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

项目摘要 最近，Sheba医学中心的Ben Borsi博士表明，一些转移性黑色素瘤患者 对抗PD-1免疫治疗无效的可通过粪便微生物区系转移(FMT)转化为应答者 来自一名对免疫治疗完全有效的黑色素瘤患者。不幸的是，其他捐赠者和接受者 组合没有成功，这意味着另一个不受控制的因素可能决定了 微生物区系对免疫治疗的调节。同时，我们一直在使用同基因的C57BL/6小鼠 测试不同的自然产生的线粒体DNA(mtDNAB6、mtDNA129和mtDNANZB) 黑色素瘤敏感性和抗PD-L1治疗。我们发现mtDNANZB小鼠对 黑色素瘤进展并对抗PD-L1治疗有强烈反应，而mtDNA129小鼠对 黑色素瘤的生长和免疫治疗的耐受性，mtDNAB6小鼠介于两者之间。这些老鼠也不同 对mtDNANZB小鼠的肠道微生物区系和代谢组学分析显示， 与肠道微生物区系精制短链脂肪酸(SCFA)的相关性。当我们表达了 线粒体靶向抗氧化酶过氧化氢酶(MCAT)在小鼠造血线粒体中的表达 细胞，我们降低了mtDNANZB小鼠的抗肿瘤免疫反应，并改变了肠道微生物区系 MtDNAB6和mtDNANZB小鼠。这些观察结果让我们得出这样的假设：肠道微生物区系 免疫系统受线粒体基因组的调节，部分是通过线粒体的反应 免疫细胞中产生的氧气(MRO)连接肠道微生物区系、肿瘤进展和 免疫疗法。为了检验这一假设，我们提出了三个具体目标。首先，我们将评估线粒体 我们三个同源菌株的功能和MRO的产生，并与它们的免疫细胞库相关 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，我们将确定哪个亚类的造血细胞负责抗肿瘤 和通过过继细胞转移(ACT)取代mtDNA129免疫细胞的支持免疫治疗反应 MtDNANZB细胞。然后我们将在功能性免疫细胞中表达MCAT，以确定这是否否定了抗- 肿瘤和前免疫治疗的反应，并改变其微生物区系。第三，我们将使用FMT来取代 MtDNA129和mtDNANZB小鼠肠道微生物区系与三个同源菌株的比较 MtDNANZB微生物群增强mtDNA129抗肿瘤和促进免疫治疗的表型 微生物区系降低了mtDNANZB的表型。为了确认这是由MRO生产促成的，我们将 在mtDNA129小鼠负责的免疫细胞中表达MCAT并证实这阻止了诱导 在mtDNANZB小鼠体内由FMT诱导的任何抗肿瘤和促进免疫治疗的表型。迅速地 将这些发现扩展到人类线粒体DNA谱系和临床服务，布尔西博士已经同意成为合作者 亚德尼博士在CMEM和SHEBA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.