抑制TIGAR介导的 CD8+T 细胞激活伴随代谢重编程在急性髓细胞白血病中的机制研究

In addition to the acute myeloid leukemia (AML) cells, effector T cells also exhibit the increased rate of aerobic glycolysis and take advantage of this metabolic pathway to generate ATP as the main energy source. T cell effector is functioned as being coupled to metabolic reprogramming processes, and that interfering with these reprogramming pathways can impair T cell responses. Tumors are also known to escape immunity via T cell dysfunction. How to eliminate AML cells and rejuvenate dysfunctional T cells are the major problems needed to be addressed. The TP53-induced glycolysis and apoptosis regulator (TIGAR) inhibits aerobic glycolysis and protects AML cells from apoptosis. We have reported high TIGAR expression in multiple leukemia cell lines, and it is an independent predictor of poor survival in AML. TIGAR is important for glycolysis of leukemia cells. Silencing TIGAR induced glycolysis through PFKFB3 upregulation and sensitized leukemia cells to glycolysis inhibition both in vitro and in HL-60 xenograft mice. Inhibition of the glycolytic pathway attenuated the activation of T cells and suppressed T cell dependent immunity. The exact mechanism of silencing TIGAR induced metabolic reprogramming in T cells is unclear. In our previous study, CTLA4 expression was aberrant up-regulated, and it was down-regulated progressively while TIGAR was suppressed in CD8+T cells from AML patients. We hypothesized that silencing TIGAR increases in the glycolytic pathway and rejuvenates dysfunction CD8+ T cells by reprogramming cellular metabolism. In order to test this hypothesis, the methods such as real time PCR, flow cytometry, bioenergetics metabolism and TIGAR knockout AML mice will be used to demonstrate the regulation mechanism of silencing TIGAR modifying metabolic and functional pathways in CD8+T cells and anti-AML activities. These changes may uncover new targets and challenges for AML immunotherapy and treatment.

AML细胞和活化的T细胞均采用糖酵解进行能量代谢。T细胞活化功能伴随代谢重编程过程，干扰代谢重编程会损害T细胞的反应。TIGAR具有抑制糖酵解和抗凋亡作用，在AML细胞中表达增高。我们曾报道TIGAR高表达是AML患者独立不佳的因素，体内外实验证实了沉默TIGAR明显促进AML细胞凋亡，并通过上调PFKFB3诱发糖酵解途径。我们前期研究发现AML患者CD8+T细胞表面的细胞毒性T淋巴细胞抗原（CTLA4）高表达，抑制TIGAR则CD8+T细胞CTLA4表达降低。为此我们提出，抑制TIGAR诱使糖酵解增加，对CD8+T细胞进行能量重编程并诱导其活化。所以我们拟构建TIGAR敲除/AML小鼠模型，采用RCR、流式细胞仪、生物能量代谢测定等，旨在明确抑制TIGAR致①AML细胞凋亡增加；②对CD8+T细胞代谢重编程；③激活CD8+T细胞抗白血病效应的作用机制，以期为AML的治疗寻找新的靶标。

急性髓细胞白血病（AML）具有高度异质性，发病机制复杂，白血病细胞的代谢变化紧伴随其分子特征的改变，从而在基因水平上调控着肿瘤细胞的恶性转化和预后，这种能量代谢的生化特征为肿瘤的靶向治疗提供了契机。我们前期的报道也证实了白血病细胞均存在利用糖酵解方式进行能量代谢，抑制糖酵解则明显增加白血病细胞的凋亡，且异常的代谢预示着不良的预后。我们前期已证实TIGAR 在多种的肿瘤疾病及恶性血液疾病中高表达，并与预后不良密切相关，是正常核型 AML 患者的独立预后不佳因素，与慢性淋巴细胞白血病患者的预后密切相关，与 CML 靶向药物伊马替尼耐药密切相关。我们之前研究表明随着 TIGAR 表达下降，PFKFB3 基因表达上调，AML 细胞的凋亡率明显增加，沉默 TIGAR 基因联合 2-DG 诱发的 AML 细胞凋亡率最为显著。抑制 TIGAR 既可以促进 AML 细胞凋亡，也可以使糖酵解增加。故我们课题拟探讨，通过抑制 TIGAR 基因表达而调控了白血病细胞的能量代谢，同时对CD8+T 细胞也进行了能量代谢重编程，起到激活 CD8+T 细胞而发挥抗白血病效应。.本课题明确TIGAR- 对于 AML 小鼠 T 细胞代谢特征的改变情况，TIGAR-能否促进 CD8+T 细胞抗 AML免疫功能的反应，对CD4+T 细胞功能有无影响。TIGAR-±2-DG 能否明显延长鼠源 AML 小鼠生存期，对小鼠的外周免疫稳态情况影响。.本课题揭示了TIGAR敲除无法恢复AML小鼠的CD4+ T细胞计数，但可显著提升CD8+ T细胞计数；CD8+ T细胞的增殖进一步增加、凋亡显著下降，可恢复CD8+ T细胞的免疫耗竭状态，从而促进CD8+ T细胞的细胞因子分泌能力和杀伤毒性。TIGAR-同时也使CD4+ T细胞分泌的IL-2和TNF-α进一步升高，TIGAR敲除联合糖酵解抑制剂可明显降低AML的肿瘤负荷，延缓AML的进展。TIGAR-小鼠能恢复初始T、记忆性T细胞和效应T细胞的比例。.本课题揭示沉默 TIGAR 在维持自身抗原的耐受下，调控了CD8+T 活化、增殖的机制，为AML的治疗寻找新的靶标。