO) 口腔癌模型。

结果

我们发现姜黄素降低了 HNSCC 中 PD-L1、PD-L2 和 Galectin-9 等 IC 配体的表达，从而调节上皮-间质转化相关的肿瘤侵袭。姜黄素还有效恢复了 CD8 ⁺细胞毒性 T 细胞裂解癌细胞的能力。为了进一步评估姜黄素对 TME 的影响，使用了 4-NQO 口腔癌模型。姜黄素增加 T 细胞增殖、肿瘤浸润淋巴细胞 (TIL) 和效应细胞因子，并降低 PD-1、TIM-3、抑制性 IC 受体及其配体（PD-L1、PD-L2 和半乳糖凝集素- 9) 在 TME 中，意味着耗尽的 CD8 ⁺ T 细胞重新焕发活力。此外，姜黄素抑制 CD4 ⁺ CD25 ⁺ FoxP3 的表达⁺ Treg 细胞以及 PD-1 和 TIM-3。

结论

这些结果表明姜黄素通过多种（PD-1 和 TIM-3）和多水平（IC 受体及其配体）IC 轴抑制重振有缺陷的 T 细胞，从而为将姜黄素与常规靶向治疗或 ICB 结合作为一种治疗提供了依据。治疗 HNSCC 患者的多方面方法。