Nutrient Condition in the Microenvironment Determines Essential Metabolisms of CD8+ T Cells for Enhanced IFNγ Production by Metformin

Abstract

Metformin (Met), a first-line drug for type 2 diabetes, lowers blood glucose levels by suppressing gluconeogenesis in the liver, presumably through the liver kinase B1-dependent activation of AMP-activated protein kinase (AMPK) after inhibiting respiratory chain complex I. Met is also implicated as a drug to be repurposed for cancers; its mechanism is believed identical to that of gluconeogenesis inhibition. However, AMPK activation requires high Met concentrations at more than 1 mM, which are unachievable in vivo. The immune-mediated antitumor response might be the case in a low dose Met. Thus, we proposed activating or expanding tumor-infiltrating CD8+ T cells (CD8TILs) in a mouse model by orally administering Met in free drinking water. Here we showed that Met, at around 10 μM and a physiologically relevant concentration, enhanced production of IFNγ,TNFα and expression of CD25 of CD8+ T cells upon TCR stimulation. Under a glucose-rich condition, glycolysis was exclusively involved in enhancing IFNγ production. Under a low-glucose condition, fatty acid oxidation or autophagy-dependent glutaminolysis, or both, was also involved. Moreover, phosphoenolpyruvate carboxykinase 1 (PCK1), converting oxaloacetate to phosphoenolpyruvate, became essential. Importantly, the enhanced IFNγ production was blocked by a mitochondrial ROS scavenger and not by an inhibitor of AMPK. In addition, IFNγ production by CD8TILs relied on pyruvate translocation to the mitochondria and PCK1. Our results revealed a direct effect of Met on IFNγ production of CD8+ T cells that was dependent on differential metabolic pathways and determined by nutrient conditions in the microenvironment.

Low glucose availability potentiates the effects of metformin on model T cell activation and exhaustion markers in vitro

“…In all of these cases (except for proliferation), there was significant statistical interaction between metformin and glucose. This is consistent with a recent report observing a reduction of IFN-γ secretion using low metformin concentration in combination with glycolysis inhibition by 2DG ( 25 ). Mechanistically, reducing the glycolytic flux with low glucose and suppressing the TCA cycle flux by blocking its main electron sink (the electron transfer chain) should result in reduced phosphoenolpyruvate levels ( 86 ), leading to a strong suppression of IFN-γ secretion as observed in our study.…”

“…Glucose has been shown to be a key nutrient determining the response to metformin in cancer cells ( 9 , 35 ) as well as being involved in regulating T cell function ( 45 , 52 ). A very recent report has also pointed to the role glucose availability might play in mouse CD8+ T cell response to metformin ( 25 ). While other nutrients can importantly impact the T cell function both directly ( 44 , 69 ) and indirectly ( 70 – 72 ), our results confirmed the key role of the glucose as opposed to the glutamine ( Figure 1 ) or pyruvate (results not shown) in survival and proliferation of Jurkat cells used as a T cell model.…”

“…The excess glucose consumption by tumor cells is a form of tumor suppression of T cell responses ( 49 , 50 ), which require T cell metabolic fitness with sufficient mitochondrial mass ( 51 ) and considerable metabolic plasticity ( 52 ) to function in such low-glucose environments. The inhibition of respiration by metformin could reduce this metabolic plasticity ( 25 ), and the combination of metformin and glycolysis inhibitor 2-deoxy-D-glucose (2DG) significantly suppressed T cell functions ( 53 ). However, metformin treatment can also lead to increased T cell mitochondrial mass ( 34 , 54 , 55 ), so it is necessary to investigate the effect of metformin on T cells at low as well as normal and elevated glucose levels.…”

“…to determine the reliance of T cells on mitochondrial metabolism to produce IFN-γ, but only low metformin concentrations and 3-4 mM versus 12 mM glucose were used ( 25 ). Moreover, this and another study using in vivo glucose supplementation with metformin treatment only used mouse CD8+ T cells ( 25 , 26 ). Additionally, despite the emerging chronic effects of diabetes and hyperglycemia on T cell function ( 65 ), the shorter term impact of high glucose concentrations (11-25 mM) routinely used in T cell culture media as opposed to physiological (~5 mM) levels has hardly been studied in a controlled in vitro setting, especially with metformin treatment.…”

Glycolysis in tumor microenvironment as a target to improve cancer immunotherapy

“…Metformin regulates metabolic signature by interacting with the PI3K-AKT-mTOR axis and HIF-1α ( Nozhat et al, 2018 ; Shao et al, 2020 ), both of which are the main regulators of Warburg effects. A bulk of studies have validated that metformin also promotes the cancer-killing functions of CD8 + T cells in metabolic regulation-dependent manners ( Pearce et al, 2009 ; Chen et al, 2021b ; Chao et al, 2022 ). Metformin treatment downregulates immune checkpoint expression and glycolytic cancer flux in a HIF-1α inhibition-dependent manner, thereby improving ICB therapy ( Chung et al, 2021 ; Song et al, 2022b ).…”