Wnt3a is a central regulator for P. gingivalis-mediated expression of PD-L1 and suppression of CD8+ T cell activity

摘要 阻断程序性死亡配体-1(PD-L1)对患者的临床疗效显著 通过恢复疲惫的CD8+T细胞反应，治疗癌症或慢性感染。然而，只有一小部分 部分患者对这种治疗反应良好，表明迫切需要深入了解 PD-L1的功能和调控机制。最近的突破性研究发现，PD-L1的表达 树突状细胞(DC)的表达对PD-L1/PD-1阻断免疫治疗的疗效至关重要。卟啉单胞菌 牙周炎(gigivalis，PG)是一种慢性牙周炎和组织损伤的病原体。肺炎支原体感染 据报道可加重肿瘤进展并促进肿瘤细胞中PD-L1的表达。但是，如果和 PG感染促进DC表面PD-L1表达及其对CD8+T细胞活性的影响 完全不为人所知。因此，人们很容易发现新的免疫信号调节器，它可以 调节PD-L1的表达并影响细胞毒性T细胞的活性，以恢复免疫为长期目标 系统在监测和消除入侵生物和癌细胞方面的作用。我们最近的研究 Pg感染可显著增加先天免疫中Wnt(Wingless s and Int-1)3a的表达 细胞和Wnt3a负向调节先天免疫反应的强度。我们的初步数据显示 PG感染(I)显著增强BMDCs上PD-L1的表达并降低同种异体骨髓细胞的细胞毒性 抗原特异性CD8+T细胞；(Ii)显著增强Wnt3a的表达，抑制Wnt3a的表达 BMDCs上PD-L1的表达；以及(Iii)导致YAP和WW-1的增加。 含有结构域的转录调节因子-1(TAZ)的表达，但上游激酶减少 树突状细胞中的磷酸化。由于YAP/TAZ被证明与PD-L1表达相关并与Wnt3a相互作用 在其他情况下，我们因此假设WNT3a是一种关键的免疫调节剂，它促进 PG刺激时PD-L1的表达及抑制CD8+T细胞活性的作用 YAP/TAZ信号及其下游转录增强因子(TEA)结构域家族的DNA- 结合因子1(TEAD1)。我们将以两个具体目标来论证这一假说：(1)：确立角色 Wnt3a作为PG诱导的PD-L1和CD8+T细胞活性受损的内源性调节因子； (2)确定Wnt3a是否通过促进PG诱导的PD-L1表达和CD8+T细胞活性受损 修饰YAP/TAZ及其下游TEAD1的活性。这一项目的成功完成将(I) 绘制了一条新的和潜在的关键信号通路(Wnt3a-YAP/TAZ-TEAD1)，并确定了更多 PD-L1表达中的干预靶点；以及(Ii)极大地有助于理解调控机制 PG感染对CD8+T细胞的杀伤作用减弱，这将为创新的发展铺平道路 通过调控WNT3-的活性控制癌症和慢性感染的免疫调节疗法 YAP/TAZ应该远远超出口腔的相关性。

Abstract Blockade of program death ligand-1 (PD-L1) is demonstrating remarkable clinical outcomes for patients with cancer or chronic infection through reinvigorating exhausted CD8+ T cell responses. However, only a small portion of patients can respond well to this treatment, indicating a dire need for an in-depth understanding of the function and regulatory mechanisms of PD-L1. Recent breakthrough findings revealed that expression of PD-L1 on dendritic cells (DCs) is critical for the efficacy of PD-L1/PD-1 blockade immunotherapy. Porphyromonas gingivalis (Pg) is a causative agent of chronic periodontal inflammation and tissue damages. Infection with Pg was reported to aggravate cancer progression and promote PD-L1 expression in tumor cells. However, if and how Pg infection promotes expression of PD-L1 on DCs and its possible effect on CD8+ T cell activity remain entirely unknown. Thus, it is tempting to identify novel immune signaling modulators that can regulate expression of PD-L1 and impact cytotoxic T cell activity, with a long-term goal of restoring the immune system function in surveillance and elimination of invaded organisms and cancer cells. Our recent studies demonstrated that Pg infection robustly increases expression of Wnt (Winglesss and Int-1) 3a in innate immune cells and Wnt3a negatively regulates the intensity of innate immune responses. Our preliminary data suggest that Pg infection (i) significantly enhances PD-L1 expression on BMDCs and impairs cytotoxicity of allogenic antigen-specific CD8+ T cells; (ii) remarkably enhances expression of Wnt3a, and inhibition of Wnt3a reduces expression of PD-L1 on BMDCs; and (iii) leads to an increase of Yes-associated protein 1(YAP) and WW- domain-containing transcription regulator-1(TAZ) expression but a decrease of an upstream kinase phosphorylation in DCs. Since YAP/TAZ was shown to associate with PD-L1 expression and interact with Wnt3a signaling in other contexts, we thus hypothesize that Wnt3a is a critical immunomodulator that promotes PD-L1 expression and suppresses CD8+ T cell activity upon stimulation with Pg by regulating activation of YAP/TAZ signaling and its downstream transcriptional enhancer factor (TEA)-domain family of DNA- binding factor 1 (TEAD1). We will demonstrate this hypothesis with two specific aims: (1): Establish the role of Wnt3a as an endogenous regulator of the Pg-induced PD-L1 and impairment of CD8+ T cell activity; (2) Establish if Wnt3a promotes Pg-induced PD-L1 expression and impairment of CD8+ T cell activity via modifying the activity of YAP/TAZ and its downstream TEAD1. Successful completion of this project will (i) maps out a novel and potentially critical signaling pathway (Wnt3a-YAP/TAZ-TEAD1) and identifies more interventional targets in PD-L1 expression; and (ii) greatly aids in understanding the regulatory mechanism that Pg infection impairs CD8+ T cell cytotoxicity, which will pave the way for the development of innovative immunoregulatory therapies for the control of cancer and chronic infections via manipulating activity of Wnt3- YAP/TAZ that should have relevance well beyond oral cavity.