Changes in T cell metabolism during graft-versus-host disease

 描述(申请人提供)：异基因血液和骨髓移植是一种治疗骨髓衰竭综合征、非恶性血液病和某些高危恶性肿瘤的方法。移植物抗宿主病(GVHD)是异基因移植后的主要毒性反应，也是异基因受者无复发死亡的主要原因。我的长期目标是成为一名独立的内科科学家，从事免疫学和新陈代谢的研究，并研究导致GVHD的T细胞特有的代谢变化。此应用程序的总体目标是增加 我在细胞代谢、代谢组分析和自噬方面的专业知识，然后应用这些新的技能和知识来定义特定的代谢变化，将介导GVHD的T细胞与参与有益和生理免疫反应的T细胞区分开来。我的初步数据支持这一中心假设，即GVHD期间T细胞的强劲刺激诱导AMP激活的蛋白激酶(AMPK)活性，进而驱动T细胞的新陈代谢适应，促进替代能源的使用，包括脂肪的氧化。然后，这种适应使T细胞能够在疾病驱动的激活需求增加的情况下存活下来。这项拟议研究的基本原理是，识别致病T细胞上调的新代谢途径将为GVHD和其他T细胞介导性疾病的治疗带来创新和意想不到的靶点。中心假设将通过追求三个具体目标来验证：1)阐明AMPK在推动同种异体反应性T细胞中的葡萄糖和脂肪酸代谢中的作用，2)确定AMPK在推动同种异体反应性T细胞自噬中的重要性，以及3)确定AMPK在抗病毒T细胞反应中的必要性。目的1将使用基于质谱学的方法来定量来自野生型(Wt)或AMPK-/-供者的同种异体反应性T细胞中的糖酵解、戊糖磷酸和TCA周期代谢。AIM 2将使用生化和成像方法的组合来测量wt或AMPK-/-T细胞的自噬，以及确定缺乏关键自噬蛋白的T细胞的GVHD潜力。AIM 3将利用AMPK充足或缺乏T细胞的小鼠的病毒感染模型来确定在病毒应答过程中AMPK的必要性。这些研究的结果有望通过确定引起GVHD的T细胞的特定代谢变化而产生积极影响，这些变化可能会导致GVHD和其他T细胞介导的免疫疾病的治疗新的治疗目标。因此，这些研究与减少疾病的使命直接相关，从而使人们能够活得更长、更有成就感。我的整个职业目标是成为一名独立的内科医生兼科学家，在 免疫学和新陈代谢的接口。在我的指导训练期结束时，我将自信地执行基于新陈代谢的实验，以揭示体内免疫反应中存在的新的新陈代谢机制。这将通过专注于四个培训领域来实现：1)提高我对细胞新陈代谢、代谢组学分析和代谢流量分析的知识，2)增加我在自噬和基于自噬的实验执行方面的专业知识，3)将我对同种异体免疫的知识扩展到GVHD之外，以及4)完善我的书面和口头陈述技能，特别是在赠款写作、口头陈述、 和手稿准备。拟议的研究将使我了解细胞代谢和代谢组谱所涉及的方法，包括研究设计、代谢物提取和质谱仪的使用。对自噬的研究以及与匹兹堡大学自噬专家的互动将增加我在基于自噬的分析方面的专业知识。托马斯·E·斯塔兹尔移植研究所和免疫系的许多研讨会、期刊俱乐部和实验室会议将拓宽我对同种异体免疫和移植免疫学的了解。在地方、地区和国家会议上展示我的发现将提高我的书面和口头陈述技能。正式的培训活动，包括关于同位素标记的研讨会，关于新陈代谢途径的课程作业，以及关于赠款撰写和领导力发展的研讨会，将补充我的动手培训。最后，我的导师和科学咨询委员会的专业知识，加上匹兹堡大学卓越的核心设施和学术环境，将确保我的科学和培训目标的成功，并帮助我确立未来作为一名独立内科科学家的职业生涯。

 DESCRIPTION (provided by applicant): Allogeneic blood and marrow transplantation is a cure for bone marrow failure syndromes, non-malignant hematologic disorders, and certain high risk malignancies. Graft-versus-host disease (GVHD) represents the major toxicity following allogeneic transplantation and is the leading cause of non-relapse mortality in allogeneic recipients. My long-term goals are to become an independent physician-scientist working at the interface of immunology and metabolism and to characterize the metabolic changes that are specific to the T cells that cause GVHD. The overall objectives of this application are to increase my expertise in cellular metabolism, metabolomic assays and autophagy, and then to apply these new skills and knowledge to define specific metabolic changes that differentiate T cells mediating GVHD from T cells involved in beneficial and physiologic immune responses. My preliminary data support the central hypothesis that the robust stimulation of T cells during GVHD induces AMP-activated protein kinase (AMPK) activity, which then drives metabolic adaptations in T cells and facilitates alternative energy use, including the oxidation of fat. Thes adaptations then allow T cells to survive the increased demands of disease-driven activation. The rationale for the proposed research is that identification of the novel metabolic pathways upregulated in pathogenic T cells will lead to innovative and unanticipated targets for the treatment of GVHD and other T cell-mediated diseases. The central hypothesis will be tested by pursuing three specific aims: 1) Elucidate AMPK's role in driving glucose