PD-L1 Intracellular RNA binding function regulates immune suppression

项目说明/摘要 促进免疫逃逸的程序性死亡配体1(PD-L1)在三重阴性乳房中过表达 癌症(TNBC)，乳腺癌的一种侵袭性亚型，其特征是预后不良。经临床批准 PD-L1抗体通过阻断细胞外PD-1/PD-L1结合增强抗肿瘤免疫。然而， 细胞内PD-L1对抗肿瘤免疫和治疗耐药的贡献仍然很低 明白了。我们发现了细胞内PD-L1作为一种RNA结合蛋白的新作用，它能促进 靶RNA的稳定性。这种新的细胞内PD-L1调节RNA表达的功能是独立的 PD-L1作为PD-1的配基在细胞外的作用。抗肿瘤免疫的活性 免疫反应是由免疫效应细胞和免疫抑制细胞之间的平衡控制的。监管T T细胞(Tregs)是一种CD4+T细胞亚群，它抑制效应细胞的活性，抑制抗肿瘤免疫，并 提高治疗抵抗力。Tregs的一个特征是转录因子Foxp3的表达，它 结合支持Treg活性的基因启动子。Foxp1是Foxp3的近亲家族成员。 越来越多的数据表明，Foxp1与Foxp3相互作用，促进Foxp3介导的转录 Treg通过在靶基因的启动子上保持Foxp3的占位来发挥作用。在我们的初步数据中，我们 已确定Foxp1是PD-L1的关键靶向RNA。我们已经发现PD-L1胞质结构域，但没有 PD-L1胞外区与Foxp1 RNA相互作用，促进Foxp1表达。此外，我们还有 发现细胞内PD-L1‘S促进foxp1的表达是正确的Treg分化所必需的， Treg功能和TNBC进展。Akt-哺乳动物雷帕霉素靶标(MTOR)途径调节 代谢重新编程以实现T细胞的正常成熟和功能。我们的初步数据显示PD-L1 和Foxp1是Akt-mTOR通路激活和代谢所必需的，但在Tregs中不是 效应T细胞，提示靶向PD-L1-Foxp1通路可能优先抑制Tregs 治疗抵抗力。我们的主要假设是细胞内PD-L1使Foxp1 RNA稳定到 促进Treg免疫抑制活性和治疗抵抗。此外，抑制细胞内 PD-L1通过阻断Treg活性促进抗TNBC免疫。这一假设将在一系列实验中得到验证。 三个目标：目标1将确定细胞内PD-L1对Foxp1 mRNA稳定性和Treg的影响 分化；目标2将确定细胞内PD-L1对Treg功能的影响；目标3将进行比较 细胞内PD-L1和细胞外PD-L1/PD-1指导TNBC治疗。清楚地描述了 细胞内PD-L1对肿瘤治疗的研究将为优化联合策略提供重要的启示 在克服免疫逃逸和治疗耐药性方面，为下一步的设计奠定了基础 生成TNBC临床试验。

PROJECT DESCRIPTION/ABSTRACT Programmed death ligand 1 (PD-L1), which promotes immune escape, is overexpressed in triple negative breast cancer (TNBC), an aggressive subtype of breast cancer characterized by poor prognosis. Clinically approved PD-L1 antibodies augment anti-tumor immunity by blocking extracellular PD-1/PD-L1 binding. However, the contribution of intracellular PD-L1 to anti-tumor immunity and therapeutic resistance has remained poorly understood. We have discovered a novel role for intracellular PD-L1 as an RNA binding protein that promotes the stability of target RNAs. This new intracellular PD-L1 function in regulating RNA expression was independent of the established extracellular role of PD-L1 as the ligand for PD-1. The activity of the anti-tumor immune response is governed by a balance between immune effector cells and immune suppressor cells. Regulatory T cells (Tregs) are a CD4+ T cell subpopulation that inhibit effector cell activity, suppress anti-tumor immunity and promote therapeutic resistance. A hallmark of Tregs is the expression of the transcription factor, Foxp3, which binds to the promoters of genes that support Treg activity. Foxp1 is a closely related family member of Foxp3. Emerging data has demonstrated that Foxp1 cooperates with Foxp3 to encourage Foxp3-mediated transcription and Treg function by maintaining Foxp3 occupancy at promoters of target genes. In our preliminary data, we have identified Foxp1 as a key PD-L1 target RNA. We have found that the PD-L1 cytoplasmic domain, but not the PD-L1 extracellular domain, interacts with Foxp1 RNA and promotes Foxp1 expression. In addition, we have discovered that intracellular PD-L1’s promotion of Foxp1 expression is necessary for proper Treg differentiation, Treg function, and TNBC progression. The Akt-mammalian target of rapamycin (mTOR) pathway regulates metabolic reprogramming for proper T cell maturation and function. Our preliminary data suggests that PD-L1 and Foxp1 are required for proper Akt-mTOR pathway activation and metabolism specifically in Tregs, but not effector T cells, suggesting that targeting the PD-L1-Foxp1 pathway may preferentially inhibit Tregs to address therapeutic resistance. Our overarching hypothesis is that intracellular PD-L1 stabilizes Foxp1 RNA to promote Treg immunosuppressive activity and therapeutic resistance. Further, inhibiting intracellular PD-L1 will promote anti-TNBC immunity by blocking Treg activity. This hypothesis will be tested in a series of three aims: Aim 1 will determine the influence of intracellular PD-L1 on Foxp1 mRNA stability and Treg differentiation; Aim 2 will determine the effect of intracellular PD-L1 on Treg function; Aim 3 will compare intracellular PD-L1 and extracellular PD-L1/PD-1 directed TNBC therapy. Clear delineation of the impact of intracellular PD-L1 on cancer therapy will provide important insight for optimizing combination strategies aimed at overcoming immune escape and therapeutic resistance, laying the foundation for the design of the next generation of TNBC clinical trials.