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Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes
Published on: June 16, 2011
T-cell development of resistance to apoptosis is driven by a metabolic shift in carbon source and altered activation
C D Bortner1, A B Scoltock1, D W Cain1
1Signal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC, USA.
Abstract:
We developed a model system to investigate apoptotic resistance in T cells using osmotic stress (OS) to drive selection of death-resistant cells. Exposure of S49 (Neo) T cells to multiple rounds of OS followed by recovery of surviving cells resulted in the selection of a population of T cells (S49 (OS 4-25)) that failed to die in response to a variety of intrinsic apoptotic stimuli including acute OS, but remained sensitive to extrinsic apoptotic initiators. Genome-wide microarray analysis comparing the S49 (OS 4-25) with the parent S49 (Neo) cells revealed over 8500 differentially regulated genes, with almost 90% of those identified being repressed. Surprisingly, our data revealed that apoptotic resistance is not associated with expected changes in pro- or antiapoptotic Bcl-2 family member genes. Rather, these cells lack several characteristics associated with the initial signaling or activation of the intrinsic apoptosis pathway, including failure to increase mitochondrial-derived reactive oxygen species, failure to increase intracellular calcium, failure to deplete glutathione, failure to release cytochrome c from the mitochondria, along with a lack of induced caspase activity. The S49 (OS 4-25) cells exhibit metabolic characteristics indicative of the Warburg effect, and, despite numerous changes in mitochondria gene expression, the mitochondria have a normal metabolic capacity. Interestingly, the S49 (OS 4-25) cells have developed a complete dependence on glucose for survival, and glucose withdrawal results in cell death with many of the essential characteristics of apoptosis. Furthermore, we show that other dietary sugars such as galactose support the viability of the S49 (OS 4-25) cells in the absence of glucose; however, this carbon source sensitizes these cells to die. Our findings suggest that carbon substrate reprogramming for energy production in the S49 (OS 4-25) cells results in stimulus-specific recognition defects in the activation of intrinsic apoptotic pathways.
Insights
We created T cells resistant to apoptosis using osmotic stress. These cells resist intrinsic apoptosis triggers but not extrinsic ones, showing altered metabolism and glucose dependence.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is crucial for T cell homeostasis.
- Understanding mechanisms of apoptotic resistance is vital for immune regulation and disease.
- Osmotic stress (OS) can be used to select for cells with altered apoptotic pathways.
Purpose of the Study:
- To develop and characterize a model system of T cells resistant to apoptosis.
- To investigate the molecular and metabolic changes associated with acquired apoptotic resistance.
- To identify novel pathways involved in T cell survival and death.
Main Methods:
- Selection of T cells (S49) resistant to apoptosis via repeated osmotic stress (OS).
- Genome-wide microarray analysis to compare gene expression between resistant (S49 (OS 4-25)) and parent (S49 (Neo)) cells.
- Assessment of intrinsic and extrinsic apoptosis signaling pathways, mitochondrial function, and cellular metabolism.
Main Results:
- Selected T cells (S49 (OS 4-25)) exhibited resistance to intrinsic apoptotic stimuli but remained sensitive to extrinsic ones.
- Apoptotic resistance was not linked to Bcl-2 family genes but involved defects in intrinsic pathway signaling (e.g., reduced reactive oxygen species, calcium, cytochrome c release, caspase activity).
- Resistant cells displayed Warburg effect metabolism, glucose dependence for survival, and altered responses to different carbon substrates.
Conclusions:
- Acquired apoptotic resistance in T cells involves significant gene expression changes and metabolic reprogramming.
- Defects in intrinsic apoptosis signaling, rather than Bcl-2 family alterations, characterize these resistant cells.
- Metabolic substrate utilization plays a critical role in T cell survival and apoptotic sensitivity, suggesting novel therapeutic targets.
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