Supporting data for “Glucose controls the antitumor activity of Vγ9Vδ2-T cells based on AMPK activation”

The attached document includes all of the figures used in my these. The details about these figures will be described clearly in my thesis.Immunometabolism is currently great research interest. Cellular metabolism controls the biological profiles, including proliferation, differentiation and functional responses of cytotoxic lymphocytes. As a chronic metabolism syndrome, type 2 diabetes mellitus (T2DMs)characterised by an abnormally elevated levels of blood glucose has a high risk and increased incidence from many types of cancers. Glucose as one of the main nutrient for supporting the normal biological activities of cytotoxic lymphocytes may play critical roles in their ability of tumor surveillance. It has well known about the effects of dysregulated metabolism, such as lipid accumulation, on the functional responses of immune cells like T cells and NK cells.However, as a promising clinic strategy in the immune cell-based immunotherapy in cancer, the biological profile of Vγ9Vδ2-T cells in T2DMs patients, and the roles of Vγ9Vδ2-T cells on the development of T2DM-related cancers still remain unknown.Vγ9Vδ2-T cells are innate-like T cells with lytic activity, their activation is unrestricted by major histocompatibility complex (MHC), which makes them kill tumor cells quickly. Indeed, as cytotoxic lymphocytes, Vγ9Vδ2-T cells have efficient abilities against different types of tumors. They can kill tumor cells by directly secreting of cytolytic molecules or indirectly regulating the cytotoxic responses of other immune cells. However, it is unknown about the impacts of dysregulated metabolism, especially hyperglycemia on the ability of tumor immune surveillance of Vγ9Vδ2-T cells. Here we found that high glucose caused an “Warburg effect”- liked abnormal bioenergetic profile of Vγ9Vδ2-T cells, leading to a high level of lactic acid accumulation. High concentrations of lactic acid accumulation further caused complete cellular metabolism “paralysis” like decreased AMPK activation in Vγ9Vδ2-T cells, which inhibited the secretion of lytic granules through the depolarization of lytic granules and MTOC at the synapse formed between Vγ9Vδ2-T cells and the target tumor cells, resulting in impaired antitumor abilities of Vγ9Vδ2-T cells against different types of tumors. Interestingly, AMPK activation by metformin and glucose control by switching glucose from high to a normal level significantly reversed all of the abnormal biological profiles of Vγ9Vδ2-T cell induced by high glucose both in vivo and ex vivo. Our results demonstrated that hyperglycemia impaired the cytotoxic abilities of Vγ9Vδ2-T cells, which could be restored by metformin treatment and glucose control. This study suggests that glucose metabolic reprogramming by targeting AMPK activation may improve tumor immunosurveillance in the patients with T2DMs.

所附文档包含本论文中使用的全部图表，相关细节将在正文中详细阐述。免疫代谢（Immunometabolism）目前是备受关注的研究领域。细胞代谢调控细胞毒性淋巴细胞的增殖、分化及功能应答等生物学特性。作为一种慢性代谢综合征，2型糖尿病（type 2 diabetes mellitus, T2DM）以血糖异常升高为核心特征，其患者罹患多种癌症的风险显著升高、发病率亦有所增加。葡萄糖作为维持细胞毒性淋巴细胞正常生物学活性的主要营养物质之一，可能在其肿瘤监视功能中发挥关键作用。目前学界已明确代谢紊乱（如脂质蓄积）对T细胞、自然杀伤细胞等免疫细胞功能应答的影响。然而，作为癌症免疫细胞治疗中极具临床应用前景的策略，2型糖尿病患者体内Vγ9Vδ2-T细胞的生物学特性，以及Vγ9Vδ2-T细胞在2型糖尿病相关癌症发生发展中的作用仍未明晰。
Vγ9Vδ2-T细胞是一类具有溶细胞活性的固有样T细胞，其激活不受主要组织相容性复合体（major histocompatibility complex, MHC）限制，可快速杀伤肿瘤细胞。作为细胞毒性淋巴细胞，Vγ9Vδ2-T细胞对多种肿瘤均具备高效杀伤能力，可通过直接分泌溶细胞分子，或间接调控其他免疫细胞的细胞毒性应答实现肿瘤清除。但目前尚不清楚代谢紊乱（尤其是高血糖）对Vγ9Vδ2-T细胞肿瘤免疫监视功能的影响。
本研究发现，高糖环境会诱导Vγ9Vδ2-T细胞产生类似“瓦伯格效应（Warburg effect）”的异常能量代谢谱，导致乳酸大量蓄积。高浓度乳酸进一步引发细胞代谢“瘫痪”，表现为Vγ9Vδ2-T细胞中腺苷酸活化蛋白激酶（AMPK）激活水平降低，通过抑制溶细胞颗粒与微管组织中心（microtubule organizing center, MTOC）在Vγ9Vδ2-T细胞与靶肿瘤细胞形成的免疫突触处的去极化，阻碍溶细胞颗粒的分泌，最终削弱Vγ9Vδ2-T细胞对多种肿瘤的抗肿瘤活性。
有趣的是，通过二甲双胍激活AMPK通路，以及将高糖环境调整至正常血糖水平，可在体内与离体实验中显著逆转高糖诱导的Vγ9Vδ2-T细胞所有异常生物学特性。本研究结果证实，高血糖会损伤Vγ9Vδ2-T细胞的细胞毒性功能，而二甲双胍干预与血糖控制可有效恢复该功能。本研究提示，靶向调控AMPK激活以重塑葡萄糖代谢，或可改善2型糖尿病患者的肿瘤免疫监视能力。