GPSM3在肿瘤诱导的髓源抑制细胞中调控其累积和功能的机制

Myeloid-derived suppressor cells (MDSCs) expand in tumor-bearing hosts and contribute to tumor-induced immunosuppression, and are therefore a significant impediment to cancer immunotherapy. However, the molecular mechanisms by which cytokines induce transcriptional cascades for the control of tumor-induced MDSCs accumulation still remain elusive. The goal of our current project is to explore the novel roles of G protein signaling modulator-3 (GPSM3) in the non-canonical model for the control of tumor-induced immunosuppression. Our preliminary data show that GPSM3 is expressed selectively in MDSCs from tumor-bearing hosts, and is up-regulated in bone marrow (BM)-derived MDSCs generated by the cytokine G-CSF, GM-CSF and IL-6. GPSM3-deficient BM progenitors display a reduced capacity to differentiate into MDSCs following G-CSF/GM-CSF stimulation. Decreased accumulation of MDSCs in GPSM3-deficient tumor-bearing hosts is linked specifically with an altered expression in the key transcriptional mediators of myeloid progenitor commitment and differentiation to the granulocytic/monocytic lineage. Moreover, the immunoregulatory activity of both tumor-induced and cytokine-induced MDSCs is dependent on GPSM3. These results have led to the novel hypothesis that GPSM3 is a master regulator of tumor-induced myelopoiesis and key driver of the accumulation of MDSCs for both immune suppression and tumor promotion. In this proposal, we will characterize further the phenotype and function of MDSCs in the tumor microenvironment, and explore the signaling pathways implicated by GPSM3 in regulating MDSCs differentiation and activation from bone marrowprogenitorsfromtumorbearersusing both gain-of-function and loss-of-function approaches. Our study will provide novel mechanisms by which GPSM3 functions as a new class of non-canonical GPCRs to fine-tune the signal duration and magnitude of key transcriptional mediators in MDSCs differentiation and activation, forming a exploratory foundation to accelerate our understanding of tumor-induced my elopoiesis. Thus, our project should have direct impact for developing new MDSC-targeted cancer therapy.

髓源抑制细胞(MDSCs)是抑制癌症患者免疫系统的主要机制之一。我们前期研究结果表明G蛋白信号传导调节因子-3(GPSM3)(一种新型非典型GPCR调节蛋白)在肿瘤来源或炎症因子诱导的MDSCs中选择性高表达。GPSM3基因缺失直接导致荷瘤小鼠中MDSCs的累积减少，并与在骨髓干祖细胞定向分化成粒/单核细胞系的关键转录调节因子表达变化密切相关。因此，我们认为GPSM3是肿瘤诱导MDSCs累积，分化和功能的一个关键调节因子。在课题中我们将进一步阐明GPSM3如何在肿瘤微环境中调节MDSCs的表型和功能，并通过基因的“功能获得”和“功能缺失”方法在临床相关小鼠肿瘤模型中去探究GPSM3介导的非典型GPCR信号通路 在调节MDSCs增殖和活化方面的作用。这些工作有助于对MDSCs在癌症发展过程中转录调控机制的理解，揭示GPSM3介导的非典型GPCR信号通路通过细胞特异性调节在抗肿瘤免疫中的作用。

背景：以往研究表明GPSM3主要在免疫细胞表达，在炎症性疾病中发挥调节作用，但是在肿瘤进展中的作用鲜有报道。本项目旨在研究GPSM3在肿瘤进展中的作用及对免疫细胞的调控。.方法：构建GPSM3缺陷型骨髓重建小鼠，对WT和GPSM3-/-小鼠分别皮下注射肿瘤细胞并监测肿瘤大小；分别从野生型MC38荷瘤小鼠和未荷瘤小鼠脾脏提取多种免疫细胞，通过qPCR检测GPSM3表达；将MC38细胞皮下接种于WT或 GPSM3-/-小鼠，通过流式细胞术检测小鼠脾脏及肿瘤中G-MDSCs和M-MDSCs的比例；WT与GPSM3-/-小鼠分别皮下注射EG7细胞，通过流式细胞术检测肿瘤浸润CD45+细胞及CD11b+细胞的比例，比较M-MDSCs与G-MDSCs的比例；将WT和GPSM-/-小鼠脾脏来源的MDSCs分别与CFSE标记的OT-I细胞进行体外培养，检测T细胞的增殖，同时用3[H]胸苷摄取量来验证；WT和GPSM3-/-小鼠分别皮下注射ID8-OVA细胞，30天后通过流式细胞术检测特异性T细胞的浸润；qPCR检测T细胞上GPSM3的表达，并对WT和GPSM3-/-小鼠脾脏来源的CD4+T细胞进行转录组学测序，分析差异基因；构建NR4A1-GFP报告小鼠，通过qPCR检测TGF-β对GPSM3-/-小鼠CD4+T细胞的调控。.结果：GPSM3的缺失可以延缓肿瘤的生长；WT小鼠无论荷瘤与否，GPSM3在MDSCs选择性高表达，在荷瘤小鼠脾脏MDSCs上的表达进一步上调，其中在G-MDSCs上表达高于M-MDSCs；肿瘤的生长过程中，小鼠脾脏及肿瘤组织中的MDSCs比例均逐渐增加，GPSM3-/-小鼠MDSCs比例低于野生型小鼠；降低GPSM3的表达可以促进EG7荷瘤小鼠中M-MDSCs细胞的增殖能力；GPSM3-/-小鼠脾脏来源的MDSCs对OT-I细胞的增殖的抑制能力减弱；GPSM3-/-小鼠中特异OVA的CD8+T细胞比明显较高；活化CD4+T细胞上GPSM3表达高于静息T细胞; TGF-β可以上调GPSM3及NR4A1的表达，GPSM3可以在TGF-β的作用下，通过调节NR4A1来调节T细胞的分化及功能。.结论：GPSM3在MDSCs选择性高表达，参与肿瘤诱导的MDSCs的聚集，调控MDSCs对T细胞的抑制功能。GPSM3的缺失可以延缓肿瘤的生长，为肿瘤的免疫治疗提供理论基础。