and fatty acid metabolism in alloreactive T cells, 2) Determine the importance of AMPK in driving alloreactive T cell autophagy and 3) Determine the necessity of AMPK during anti-viral T cell responses. Aim 1 will use a mass spectrometry-based approach to quantitate glycolytic, pentose phosphate, and TCA-cycle metabolism in alloreactive T cells from wildtype (wt) or AMPK-/- donors. Aim 2 will use a combination of biochemical and imaging approaches to measure autophagy in wt or AMPK-/- T cells, as well as determine the GVHD potential of T cells lacking key autophagy proteins. Aim 3 will utilize a viral infection model in mice with either AMPK sufficient, or deficient, T cells to determine the necessity of AMPK during a viral response. The outcomes from these studies are expected to have a positive impact by defining specific metabolic changes in GVHD-causing T cells that will likely lead to new therapeutic targets for the treatment of GVHD and other T cell-mediated immune disorders. Thus, these studies are directly relevant to the mission of reducing disease so that people can live longer and more fulfilling lives. My overall career goal is to become an independent physician-scientist working at the interface of immunology and metabolism. By the end of my mentored training period, I will confidently execute metabolism-based experiments to uncover novel metabolic mechanisms present during immune responses in vivo. This will come about through dedicated attention to four training areas: 1) Improve my knowledge of cellular metabolism, metabolomic analysis and metabolic flux analysis, 2) Increase my expertise in autophagy and the execution of autophagy-based experiments, 3) Extend my knowledge of alloimmunity beyond GVHD, and 4) Refine my written and oral presentation skills, particularly in regards to grant writing, oral presentations, and manuscript preparation. The proposed studies will expose me to cellular metabolism and the methods involved in metabolomic profiling, including study design, metabolite extraction, and mass spectrometer utilization. Investigation of autophagy and interactions with autophagy experts at the University of Pittsburgh will increase my expertise in autophagy-based assays. The many seminars, journal clubs, and lab meetings at the Thomas E. Starzl Transplantation Institute, and within the Department of Immunology, will broaden my knowledge of alloimmunity and transplantation immunology. Presentation of my findings at local, regional, and national conferences will increase my written and oral presentation skills. Formal training activities, including workshops on isotope labeling, coursework on metabolic pathways, and seminars on grant writing and leadership development will supplement my hands-on training. Finally, the expertise of my mentor and scientific advisory committee, in combination with the outstanding core facilities and academic environment at the University of Pittsburgh, will ensure the success of my scientific and training goals and help establish my future career as an independent physician scientist.