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NAD+ Supplementation Hinders STING-Triggered Senescence in CD8+ T Cells Through Mitochondrial Homeostasis Enhancement

NAD+ Supplementation Hinders STING-Triggered Senescence in CD8+ T Cells

Researchers Bin Ye, Ning Ma, and Yanfen Zhang from Harbin Medical University investigated whether improving mitochondrial homeostasis could alleviate the aging phenotype. Their findings demonstrated that mitochondrial dysfunction and a senescence-associated secretory phenotype (SASP) are present in aged T cells and tumor-bearing mice. Enhancing NAD+ levels through NMN supplementation promotes mitochondrial autophagy, preventing cellular aging and SASP. Additionally, NMN treatment can suppress neuroinflammation and extend the lifespan of mice.

According to research, NMN supplementation can increase NAD+ levels and reduce T-cell aging signs. Additionally, cells can remove damaged mitochondria through mitophagy. Additionally, NMN has been shown to slow the aging process, decrease inflammation in the brain, and prolong life expectancy in mouse models. These findings suggest that mitophagy could be a promising therapeutic avenue for preserving T-cell function and promoting longevity.


As we age, our physical and physiological capabilities tend to decline. On a cellular level, this manifests as cellular senescence—the progressive deterioration of essential cellular functions such as communication and replication, leading to the death or removal of aging cells by their healthier counterparts. This cellular turnover is a vital aspect of life at the microscopic scale and mirrors the aging process of individual cells.

As individuals age, an imbalance emerges between proinflammatory and anti-inflammatory factors, giving rise to a persistent, low-grade inflammatory state known as inflammation. This condition gradually intensifies and is intricately linked to immune senescence, which refers to the diminished efficiency and accuracy of the aging adaptive immune system. The natural deterioration of the thymus gland, vital to developing and functioning T cells, B cells, NK cells, and monocytes, is responsible for decreasing the immune system’s efficiency.

Did you know that our body’s immune system changes as we age? Specifically, the T-cells, those essential cells that fight off diseases and infections, undergo some significant changes. It’s fascinating to think about how our body adapts over time. The substantial reduction in the number and effectiveness of T lymphocytes is a critical factor in the weakened immune responses observed in older people. Consequently, exploring the connection between T lymphocytes and aging is becoming a vital area of research to understand the immunological mechanisms behind the aging process.

As individuals age, memory T cells become highly differentiated, which results in the decreased expression of costimulatory molecules such as CD27 and CD28. The reduction causes T cell senescence and accompanies molecular indicators of aging, such as mitochondrial dysfunction and epigenetic alterations.T cell senescence is also associated with nuclear DNA damage, the release of mitochondrial DNA (mtDNA), telomere shortening, and the activation of aging-related signaling pathways.

As T cells age, a specific outcome is the appearance of cytoplasmic nuclear DNA. This DNA can activate the innate immune response via the DNA-sensing pathway, which involves the cyclic GMP-AMP synthase (cGAS)-STING (stimulator of interferon genes). This pathway detects cytosolic DNA and triggers interferon production, leading to an inflammatory response to combat perceived foreign entities. The activation of the cGAS-STING pathway has been observed in various senescent primary cells and is a hallmark of cellular senescence.

Senescent cells often exhibit a senescence-associated secretory phenotype (SASP), which can attract and modulate immune cells while transforming the tissue microenvironment. In age-related diseases such as ataxia and progeria, cells display extranuclear DNA aggregates that initiate an innate immune response through the DNA-sensitive cGAS-STING pathway. This pathway’s activation promotes inflammation, induces cell cycle arrest, and accelerates cellular aging. This process occurs more rapidly than the gradual cell cycle halt caused by telomere attrition.

In models of Alzheimer’s disease, impaired mitochondrial autophagy in the brain leads to the accumulation of mtDNA in the cytoplasm, which activates the cGAS-STING pathway, further contributing to inflammation and aging. These findings suggest that targeting STING signaling could be a potential strategy for mitigating immune cell senescence.

