Mechanisms of B cell specific IL-35 expression in cancer.

项目摘要 胰腺导管腺癌(PDA)是胰腺癌中最常见的诊断形式(~90% 令人沮丧的5年存活率约为8%。PDA具有显著的促肿瘤微环境，富含 抑制抗肿瘤免疫功能的免疫抑制细胞。目前的免疫治疗方法 在改善PDA患者预后方面都不成功，因此需要更好地了解 胰腺肿瘤微环境中的免疫调节信号机制。我们已经确定了 调节性B细胞通过其在胰腺癌中的表达成为胰腺肿瘤发生的关键因素 调节性细胞因子IL-35，直接抑制抗肿瘤免疫反应。导致这种现象的机制 B细胞在癌症中产生IL-35并促进胰腺肿瘤生长尚不清楚。使用鼠标模型 携带单克隆性固定B细胞受体(BCR)的，或具有B细胞特异性Toll样缺失的小鼠 受体(TLR)接头蛋白MyD88，我发现原位移植的胰腺肿瘤 与WT小鼠相比，小鼠的体积缩小了。我还进行了TLR和BCR激活的体外分析和 研究发现，共刺激内体TLR和BCR可显著增加B细胞IL-35表达 细胞。根据我的初步数据，我假设癌症驱动的B细胞中IL-35的上调依赖于 BCR和内体TLRs之间的串扰，通过促进胰腺肿瘤生长 免疫抑制。我提出了两个具体目标来检验我的假设。在目标1中，我将确定BCR和 布鲁顿酪氨酸激酶(BTK)介导的内体TLR信号参与B细胞IL-35的表达 细胞，通过分析初级调节B细胞和已建立的B细胞系中的信号通路。在目标2中，我 将研究B细胞中BCR和TLR信号的激活如何促进体内PDA肿瘤的生长。至 完成这项任务，我将分析表达固定BCR特异性的小鼠模型，无论是否具有特定的 抗原暴露，B细胞缺乏MyD88信号的小鼠模型，以及两种模型的交叉。我们的 拟议的研究将提供对B细胞介导的一个以前未描述的方面的理解 在PDA中发挥作用并使用最先进的PDA小鼠模型来测试阻断免疫抑制的策略 小路。最终，我预计我的发现将揭示一种有针对性的机制来抑制B细胞介导的 胰腺肿瘤的免疫抑制，并提供多方面的训练经验，以帮助推进我的 科学事业。

Project Abstract Pancreatic Ductal Adenocarcinoma (PDA) is the most commonly diagnosed form (~90%) of pancreatic cancer with a dismal 5-year survival rate of ~8%. PDA features a prominent pro-tumorigenic microenvironment rich in immunosuppressive cells that inhibit functions of anti-tumor immunity. Current immunotherapeutic approaches have been unsuccessful in improving PDA patient outcomes, leaving a need for a better understanding of the immunomodulatory signaling mechanisms within pancreatic tumor microenvironment. We have identified infiltrating regulatory B cells as being key contributors to pancreatic tumorigenesis through their expression of the regulatory cytokine IL-35, which directly inhibits anti-tumor immune responses. The mechanisms that cause B cells to produce IL-35 in cancer and promote pancreatic tumor growth are not known. Using a mouse model harboring a monoclonal fixed B cell receptor (BCR), or mice with a B cell-specific deletion of the Toll-Like Receptor (TLR) adaptor protein MyD88, I found that orthotopically implanted pancreatic tumors were drastically reduced in size as compared to WT mice. I have also conducted in vitro analysis of TLR and BCR activation and found that co-stimulation of endosomal TLRs and the BCR leads to a robust increase of IL-35 expression in B cells. Based on my preliminary data, I hypothesize that cancer-driven upregulation of IL-35 in B cells is dependent on crosstalk between BCR and endosomal TLRs, which promotes pancreatic tumor growth through immunosuppression. I propose two specific aims to test my hypothesis. In Aim 1, I will determine how BCR and endosomal TLR signaling via Bruton's tyrosine kinase (BTK) contributes to expression of IL-35 expression in B cells, using analysis of signaling pathways in primary regulatory B cells and established B cell lines. In Aim 2, I will investigate how activation of both BCR and TLR signaling in B cells promotes PDA tumor growth in vivo. To accomplish this task, I will analyze mouse models expressing a fixed BCR specificity with or without specific antigen exposure, mouse models lacking MyD88 signaling in B cells, as well as a cross of the two models. Our proposed research will provide an understanding of a previously uncharacterized facet of B cell-mediated function in PDA and use state-of-the-art PDA murine models to test strategies that block immune suppressive pathways. Ultimately, I anticipate my findings will reveal a targetable mechanism to inhibit B cell-mediated immunosuppression in pancreatic tumors and provide a multi-faceted training experience to help advance my scientific career.