Metabolism of CD4+ Th1 and Th2 cells Público
Wojdylo, Julia (2014)
Published
Abstract
The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that integrates diverse environmental inputs and regulates cell survival, proliferation, protein synthesis, and metabolism. mTOR, a member of the phosphoinositide 3-kinase related kinase family, associates with a variety of adapter proteins to form two distinct signaling complexes, mTORC1 and mTORC2, with disparate upstream regulators and downstream effectors. T-lymphocytes are a critical component in cell-mediated immunity. T-cell receptor recognition of antigen alone fails to activate T-lymphocytes; a second antigen-presenting cell-derived costimulatory signal is necessary to prevent anergy and cell death. The immune microenvironment helps to guide CD4+ T-lymphocytes to an effector lineage commitment, which is associated with dynamic changes in the function and metabolic program of the cell. As a central signal integrator and regulator of many cellular metabolic programs, mTOR signaling is intimately associated with T-lymphocyte metabolism in the development, homeostasis, activation, and differentiation of T-lymphocytes. In the absence of mTOR, CD4+ T cells fail to differentiate into effector cells and become Foxp3+ regulatory T-cells. In addition, mTORC1 deficient cells fail to differentiate into Th1 or Th17 cells and mTORC2 deficient cells fail to differentiate into Th2 cells under appropriate skewing conditions. Previous studies have shown that mTORC1 deficient cells are not highly glycolytic, yet the highly glycolytic Th2 cells can differentiate from mTORC1 deficient cells. To determine bioenergetic and biosynthetic differences between Th1 and Th2 cells, CD4+ T cells were isolated from 5C.C7 mice, stimulated for 48 hours under Th1 and Th2 skewing conditions, and then expanded with IL-2 for 5 days. Following expansion, cells were rechallenged with plate-bound anti-CD3 and anti-CD28 for 12 hours. Metabolic profiles of the cells were examined during the induction, resting, and rechallenge phase of Th1 and Th2 cells using RT-PCR and XF96 Analyzer. During induction and upon restimulation of Th1 and Th2 cells, it was found that both Th1 and Th2 cells upregulate glycolytic and pentose phosphate pathway machinery, and downregulate machinery involved in fatty acid oxidation. However, differences in levels of metabolic gene expression observed in Th1 and Th2 cells upon rechallenge suggest a more memory-like role for Th2 cells.
Table of Contents
LITERATURE REVIEW. 1
Identification of mTOR. 1
mTOR Structural Domains. 1
mTOR Signaling Complexes. 2
mTORC1 Signaling Pathway. 5
mTORC2 Signaling Pathway. 11
Regulation of T cell Differentiation, Activation, and Function by mTOR. 12
Naïve T cells. 13
T cell Activation. 13
EXPERIMENTAL PROCEDURES. 20
Mice. 20
Isolation of CD4+ T lymphocytes. 20
T cell Stimulation and Skewing. 21
Ficoll Extraction. 22
Total RNA Extraction. 22
cDNA Synthesis. 23
Real-Time PCR. 24
Seahorse Assay. 24
EXPERIMENTAL RESULTS. 26
Establishing the Generation and Kinetics of Th1 and Th2 cells. 26
Metabolism During Induction Phase of Th1 and Th2 cells. 27
Metabolism Upon Rechallenge of Resting Th1 and Th2 cells. 32
Effect of mTOR Kinase Inhibitors Upon Rechallenge of Resting Th1 and Th2 Cells. 37
DISCUSSION. 39
LITERATURE CITED. 44
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