This study reveals that T-cell senescence provoked by malignant tumor cells is a common characteristic within the suppressive tumor microenvironment. The research found that compromised mitochondria cause an accumulation of cytoplasmic DNA, leading to a STING-dependent senescence response in the brain and in vitro. Enhancing NAD+ levels using nicotinamide mononucleotide (NMN) facilitates the clearance of damaged mitochondria through mitophagy, thereby averting senescence and the senescence-associated secretory phenotype (SASP) in aging models. These insights directly connect the tumor microenvironment and the processes of senescence, SASP, and disrupted mitochondrial balance.


The development of T cell senescence is a general feature of the tumor microenvironment.

The suppression and dysfunction of tumor-reactive T cells within the tumor microenvironment pose significant challenges to the effectiveness of tumor immunotherapy. This study explored the presence of senescent T cell populations within the tumor microenvironment in vivo. We established a murine melanoma model by inducing tumor growth using the B16F0 melanoma cell line. Once the tumors grew to a diameter of 10-15 mm, we isolated T cells from the spleens of mice and analyzed them.

Mice bearing B16F10 melanoma tumors exhibited increased levels of senescence-associated beta-galactosidase-positive (SA-β-gal+) CD8+ T cells, whereas this feature was not observed in tumor-free mice used as control. The T cells that were senescent and associated with SASP showed a lower ratio of CD4+ to CD8+ T cells. CD8+ T cells from mice with B16F10 melanoma tumors expressed significantly higher levels of senescence markers than those from tumor-free mice. Additionally, CD8+ T cells from the spleens of melanoma-bearing mice showed heightened mRNA expression of proinflammatory cytokines, including IL1-β, IL-6, TNF-α, and IFN-γ, relative to their tumor-free counterparts.

Protein levels of senescence markers associated with the SASP were also quantified using enzyme-linked immunosorbent assays (ELISA). The data suggest that the induction of T cell senescence may be a strategic mechanism cancers utilize to impair T cell function and evade immune surveillance.

Mitochondrial dysfunction in senescent CD8+ T cells

Cellular aging compromises various functions, including the critical role of mitochondria as the cell’s powerhouses. The main focus of this study was to investigate the influence of senescent CD8+ T cell dysfunction on mitochondrial performance. The study results are consistent with the recent findings that link cellular senescence to mitochondrial anomalies. Senescent cells were found to have higher levels of reactive oxygen species (ROS), reduced ATP production, and depolarized mitochondrial membrane potential.

Given the significance of NAD+ in DNA repair, mitochondrial stability, senescence, and longevity across various organisms, the study also assessed whether senescent CD8+ T cells experience disturbances in NAD+ concentration and the NAD+/NADH ratio. The results showed that senescent cells had lower levels of both than controls.

Furthermore, considering the role of cytosolic mtDNA from impaired mitochondria as a potential inflammatory agent, the study measured mtDNA release in senescent CD8+ T cells. The study quantified specific mitochondrial and nuclear genes through cytosolic and nuclear fractionation, followed by qPCR analysis. The cytoplasmic DNA was confirmed to be highly pure, with negligible nucleolysis. The findings showed a significant increase in free mtDNA levels in the cytoplasm of senescent CD8+ T cells compared to controls, suggesting that mtDNA leakage from damaged mitochondria could trigger inflammation and further senescence in CD8+ T cells.

Development of T cell senescence and tumor microenvironment

Mitochondrial dysfunction in senescent CD8+ T cells

The cGAS‐STING‐IRF3 pathway was activated in senescent CD8+ T cells.

Previous research has shown that senescent cells can release damaged DNA into the cytoplasm, potentially activating the STING pathway and promoting the production of inflammatory factors that contribute to senescence. This study aimed to verify whether the cGAS-STING pathway is active in senescent CD8+ T cells. Elevated mRNA levels of cGAS, STING, TBK1, and IRF3 were confirmed in these cells, along with increased expression of proinflammatory cytokines.

