髓系来源抑制细胞（myeloid-derived suppressor cell，MDSC）具有强大的抑制T细胞激活和功能的免疫活性，在癌症的扩散和转移中发挥重要作用。MDSC由一群异质性髓系细胞组成，包括巨噬细胞、粒细胞和发育未成熟阶段的树突状细胞。稳态状态下不存在MDSC，但病理条件下，MDSC被诱导产生。在癌症、自身免疫和感染性疾病中，循环MDSC的频率显著增加，且总与疾病的严重程度相关，并降低生存率。MDSC在骨髓或紧急造血域中由造血干细胞（hematopoietic stem cell，HSC）逐步发育分化而来，MDSC可以从骨髓释放到外周血中，并进一步迁移到病灶区域发挥作用。MDSC可以通过分泌抑炎细胞因子，高表达抑制性受体相应配体从而抑制T细胞活性行使其细胞抑制功能。而MDSC的发育分化稳态调控是其功能行使的基础。目前，对于调控MDSC分化和功能活性的机制仍不完全清楚。Sirtuin蛋白2（sirtuin 2，SIRT2）作为去乙酰化酶，其催化活性依赖烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide，NAD⁺）。许多研究显示SIRT2参与多种重要生理生化活动，如糖脂代谢和细胞凋亡等。研究显示，Sirt2缺失诱发基因不稳定和肿瘤发生。研究组前期工作也显示，去乙酰化酶SIRT1负性调控MDSC分化和功能并在肿瘤免疫中发挥调控效应。基于前期工作，本论文主要研究了SIRT2对MDSC发育分化和功能的调控效应及机制，这将为靶向MDSC介导的癌症诊断和治疗提供新的实验室依据。

为研究SIRT2对MDSC的发育分化的调控作用，本研究选用了髓系细胞条件缺失Sirt2基因的小鼠（以下简称Sirt2^Δmye小鼠）开展研究。对小鼠进行小鼠结肠癌（MC38）皮下荷瘤，发现Sirt2^Δmye小鼠的肿瘤的生长速度明显增快，小鼠肿瘤局部浸润MDSC的数量明显增多，而且CD8⁺ T细胞浸润也明显增多。这提示，髓系Sirt2缺失促进MDSC集聚，抑制机体抗肿瘤免疫，有助于肿瘤生长。进一步分析肿瘤局部、骨髓、外周血和脾脏MDSC的数量和抑制功能，发现Sirt2^Δmye小鼠中全身各器官中MDSC的比例均显著上调，且表达促炎细胞因子TNFα能力下降而抑炎细胞因子Arg1能力上调。而且，小鼠腹腔瘤研究，Sirt2^Δmye小鼠也显示相似的MDSC改变。这综合说明，髓系缺失Sirt2明显促进MDSC发育分化并使其抑制功能活性增强。MDSC发育分化是从髓系前体细胞而来。进一步研究显示，Sirt2^Δmye荷瘤小鼠粒-单核系祖细胞（granulocyte-monocyte progenitor，GMP）明显增多，而不影响长时程造血干细胞（long-term HSC，LT-HSC）、共同髓样祖细胞（common myeloid progenitor，CMP）、多潜能前体细胞（multipotent progenitor，MPP）和巨核-红系祖细胞（megakaryocyte-erythrocyte progenitor，MEP）等比例变化。这提示，荷瘤小鼠髓系Sirt2缺失可能通过影响骨髓前体细胞GMP发育分化调控MDSC发育分化和功能。进一步，腹腔过继转移Sirt2^Δmye和对照小鼠的Lin^- HSC，并观察其对小鼠腹腔肿瘤的影响。结果显示，Sirt2^Δmye的Lin^- HSC过继转移入腹腔瘤小鼠，明显改变腹腔GMP分化百分率，并促进了MDSC发育分化和功能改变。相应的，CD8⁺ T细胞功能也明显下调，显示出免疫抑制的肿瘤微环境。而且，体外分选Sirt2^Δmye和对照小鼠的Lin^- HSC进行体外MDSC克隆形成实验分析。结果显示，Sirt2^Δmye的Lin^- HSC明显促进GMP及MDSC发育分化和功能。这说明，Sirt2缺失是通过调控髓系前体GMP细胞影响MDSC发育分化和功能。

