Metabolic Regulation ofPD-L1 in CD11c+ Cells

项目摘要/摘要 长期目标是找到更好的干预方法来阻止癌症的发生、进展和 转移。PD-L1/PD-1信号通路是肿瘤利用的一种重要的免疫检查点机制 逃避抗肿瘤免疫反应。PD-L1与活化T细胞上的PD-1结合介导抑制信号 通过表达PD-1的T细胞。相比之下，PD-L1在髓系中的表达在功能上的作用 车厢不太清楚。CD11c+树突状细胞因其捕获肿瘤抗原和 将这些抗原交叉呈现给肿瘤引流淋巴中的T细胞，导致产生 导致肿瘤排斥反应的肿瘤特异性细胞毒性T淋巴细胞(CTL)。有趣的是，我们已经展示了 从溶酶体酸性脂肪酶基因敲除(LAL-/-)小鼠血液中分离的CD11c+细胞 在没有抗原提呈的情况下，免疫抑制，并在体外直接刺激肿瘤增殖和 肿瘤在体内生长。同时，在LAL-/-CD11c+细胞中，PD-L1的表达增加，这是 免疫抑制和肿瘤刺激。我们的观察表明，LAL缺乏诱导代谢 Switch引导CD11c+细胞产生肿瘤刺激而不是排斥反应。基于这些观察，这一点 提案将侧重于在LAL-/-CD11c+细胞中表达PD-L1如何有助于免疫抑制和肿瘤 Lal缺乏时的刺激。LAL是一种分解胆固醇酯和甘油三酯的关键酶。 细胞中产生游离脂肪酸和胆固醇的溶酶体。游离脂肪酸的代谢物是 核转录因子过氧化物酶体增殖物激活受体(PPAR)是PPAR的负性调节因子 发炎。由于LAL与脂肪酸新陈代谢密切相关，拟议中的研究将进一步阐明 LAL-/-CD11c+细胞影响肿瘤免疫刺激的潜在代谢机制 通过PD-L1的表达。RNAseq和流式细胞术表明PD-L1的表达受 在LAL-/-CD11c+细胞中的葡萄糖/谷氨酰胺代谢过程和雷帕霉素的哺乳动物靶点(MTOR) 处于PPAR的控制之下。因此，目前的提议将检验一个核心假设，即 LAL/PPAR/mTOR轴调控的PD-L1在LAL-/-CD11c+细胞免疫抑制中的关键作用 以及通过代谢重新编程来刺激肿瘤。为了实现我们的目标，我们将使用独特的Lal-/- 小鼠模型和条件c-FMS-RTTA/(Teto)7-CMV-Hlal；LAL-/-三重小鼠模型(c-FMS-Tg/KO)，其中 Hal在Lal-/-小鼠的髓鞘中特异表达。制定了以下具体目标 要检验中心假设：1)表征PD-L1表达的发育和代谢调节 CD11c+细胞中PD-L1的表达及其在调节T细胞增殖中的作用 CD11c+细胞在肿瘤刺激过程中PD-L1的表达特征。成功的结果将是 帮助设计更有效的方案，通过操纵提高PD-L1肿瘤免疫治疗的疗效 代谢途径和免疫检查点途径。

Project Summary/Abstract The long-term goal is to find better intervening approaches to interdict cancer initiation, progression and metastasis. The PD-L1/PD-1 signaling pathway is a critical immune checkpoint mechanism utilized by tumor to escape antitumor immune responses. PD-L1 binds to PD-1 on activated T cells to mediate an inhibitory signal through PD-1-expressing T cells. In comparison, the functional roles of PD-L1 expression in the myeloid compartment are less clear. CD11c+ DCs are well known for their function to capture tumor antigens and cross-present these antigens to T cells in tumor-draining lymph nodes, resulting in the generation of tumor-specific cytotoxic T lymphocytes (CTLs) that contribute to tumor rejection. Interestingly, we have shown that CD11c+ cells isolated from the blood of lysosomal acid lipase (LAL) knockout (lal-/-) mice possess immunosuppression in the absence of antigen presentation, and directly stimulate tumor proliferation in vitro and tumor growth in vivo. Concomitantly, PD-L1 expression was increased in lal-/- CD11c+ cells, which is required for immunosuppression and tumor stimulation. Our observations suggest that the LAL deficiency-induced metabolic switch leads CD11c+ cells to confer tumor stimulation rather than rejection. Based on these observations, this proposal will focus on how PD-L1 expression in lal-/- CD11c+ cells contributes to immune suppression and tumor stimulation during LAL-deficiency. LAL is a key enzyme that hydrolyzes cholesteryl esters and triglycerides in the lysosome of cells to generate free fatty acids (FFAs) and cholesterol. The metabolites of FFAs are ligands for nuclear transcription factor peroxisome proliferator activated receptor  (PPAR), a negative regulator of inflammation. Since LAL is critically involved in fatty acid metabolism, the proposed study will further elucidate the underneath metabolic mechanism by which lal-/- CD11c+ cells influence tumor immunity and stimulation through PD-L1 expression. RNAseq and flow cytometry demonstrated that PD-L1 expression is regulated by glucose/glutamine metabolic processes and mammalian target of rapamycin (mTOR) in lal-/- CD11c+ cells, which is under the control of PPAR. Therefore, the current proposal will test a central hypothesis that LAL/PPAR/mTOR axis-regulated PD-L1 expression plays a key role in lal-/- CD11c+ cells’ immune suppression and tumor stimulation through metabolic reprogramming. To achieve our goals, we will use the unique lal-/- mouse model and the conditional c-fms-rtTA/(TetO)7-CMV-hLAL;lal-/- triple mouse model (c-fms-Tg/KO) in which hLAL is specifically expressed in the myeloid compartment of lal-/- mice. The following specific aims are designed to test the central hypothesis:1) Characterizing the developmental and metabolic regulation of PD-L1 expression in CD11c+ cells; 2) Characterizing PD-L1 expression of CD11c+ cells in regulating T cell proliferation and functions; 3) Characterizing PD-L1 expression of CD11c+ cells in tumor stimulation. The successful outcome will help to design a more efficient regimen to increase the efficacy of PD-L1 cancer immunotherapy by manipulating the metabolic pathway and the immune checkpoint pathway.