Immunofluorescence analysis indicated that STING expression was heightened and clustered around the nucleus in senescent CD8+ T cells. The phosphorylation of TBK1 and IRF3 was also observed, suggesting their activation. This was further supported by the upregulation of mRNA for interferon-stimulated genes (ISGs) downstream of cGAS/STING, such as Ifit1, Isg15, and Ccl5.

Interestingly, the study also noted a significant increase in the expression of PINK1, a kinase involved in regulating mitochondrial dysfunction and initiating mitophagy, whose role in CD8+ T cell aging is not yet fully understood.

Inhibition of STING inhibits activation of the cGAS‐STING and alleviates cellular senescence.

The study investigated whether the tumor microenvironment influences the aging of CD8+ T cells via the cGAS-STING pathway. Using H-151, a specific STING inhibitor, the research found that inhibiting STING reduced the presence of senescence markers in CD8+ T cells. The treated cells showed reduced STING enrichment, as demonstrated by immunofluorescence. Further analysis found that while cGAS expression remained high, the inhibitor significantly suppressed STING activation and its downstream effects.

Protein and mRNA assessments confirmed that the cGAS-STING pathway is involved in aging. However, H-151 treatment notably decreased the expression of senescence signaling mediators and the production of SASP factors, as measured by ELISA and qPCR. Additionally, a senescence-associated β-galactosidase (SA-β-gal) assay indicated that H-151 treated cells had fewer SA-β-gal-positive cells compared to untreated senescent CD8+ T cells, suggesting that STING activation is critical for the development of senescence phenotypes.

NMN ameliorates STING‐mediated senescence by improving mitochondrial functions and reduces cytoplasmic mtDNA

The study explored whether increasing intracellular NAD+ levels with NMN (Nicotinamide Mononucleotide) could counteract senescence in CD8+ T cells. NMN supplementation was found to restore NAD+ levels, improve the NAD+/NADH ratio, and reduce mitochondrial DNA (mtDNA) in the cytoplasm of senescent cells.

When given, NMN reduces the amount of harmful reactive oxygen species in the body and decreases the energy levels in the mitochondria. The treatment also positively affected ATP levels in the cells, enhancing mitochondrial function.

Additionally, NMN appeared to suppress the activation of the cGAS-STING pathway, similar to the effects observed with the STING inhibitor H-151. This was evidenced by reduced expression of cGAS, STING, and the phosphorylated forms of TBK1 and IRF3. NMN also significantly lowered the secretion of inflammatory cytokines such as IL-1β, IL-6, TNF-α, and IFN-γ and decreased the number of senescence-associated β-galactosidase (SA-β-gal) positive cells, indicating a reduction in cellular senescence.

NMN prevents neuroinflammation and senescence in mice

Studies suggest preventing senescence in tumor-specific T cells is critical for antitumor immunity. Current in vitro studies indicate that the cGAS-STING pathway serves as a crucial checkpoint for regulating T cell senescence and function that are mediated by tumor cells. An intraperitoneal injection of NMN (500 mg/kg per mouse) once every three days in tumor-induced immunosenescence mice for 21 days showed that NMN treatment inhibited tumor growth and extended survival.

cGAS‐STING‐IRF3 pathway was activated in senescent CD8+ T cells

Tumor growth rates were significantly slower in NMN-treated mice compared to controls. NMN treatment also reduced spleen enlargement associated with tumor-induced senescence. Molecular analysis revealed that NMN inhibited the mRNA expression of inflammatory and senescence markers like IL-1β, IL-6, IFN-γ, and P21 in the brain, liver, and spleen. Furthermore, NMN treatment increased the fractions of CD4+ and CD8+ T cells in the spleen.

The NAD+/NADH ratio, disrupted by tumor presence, improved in the brain and spleen with NMN treatment. NMN also decreased the activation of the cGAS-STING pathway and prevented cellular senescence in the spleen.

Inhibition of STING inhibits activation of the cGAS

These findings suggest that NMN treatment can enhance antitumor activity, reduce neuroinflammation, improve aging conditions, and potentially prolong the survival of mice by modulating the STING pathway. This research points to the potential of NMN in cancer therapy and age-related disease management.


