Date: Monday, June 3, 2019
Session Time: 2:30pm-4:00pm
Presentation Time: 2:42pm-2:54pm
Location: Room 311
*Purpose: Mechanistic target of rapamycin complex 2 (TORC2) deficiency in conventional dendritic cells (DC) results in their enhanced pro-inflammatory and T cell allostimulatory activity; however, underlying mechanisms remains unclear.
*Methods: A Seahorse XFe96 Bioanalyzer was utilized to measure metabolic flux in real-time for bone marrow-derived DC generated from wild-type control (Ctrl) C57BL/6 (B6) or CD11c-CreRictorf/f (herein referred to as TORC2-/- DC) B6 mice, in the absence or presence of rapamycin. ATP concentrations and mitochondrial mass were determined using an ATP determination kit and flow cytometry. The Golgi apparatus was visualized by confocal and transmission electron microscopy. A mouse nCounter immunology panel was used to analyze the transcriptional profiles of TORC2-/- DC versus Ctrl DC.
*Results: TORC2-/- DC used an altered metabolic program compared to Ctrl DC, characterized by enhanced baseline glycolytic function, increased dependence on glycolytic ATP production and higher viability following LPS stimulation. TORC2-/- DC exhibited an increased spare respiratory capacity (SRC) compared to Ctrl DC. This metabolic phenotype corresponded with increased mitochondrial mass and failure of TORC2-/- DC mitochondria to depolarize following stimulation. TORC2-/- DC displayed more compact Golgi stacks with less perinuclear localization compared with Ctrl DC. Rapamycin-mediated inhibition of mTORC1 activity in TORC2-/- DC led to loss of their enhanced SRC and glycolytic activity. The altered metabolic activity of TORC2-/- DC could be ascribed to enhanced TORC1 activity, namely increased expression of multiple genes upstream of Akt/TORC1 activity, including the integrin alpha IIb, protein tyrosine kinase 2/focal adhesion kinase, IL-7R and Janus kinase 1, culminating in increased expression of the transcription factors peroxisome proliferator-activated receptor gamma and sterol regulatory element-binding transcription factor 1.
*Conclusions: These findings suggest a novel role for TORC2 in restricting TORC1-driven metabolic functions and mitochondrial regulation in DC and have implications for the impact of TORC2 inhibition on immune reactivity.
To cite this abstract in AMA style:Dai H, Watson A, Menk A, Stolz D, Delgoffe G, Thomson A. mTORC2 Restricts mTORC1 Metabolic Function in Dendritic Cells to Limit Their Allostimulatory Activity [abstract]. Am J Transplant. 2019; 19 (suppl 3). https://atcmeetingabstracts.com/abstract/mtorc2-restricts-mtorc1-metabolic-function-in-dendritic-cells-to-limit-their-allostimulatory-activity/. Accessed November 15, 2019.
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