SIRT2是如何调控GMP分化和功能呢？已显示，相关转录因子等在MDSC前体细胞分化中发挥重要作用。进一步体外GMP分化研究显示，粒细胞-巨噬细胞集落刺激因子受体（granulocyte-macrophage colony stimulating factor receptor，GM-CSFR）的表达水平显著上调。但是不影响巨噬细胞集落刺激因子受体（macrophage colony stimulating factor receptor，M-CSFR）以及粒细胞集落刺激因子受体（granulocyte colony stimulating factor receptor，G-CSFR）的表达水平。检测发现 spi-1原癌基因（spi-1 proto-oncogene，PU.1）, CCAAT 增强子结合蛋白家族α（CCAAT enhancer binding protein α，C/EBPα）表达上调，干扰素调节因子8（interferon regulatory factor 8，IRF8）表达下调，而不影响CCAAT 增强子结合蛋白家族β（CCAAT enhancer binding protein β，C/EBPβ）表达。这提示，SIRT2可能是通过调节GM-CSFR及其下游的PU.1, C/EBPα和IRF8等指导MDSC发育分化和功能。

研究已经显示，NAD⁺ 水平及线粒体氧化磷酸化代谢活性等调控SIRT2表达和活性。琥珀酸脱氢酶（succinate dehydrogenase，SDH）是调控线粒体代谢的关键分子。采用GMP体外分化MDSC体系，研究MDSC的分化和功能。研究发现，Sirt2^Δmye明显上调SDHA和SDHB的表达水平。而采用琥珀酸脱氢酶抑制剂丙二酸二甲酯（dimethylmalonate，DMM）阻断SDH表达和活性明显恢复Sirt2^Δmye导致的MDSC发育分化和功能的改变。采用烟酰胺磷酸核糖转移酶抑制剂达珀利奈（daporinad，FK866）处理阻断NAD⁺ 合成关键调控分子烟酰胺磷酸核糖转移酶（nicotinamide phosphoribosyl transferase，NAMPT）表达，明显恢复了Sirt2^Δmye导致的MDSC发育分化和功能变化。这综合说明，髓系Sirt2缺失调控GMP发育分化为MDSC是通过NAD⁺-SDH介导的氧化磷酸化（oxidative phosphorylation，OXPHOS）途径。

综上所述，本课题较系统地阐明了代谢调控子SIRT2靶向髓系前体细胞GMP，可通过GM-CSFR及PU.1、C/EBPα及IRF8等结合NAD⁺-SDH-OXPHOS途径调控MDSC的发育分化和功能，在肿瘤免疫调控中发挥重要作用。这些结果将为靶向SIRT2抗肿瘤免疫治疗策略研究提供新的实验依据。

Myeloid-derived suppressor cells (MDSC) have potent T-cell activation and functional immunosuppressive activities, and play an important role in cancer dissemination and metastasis. MDSC consists of a heterogeneous population of myeloid cells, including macrophages, granulocytes, and dendritic cells at the immature stages of development. MDSC is not present in the steady state, but in pathological conditions, MDSC is induced. The frequency of circulating MDSC was significantly increased in cancer, autoimmune and infectious disease, and worsened survival. MDSC is gradually differentiated from hematopoietic stem cells (HSC) in the bone marrow or emergency hematopoietic domain, and can be released from the bone marrow into the peripheral blood and further migrate to the focal area. MDSC can exercise its cytostatic function by secreting anti-inflammatory cytokines and highly expressing the corresponding ligands of inhibitory receptors to suppress T cell activity. The homeostatic regulation of MDSC development and differentiation is the basis of its function. Currently, the mechanisms that regulate the differentiation and functional activity of MDSC are still not fully understood. The mechanisms that regulate MDSC differentiation and functional activity are still not fully understood. Sirtuin protein 2 (SIRT2) acts as a deacetylase and its catalytic activity is dependent on nicotinamide adenine dinucleotide (NAD⁺). Many studies have shown that SIRT2 is involved in a variety of important physiological and biochemical activities, such as glycolipid metabolism and apoptosis. Studies have shown that Sirt2 deficiency induces genetic instability and tumorigenesis. Previous work by the research group also showed that the deacetylase SIRT1 negatively regulates MDSC differentiation and function and exerts regulatory effects in anti-tumor immunity. Based on the previous work, this thesis focuses on the regulatory effects and mechanisms of SIRT2 on MDSC developmental differentiation and function, which will provide a new laboratory basis for targeting MDSC-mediated cancer diagnosis and therapy.

