Anti-tumor immunity and intestinal microbiota are modulated by mitochondrial DNA

项目摘要 最近，Sheba医学中心的Ben Boursi博士表明，一些转移性黑色素瘤患者， 抗PD-1免疫治疗难治的患者可以通过粪便微生物群转移（FMT）转化为应答者 免疫治疗完全有效的黑色素瘤患者。不幸的是，其他捐助者和受援者 组合不成功，这意味着一个额外的不受控制的因素可能决定了 免疫疗法的微生物群调节。与此同时，我们一直在使用同源C57 BL/6小鼠， 不同的天然存在的线粒体DNA（mtDNAs）（mtDNAB 6，mtDNA 129和mtDNANZB）来测试 黑色素瘤敏感性和抗PD-L1治疗。我们发现，mtDNANZB小鼠对 黑色素瘤进展，并强烈响应抗PD-L1治疗，而mtDNA 129小鼠是允许的， 黑色素瘤生长和免疫治疗难治，mtDNAB 6小鼠介于两者之间。这些老鼠也不同 在他们的肠道微生物群和代谢组学分析的mtDNANZB小鼠显示受损的脂肪酸氧化， 与肠道微生物群对短链脂肪酸（SCFA）的加工相关。当我们表达了 小鼠造血干细胞线粒体中的靶向抗氧化酶过氧化氢酶（mCAT） 细胞，我们减少了mtDNANZB小鼠的抗肿瘤免疫反应，并改变了小鼠的肠道微生物群。 mtDNAB 6和mtDNANZB小鼠。这些观察结果使我们提出了这样的假设：肠道微生物群 和免疫系统是由线粒体基因组调节的，部分是通过线粒体反应， 免疫细胞中的氧物质（mROS）产生与肠道微生物群、肿瘤进展和 免疫疗法为了验证这一假设，我们提出了三个具体目标。首先，我们将评估线粒体 功能和mROS的产生，并将其与免疫细胞库相关联 和功能然后，我们将确定这些同源菌株是否对其他菌株表现出相同的反应范围。 肿瘤类型其次，我们将确定造血细胞的哪个亚类负责抗肿瘤作用。 和通过使用过继细胞转移（ACT）以用 mtDNANZB细胞。然后，我们将在功能性免疫细胞中表达mCAT，以确定这是否会否定抗- 肿瘤和促免疫治疗反应并改变其微生物群。第三，我们将用裂变材料条约取代 将mtDNA 129和mtDNANZB小鼠的肠道微生物群与三种同类品系的肠道微生物群进行比较，以确定 mtDNANZB微生物群增强mtDNA 129抗肿瘤和促免疫治疗表型，如果mtDNA 129 微生物群减少了mtDNANZB表型。为了证实这是由mROS产生介导的，我们将 在mtDNA 129小鼠的责任免疫细胞中表达mCAT，并证实这阻断了诱导 由FMT从mtDNANZB小鼠诱导的任何抗肿瘤和促免疫治疗表型。尽快 为了将这些发现扩展到人类mtDNA谱系和临床服务，Boursi博士同意成为合作者 亚德尼博士在CMEM和舍巴都安排了职位

Project Summary Recently, it was shown by Dr. Ben Boursi, Sheba Medical Center, that some metastatic melanoma patients who are refractory to anti-PD-1 immunotherapy can be converted to responders by fecal microbiota transfer (FMT) from a melanoma patient that had a complete response to immunotherapy. Unfortunately, other donor-recipient combinations were unsuccessful implying that an additional uncontrolled factor may determine the effects of microbiota modulation of immunotherapy. Concurrently, we have been using congenic C57BL/6 mice harboring different naturally occurring mitochondrial DNAs (mtDNAs) (mtDNAB6, mtDNA129, and mtDNANZB) to test melanoma sensitivity and anti-PD-L1 therapy. We discovered that the mtDNANZB mice are highly resistant to melanoma progression and strongly respond to anti-PD-L1 therapy, while mtDNA129 mice are permissive for melanoma growth and refractory to immunotherapy, with mtDNAB6 mice being in between. These mice also differ in their gut microbiota and metabolomic analysis of the mtDNANZB mice revealed impaired fatty acid oxidation of relevance to the elaboration of short chain fatty acids (SCFAs) by the gut microbiota. When we expressed the mitochondrially-targeted antioxidant enzyme catalase (mCAT) in the mitochondria of the mouse hematopoietic cells, we diminished the anti-tumor immune response of the mtDNANZB mice and changed the gut microbiota of both the mtDNAB6 and mtDNANZB mice. These observations led us to the hypothesis that: Both the gut microbiota and the immune system are modulated by the mitochondrial genome, in part through mitochondrial reactive oxygen species (mROS) production in immune cells linking the gut microbiota, tumor progression, and immunotherapy. To test this hypothesis, we propose three specific aims. First, we will evaluate mitochondrial function and mROS production in our three congenic strains and correlate this with their immune cell repertoire and function. Then, we will determine if these congenic strains show the same range of responses to other tumor types. Second, we will determine which subclass of hematopoietic cells are responsible for the anti-tumor and pro-immunotherapy response by using adoptive cell transfer (ACT) to replace mtDNA129 immune cells with mtDNANZB cells. We will then express mCAT in the functional immune cells to determine if this negates the anti- tumor and pro-immunotherapy response and changes their microbiota. Third, we will use FMT to replace the gut microbiota of the mtDNA129 and mtDNANZB mice with that of the three congenic strains to determine if mtDNANZB microbiota enhances the mtDNA129 anti-tumor and pro-immunotherapy phenotype and if mtDNA129 microbiota diminish the mtDNANZB phenotype. To confirm that this is mediated by mROS production, we will express mCAT in the responsible immune cells of the mtDNA129 mice and confirm that this blocks the induction of any anti-tumor and pro-immunotherapy phenotype induced by FMT from mtDNANZB mice. To expeditiously extend these findings to human mtDNA lineages and clinical service, Dr. Boursi has agreed to be a collaborator and Dr. Yardeni has arranged positions at both CMEM and Sheba.