Why do the elderly get sick more than younger folks? Fight Aging has a good lay friendly piece on the mitochondrial aspects of T cell exhaustion. The following paper is cool enough to be published in the prestigious journal Cell. The authors found that hypoxia does something bad to the mitochondria that prevents them from producing cytokines needed to keep chronic virus infections contained. We’ve know for a long time that the real damage to cardiac mitochondria occurs when oxygen is restored. The mitochondria turn into superoxide generating machines. We might be done with the pandemic proper, but older folk are still getting Covid, the flu, and so on. Then there is the chicken pox/ shingles virus.
Schurich A, Pallett LJ, Jajbhay D, Wijngaarden J, Otano I, Gill US, Hansi N, Kennedy PT, Nastouli E, Gilson R, Frezza C, Henson SM, Maini MK. Distinct Metabolic Requirements of Exhausted and Functional Virus-Specific CD8 T Cells in the Same Host. Cell Rep. 2016 Aug 2;16(5):1243-1252. PMC free article
- Figure 1. HBV specific CD8+ T Cells express more Glut 1
- Niacin and the role of NADH/NAD+
- Figure 2. CD8+ T Cells may become hypoxic in the liver.
- Figure 3 HBV T cells, glycolysis, “immediate effector function”
- Figure 4 IL-12 for exhausted T cells not producing IFNγ?
- Cu(I)NA2 comes to the rescue
- Conclusion
This publication asked the question of why we humans might have some chronic virus infections like cytomegalovirus that are usually well contained and generally harmless. Others, like hepatitis B virus, are far from harmless. The above publication compared CMV and HBV specific T cells from the same patients. The lay reader not particularly intersected in scientific experiments is encouraged to just glance at the images to take in the hard work that goes into understanding out our bodies work.
The patients in this study had both CMV and HBV reactive T cells. Peripheral blood mononuclear cells (PBMC) were isolated from patients positive for HLA-A2+ , one of many variants of the MHC complex of T cell receptors. According to these authors, naïve T cells rely on mitochondrial oxidative phosphorylation of fatty acids. Activated T cells switch to glycolysis as a quicker source of energy even though it is less efficient.
The authors used fluorescent probe labeled peptides from HP,KLHLYSHPI, from Proimmune. As the affinity of isolated, soluble monomeric MHC-peptide complexes for their specific TCR is weak, and the interaction between the MHC-peptide and associated TCR has a half-life of approximately 10 seconds, multimerization is applied to enhance detection sensitivity. The detection sensitivity increases with the amount of MHC monomers complexed onto a multimer. For enhanced detection sensitivity, the Dextramer® technology holds an optimized number of MHC-peptides that enable efficient and sensitive detection of antigen-specific T cells. Read more about the sensitivity of the Dextramer® reagents here.
Figure 1. HBV specific CD8+ T Cells express more Glut 1
The authors are building their case that cytotoxic T cells that express the CD8 marker also express more Glut1 because they are more dependent on glycolysis. Virus specific dextramer was used to activate these T cells into cytokine production. The center histogram graph is perhaps the easiest to understand. The CMV specific CD8+ T cell population has a large number of members that just express a few Glut1 transporters on their cell membranes. The implication is that they are more reliant on fatty acid oxidation and OxPhos to meet their energy needs.
This post is skipping some of the data in Figure 1. Panel 1E asks the question of whether the Glut1 glucose transporters are actually operational. 2-NBDG is glucose with a fluorescent tag NBD. Not only are more Glut1 on the surface of HBV T cells, but they are also operational.
The argument for a copper supplement is that the ATP yield from one molecule is 38 with a copper replete electron transport chain versus 2 ATP from glycolysis alone. The Schurich group has published an excellent review on T cell metabolic requirements in chronic infections. Cholesterol and fatty acid synthesis is needed for T cell proliferation. Fatty acid oxidation was given limited mention. For now we are left with the hypothesis that Glut1 is there because O2 is not available as an electron accept in the electron transport chain. We have to remember that glucose can also be a source of electrons for OxPos ATP production.
Niacin and the role of NADH/NAD+
Our programmed death receptor 1 PD-1 in Long Covid journey began in a previous post. A follow up post examined the role of PD-1 andTim-3 in chronic versus acute LCMV infections. These cell surface receptors shut down transcription of genes for fatty acid metabolism. Sometimes we need to remember niacin, the other two thirds of BioCu1/ Copper One.
Figure 2. CD8+ T Cells may become hypoxic in the liver.