In order to investigate the regulatory role of SIRT2 on the developmental differentiation of MDSC, mice conditionally deficient in Sirt2 gene in myeloid cells (hereinafter referred to as Sirt2^Δmye mice) were selected for this study. The mice were subjected to subcutaneous loading of mouse colon carcinoma (MC38), and it was found that the growth rate of tumors in Sirt2^Δmye mice was significantly increased, and the number of mice tumors locally infiltrated with MDSC was significantly increased, as well as the infiltration of CD8⁺ T cells was also significantly increased. This suggests that myeloid Sirt2 deficiency promotes MDSC clustering, suppresses the body's anti-tumor immunity and contributes to tumor growth. Further analysis of the number and inhibitory function of MDSC in the local tumor, bone marrow, peripheral blood and spleen revealed that the proportion of MDSC in all organs throughout the body was significantly up-regulated in Sirt2^Δmye mice, and the ability to express the pro-inflammatory cytokine TNFα was decreased whereas the ability to inhibit the inflammatory cytokine Arg1 was up-regulated. Moreover, mouse peritoneal tumor studies, Sirt2^Δmye mice also showed similar MDSC alterations. This collectively suggests that myeloid deletion of Sirt2 markedly promotes MDSC developmental differentiation and results in enhanced inhibitory functional activity. MDSC developmental differentiation is derived from myeloid precursor cells. Further studies showed that granulocyte-monocyte progenitor (GMP) was significantly increased in Sirt2^Δmye tumor-bearing mice without affecting long-time haematopoietic stem cells (LT-HSC), common myeloid progenitor (CMP), multipotent progenitor (MPP) and megakaryocyte-erythrocyte progenitor (MEP). This suggests that myeloid Sirt2 deletion in tumor-bearing mice may regulate MDSC developmental differentiation and function by affecting bone marrow precursor cell GMP developmental differentiation. Further, Lin^- HSC from Sirt2^Δmye and control mice were intraperitoneally overtransferred and their effects on abdominal tumors in mice were observed. The results showed that Sirt2^Δmye of Lin^- HSC overtransferred into peritoneal tumor mice significantly altered the per cent of peritoneal GMP differentiation and promoted MDSC developmental differentiation and functional changes. Correspondingly, CD8⁺ T cell function was significantly down-regulated, demonstrating an immunosuppressive tumor microenvironment. Moreover, Lin^- HSC from Sirt2^Δmye and wide type mice were sorted in vitro to be analyzed for in vitro MDSC clone formation assay. The results showed that Lin^- HSC of Sirt2^Δmye significantly promoted GMP and MDSC developmental differentiation and function. This suggests that Sirt2 deletion affects MDSC developmental differentiation and function by regulating myeloid precursor GMP cells.

How does SIRT2 regulate GMP differentiation and function? It has been shown that related transcription factors, among others, play an important role in MDSC precursor cell differentiation. Further in vitro GMP differentiation studies showed that the expression level of granulocyte-macrophage colony stimulating factor receptor (GM-CSFR) was significantly upregulated. However, it did not affect the expression levels of macrophage colony stimulating factor receptor (M-CSFR) and granulocyte colony stimulating factor receptor (G-CSFR). The spi-1 proto-oncogene (PU.1), CCAAT enhancer binding protein α (C/EBPα) expression was up-regulated and interferon regulatory factor 8 (IRF8) expression was down-regulated without affecting CCAAT enhancer binding protein β (C/EBPβ) expression. This suggests that SIRT2 may be directing MDSC developmental differentiation and function by regulating GM-CSFR and its downstream PU.1, C/EBPα and IRF8.

Studies have shown that NAD⁺ levels and mitochondrial oxidative phosphorylation metabolic activity, among others, regulate SIRT2 expression and activity. Succinate dehydrogenase (SDH) is a key molecule in the regulation of mitochondrial metabolism. The GMP in vitro differentiation MDSC system was used to study the differentiation and function of MDSC. It was found that Sirt2^Δmye significantly upregulated the expression levels of SDHA and SDHB. And blocking SDH expression and activity using dimethylmalonate (DMM), a succinate dehydrogenase inhibitor, significantly restored the changes in MDSC developmental differentiation and function caused by Sirt2^Δmye. Treatment with the nicotinamide phosphoribosyl transferase inhibitor daporinad (FK866) to block the expression of nicotinamide phosphoribosyl transferase (NAMPT), a key regulatory molecule of NAD⁺ synthesis, significantly restored the Sirt2^Δmye-induced MDSC developmental differentiation and functional changes. Together, this suggests that medullary Sirt2 deletion regulates the developmental differentiation of GMP into MDSC via the NAD⁺-SDH-mediated oxidative phosphorylation (OXPHOS) pathway.

In summary, the present work has more systematically elucidated that the metabolic regulator SIRT2 targets myeloid precursor cell GMP, which can regulate the developmental differentiation and function of MDSC through GM-CSFR, PU.1, C/EBPα and IRF8 in combination with the NAD⁺-SDH-OXPHOS pathway, and plays an important role in tumor immunoregulation. These results will provide a new experimental basis for the study of anti-tumor immunotherapy strategies targeting SIRT2.