Lactate transporter MCT1 is required for the high suppressive function of tumor infiltrating regulatory T cells

乳酸转运体MCT1是肿瘤浸润性调节性T细胞高抑制功能所必需的 摘要 癌症免疫疗法彻底改变了我们治疗癌症的方式，但大多数患者由于 几种耐药机制，包括调节性T(Treg)的募集、增殖和分化 肿瘤微环境中的细胞。而细胞毒性效应T(Tef)细胞则因 TME，Treg细胞的天然免疫抑制功能保持不变。Treg和TJeff细胞表现出不同的 新陈代谢，这可能解释了它们在TME内的功能差异。在新陈代谢方面，TME是 以低氧、低pH和限制代谢产物为特征，如葡萄糖和氨基酸。而高度的 糖酵解TJeff细胞与肿瘤直接竞争葡萄糖，最近的证据表明Foxp3，即 确定Treg细胞转录因子的谱系，可以重新编程Treg细胞的新陈代谢，使其在高乳酸条件下发挥作用， 低糖环境。假设Treg细胞是由TME内的乳酸支持的，我们培育了 乳酸转运蛋白MCT1(Slc16a1f/fFoxp3YFPCre)Treg特异性缺失的小鼠接种 他们患有B16黑色素瘤。MCT1的Treg特异性缺失导致肿瘤生长减慢和生存增加 不会导致全身性自身免疫。测量乳酸浓度，我们观察到体内的高水平 B16肿瘤相对于外周淋巴组织。虽然MCT1主要被认为是运输乳酸， 它还有其他几种底物，如丙酸，对结肠Treg细胞的研究表明，丙酸可以增强 Treg细胞的功能和分化。我们实验室的初步数据证实了这些结果，显示了Treg细胞 在丙酸条件下，Treg标记Nrp1和Helios的表达增加。然而，从这些 观察产生了两个问题，1)MCT1是否是Treg细胞功能所必需的，只有在乳酸水平高的情况下才需要，以及2)如何 非乳酸MCT1底物是否影响Treg细胞代谢？我们假设MCT1是 富含乳酸的TMEs瘤内Treg细胞功能及其底物促进氧化 新陈代谢和Treg细胞特征基因的表达。为了解决这一假设，我们首先将(1) 确定代谢不同的TME中MCT1对瘤内Treg细胞功能的需求。我们会 用代谢性不同的黑色素瘤细胞系接种Slc16a1f/f Foxp3YFPCre小鼠并测量其影响 通过海马对瘤内Treg细胞的代谢进行研究，并通过抑制实验来实现其功能。第二，我们将(2) 测定非乳酸MCT1底物丙酸对Treg细胞代谢的影响。使用Foxp3YFPCre 我们将分离小鼠的Treg细胞，然后在含有丙酸的培养液中培养它们，并测量对 并应用同位素通量分析来确定Treg细胞是如何利用丙酸的。通过 了解MCT1及其底物对Treg细胞代谢和功能的作用，我们可以更好地设计 专门抑制肿瘤内Treg细胞和提高癌症免疫疗法的治疗方法。这次培训将 通过提高我的能力，让我为学术博士后和最终的独立研究员职位做好准备 研究和沟通能力，加深了我对癌症中Treg细胞的了解。

Lactate transporter MCT1 is required for the high suppressive function of tumor infiltrating regulatory T cells Abstract Cancer immunotherapy has revolutionized the way we treat cancer, but most patients fail to respond due to several resistance mechanisms including the recruitment, proliferation, and differentiation of regulatory T (Treg) cells in the tumor microenvironment (TME). While cytotoxic effector T (Teff) cells are rendered dysfunctional by the TME, the natural immunosuppressive function of Treg cells remains intact. Treg and Teff cells exhibit distinct metabolisms which may explain the discrepancy in their function within the TME. Metabolically, the TME is characterized by hypoxia, low pH, and limiting metabolites such as glucose and amino acids. While the highly glycolytic Teff cells are in direct competition with the tumor for glucose, recent evidence suggests Foxp3, the lineage defining transcription factor of Treg cells, can reprogram metabolism of Treg cells to function in high lactate, low glucose environments. Hypothesizing that Treg cells were supported by lactic acid within the TME, we bred a mouse with a Treg specific deletion of the lactate transporter MCT1 (Slc16a1f/f Foxp3YFPCre) and inoculated them with B16 melanoma. Treg specific loss of MCT1 resulted in slowed tumor growth and increased survival without leading to systemic autoimmunity. Measuring lactic acid concentration, we observed high levels within B16 tumors relative to peripheral lymphoid tissues. While MCT1 is predominantly recognized to transport lactate, it has several other substrates, such as propionate and studies on colonic Treg cells suggest propionate enhances Treg cell function and differentiation. Preliminary data from our lab corroborate these results, showing Treg cells conditioned in propionate increased expression of Treg markers Nrp1 and Helios. However, from these observations two questions arise, 1) is MCT1 required for Treg cell function only in lactate high TMEs, and 2) how do non-lactate MCT1 substrates influence Treg cell metabolism? We hypothesize that MCT1 is required for intratumoral Treg cell function in lactate rich TMEs and that its substrates promote an oxidative metabolism and the expression of Treg cell signature genes. To address this hypothesis, first we will (1) determine the requirement of MCT1 for intratumoral Treg cell function in metabolically distinct TMEs. We will inoculate Slc16a1f/f Foxp3YFPCre mice with metabolically distinct melanoma cell lines and measure the impact on intratumoral Treg cell metabolism via Seahorse, and function via suppression assay. Second, we will (2) determine the impact of non-lactate MCT1 substrate propionate on Treg cell metabolism. Using Foxp3YFPCre mice we will isolate Treg cells then condition them in media containing propionate and measure the impact on metabolism via Seahorse and apply isotopic flux analysis to identifying how Treg cells utilize propionate. By understanding the role of MCT1 and its substrates for Treg cell metabolism and function we can better design therapies that specifically dampen intratumoral Treg cells and improve cancer immunotherapies. This training will prepare me for an academic post-doctoral and ultimately independent investigator position by enhancing my research and communication skills and deepening my knowledge of Treg cells in cancer.