The next figure compares virus killing cytotoxic T cells from peripheral blood mononuclear populations (PBMC) to intrahepatic liquid (IHL). These cells were stimulated with the HBV dextramer. The low affinity binding (box) is really not that much different. The expression of the Glut1 passive glucose transporter is greater at p<0.05 level of confidence. The authors think that the hepatic environment may be hypoxic. If the liver is hypoxic, could the same situation exist in GI epithelial cells and the SARS-Cov2 virus?
The story continues with PBMC cultured under normoxic and hypoxic conditions. Regardless of the virus specificity, growth in hypoxic conditions increases the expression of Glut1.
This post makes use of a SeaHorse cartoon illustrating the strategy of gradually adding electron transport chain inhibitors and their use to calculate the spare and maximal respiratory capacity. The spare reserve capacity (SRC) is the maximum O2 consumption minus that which is coupled to ATP production.
Note that the PD-1+ T cells have some sort of electron transport chain defect between complex I and V.
Figure 3 HBV T cells, glycolysis, “immediate effector function”
Part of the reasoning behind these experiments was a previous study that Schurich and coauthors cited suggesting that galactose as a sugar source slows down glycolysis. It probably slows down oxidative phosphorylation (ATP production with reduction of oxygen to water. NADH is reduced to NAD+) and the ETC if fatty acid beta oxidation is not a choice.. If sugars are the only source of energy, there is still the additional step required. Glucose to glucose 6-phosphate is the first step in glycolysis. Galactokinase and Gal-1-P uridyltransferase are an additional two steps that slow down glycolysis, but still permit it.
The boxed in areas on the right hand side of the 3A charts are CD8+ T cells that contain a large amount of IFNγ cytokine, presumably in secretory vesicles. Note that T cells that have receptors for CMV have a tendency to express large amounts of IFNγ whether or not the sugar in the growth medium is glucose or galactose. Panel 3B puts numbers to go with the flow plots. The same trend for greater TNF cytokine production is also seen for CMV vs HBV.
MitoTracker stains healthy and unhealthy mitochondria
The fluorescent dye MitoTracker stains mitochondria regardless of whether or not they have a resting membrane potential. In some pathologies postulated by the authors, mitochondria may become enlarged. The possibility of mitochondrial proliferation was not discussed but could potentially be detected in this assay.
In panel D we see a larger population of HBV specific T cells have more MitoTracker dye uptake. Is this pathology or simply a failed attempt to keep up with increased energy demands. When the T cells are activated with HBV dentomers, the difference is lost.
JC-1 measures membrane potential of healthy mitochondria.
The JC-1 mitochondrial membrane potential dye was used to look at functionality.
When the membrane potential is inside negative, JC-1 enters the healthy mitochondria and aggregates in such a way to yield red fluorescence. In HBV virus specific T cells, cells with 50/50 green/red fluorescence predominate, 63.2 %. In CMV specific cells.
Figure 4 IL-12 for exhausted T cells not producing IFNγ?
This post is skipping the work with IL-12. In so many ways Anna Schurich and fellow researches published an interesting study that raises more questions about what is going on in the mitochondria in T cells, or any cells, during hypoxia. Bumping up production of INFγ with IL-12 is interesting. That IL-12 also seems to increase the output of mitochondria is more interesting and leaves us searching for an explanation of why this is.
- Why is the liver hypoxic and the many organs that CMV infects not hypoxic?
- Is the liver always hypoxic or just when the metabolic load of a meal arrives via the portal vein or when the patient takes a drug that requires metabolism by cytochrome P450?
- Could the real mitochondria damage occur when O2 is restored?
Cu(I)NA2 comes to the rescue
Marin W. et al. (2021) Mitochondria as a therapeutic target for cardiac ischemia‑reperfusion injury (Review) Int, J. Mol Med. 47: 485-499, 2021 PMC free article
The review summarizes several decades of research towards the understanding of how cardiac mitochondria become generators of superoxide when ischemia that results from a myocardio infarction is reversed too quickly by reoxygenation. Complex I becomes a generator of super oxide. The Marin review discusses therapeutic interventions to restore the NAD+ that is depleted in the ischemic heart. Nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN) are the natural precursors of NAD+. Copper is only given brief mention in this review as a cofactor in Cu/Zn SOD 1 that removes the superoxide generated by the ischemia reperfusion injured cardiac mitochondria.
Conclusion
The pandemic might be in the rear view mirror for many, but the elderly are still coming down with Covid and many other viral infections that are less of a problem for the young. The lay reader is not expected to understand all of the experiments leading to T cell mitochondria as being the smoking gun for T cell exhaustion in viral infections. We think that the combination of niacin and copper might one day prove to be therapeutic.
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