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.
Allogeneic blood and marrow transplantation continues to be a curative modality for many diseases (e.g. high-risk leukemia, bone marrow failure syndromes, and immunodeficiency), but the widespread application of allogeneic transplantation remains limited by the significant morbidity and mortality of graft-versus-host disease (GVHD). The research and training proposed in this application is relevant to public health because an improved understanding of GVHD pathogenesis, through increased training in metabolic analysis and an innovative study of T cell metabolism, has the potential to uncover novel therapies to treat GVHD and other T cell-mediated immune disorders. This advancement in understanding will increase the safety and accessibility of allogeneic transplantation and thus fulfill the NIH mission of developing fundamental knowledge to reduce the burdens of human illness.
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