As life expectancy increases, it’s crucial to understand how aging leads to more significant disease susceptibility. The study finds that mitochondrial dysfunction contributes to the aging of CD8+ T cells, with damaged mitochondria releasing mtDNA into the cytoplasm, activating the STING pathway, and exacerbating senescence and the senescence-associated secretory phenotype (SASP). Boosting NAD+ levels can improve mitochondrial function, reduce cytoplasmic DNA, and inhibit STING activation, which enhances motor function, reduces inflammation, and slows aging in CD8+ T cells.

Mitochondrial dysfunction is linked to senescence and neurodegenerative diseases. Inflammatory cytokines drive senescence and the immune response that clears senescent cells. Aging is often accompanied by inflammation and autoimmune conditions. Cellular senescence, caused by growth arrest due to telomere shortening or stress responses like DNA damage, is a significant factor in age-related dysfunction. DNA damage responses cause permanent cell cycle arrest to maintain genomic stability. T cells can become senescent due to repeated stressors, with naive T cells shortening telomeres through proliferation and memory T cells having shorter telomeres from extensive replication. Additionally, DNA damage responses in proliferating T cells can impair vaccine responses in older individuals.

NMN ameliorates senescence by improving mitochondrial functions

The study highlights the role of inflammatory responses in aging, which may be connected to increased STING pathway activity. In vitro observations showed cytoplasmic dsDNA accumulation activating STING, leading to inflammation, senescence, and a decline in health span in model mice. Inhibiting STING reversed senescence phenotypes, pointing to STING as a critical regulator in aging.

Aging is associated with a decline in the cellular NAD+ pool, leading to mitochondrial dysfunction and senescence in CD8+ T cells. Damaged mitochondria release mtDNA, triggering inflammation and contributing to senescence. Enhancing mitochondrial function prevents senescence and the associated secretory phenotype (SASP).

Research shows that mitochondrial stress elevates proinflammatory cytokines and senescence. In tumor-bearing mice, impaired mitochondria release mtDNA into the cytoplasm, promoting senescence. Increasing NAD+ levels can activate mitophagy to remove damaged mitochondria, suggesting that treatments like NMN can prevent senescence in such models.

Furthermore, NMN treatment can reduce the activation of the cGAS-STING pathway in the brain, lowering neuroinflammation and improving DNA repair through NAD+/SIRT1 signaling. Other sirtuins, like SIRT6 and SIRT3, also enhance DNA repair with NAD+ supplementation.

NMN treatment altered protein levels in the spleens of senescent mice, reducing cGAS and STING proteins. Age-related NAD+ level differences and localized effects on specific tissues might influence these changes. In experiments with tumor-bearing mice, NMN led to stable weight, slower tumor growth, and more prolonged survival. However, excessive NAD+ supplementation could disrupt metabolic balance, and its effects on other tissues remain uncertain.

In senescent mice, NAD+ levels significantly drop compared to young mice. Since NAD+ is vital for DNA repair, mitochondrial function, and cellular senescence, supplementation is beneficial where NAD+ is depleted, like in aging or tumor-bearing conditions. The appropriateness of NAD+ supplementation for healthy young individuals needs further investigation.

The study links the cGAS-STING pathway to inflammation and senescence, with activation partly due to mtDNA from damaged mitochondria in senescent CD8+ T cells. NAD+ supplementation can promote mitophagy, reducing inflammation and senescence. These findings suggest targeting mitochondrial quality maintenance to help prevent senescence and inflammation in age-related diseases.

NMN prevents neuroinflammation and senescence in mice


The subheadings of the materials and methods are listed below. If you want to check the details, please visit the full PDF file here.

  • Animals
  • Cell culture and treatment
  • SA‐β‐gal staining and quantitative assay
  • NAD+ detection
  • ATP detection
  • Flow cytometry analysis
  • Intracellular ROS production assays
  • Mitochondrial membrane potential detection
  • Immunofluorescence staining of STING
  • RNA extraction and quantitative reverse transcription polymerase chain reaction (PCR) analyses
  • Quantification of mtDNA in cytosolic extracts
  • West blotting analysis
  • Statistical analysis


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