Background Introduction

Tumor immunotherapy has brought revolutionary changes to cancer treatment, providing new hope for patients with various types of cancer. The effector function of CD8⁺ T cells, or their cytotoxicity, is central to cancer immunotherapy. However, many patients respond poorly to immunotherapy, with one major reason being that the tumor microenvironment paralyzes anti-tumor immune responses by limiting T cell infiltration, impairing T cell maintenance, and suppressing effector functions

Recent studies suggest that ionic imbalance in the tumor microenvironment may be an important factor limiting T cell effector functions. For example, it has been found that K⁺ accumulates in necrotic tumor microenvironments, indicating that K⁺ may act as a tumor-induced ionic checkpoint affecting T cell effector functions. Elevated Na⁺ concentrations have been shown to strongly promote the differentiation of Th17 cells and the response of Th2 cells to the mTORC2 signaling pathway. Recently, Na⁺ has also been demonstrated to disrupt the immunosuppressive functions of regulatory T cells by perturbing mitochondrial respiration. However, the direct effects of Na⁺ on CD8⁺ T cells and its impact on their anti-tumor cytotoxicity remain unclear. Therefore, a team led by Professor Christina E. Zielinski from the Leibniz Institute for Natural Product Research & Infection Biology studied this unknown mechanism and found that increased NaCl in the tumor microenvironment enhances T cell metabolic adaptability, thereby enhancing cytotoxicity.

Research Findings

The article first discovered that in breast cancer patients, the sodium and potassium ion concentrations in cancer tissues were significantly higher than in adjacent normal tissues. They isolated CD8⁺CD45RA^- T cells from the peripheral blood of healthy donors and treated them with high and low sodium chloride, naming the upregulated gene set as the "sodium chloride signature." They found that the "sodium chloride signature" was enriched in over twenty types of solid tumors and in CD8⁺ tumor-infiltrating lymphocytes in breast cancer, indicating that a high sodium chloride state indeed leaves a transcriptional imprint on TIL cells.

Figure 1: NaCl is highly enriched in solid tumors

They then explored the effects of high salt on T cell activation and effector functions. Whole-genome sequencing results showed that high salt altered the overall transcriptome levels of CD8⁺CD45RA^- T cells, with multiple genes related to T cell activation, metabolism, and effector functions significantly upregulated. Single-cell sequencing results indicated that high salt-treated T cells clustered in "activation, proliferation, differentiation, and effector functions," with T cell activation and TCR signaling pathways and their downstream effects significantly enhanced. High salt also promoted enhanced calcium ion influx induced by TCR stimulation.

Figure 2: NaCl enhances the activation state of human CD8⁺ memory T cells.

They continued to analyze the transcriptome data and found that under high NaCl conditions, CD8⁺ T cells significantly transitioned to effector cells, while their stemness decreased. Trajectory inference analysis at the single-cell level showed the differentiation trajectory of CD8⁺ T cells from low salt to high salt conditions. Additionally, CD8⁺ T cells under high salt conditions secreted more cytokines and cytotoxic molecules.

Figure 3: NaCl enhances CD8⁺ T cells effector functions and cytotoxicity.

They hypothesized that metabolic reprogramming might be a potential mechanism and continued to investigate, discovering that high sodium chloride conditions promote ATP production, enhance the transport of several key nutrients, increase the expression of transport proteins, and improve mitochondrial states. The mTOR pathway was also significantly activated under high sodium chloride conditions.

Figure 4: NaCl potentiates the metabolic fitness of CD8⁺ T cells

They subsequently found that the activation of Na⁺/K⁺-ATPase induced by high Na⁺ led to relative hyperpolarization of the cell membrane, enhancing calcium ion influx induced by TCR activation. The use of the Na⁺/K⁺-ATPase inhibitor Ouabain weakened calcium ion flux and reduced mTOR signaling activation and cytotoxic factor release in T cells in the presence of sodium chloride. They concluded the following mechanism: Under high NaCl conditions, Na⁺ influx increases, leading to elevated intracellular Na⁺ concentrations, which enhances Na⁺/K⁺-ATPase activity, helping to maintain the hyperpolarized state of the cell membrane, thereby enhancing calcium ion influx during TCR activation. Calcium ions induce NFAT activation and activate the mTOR signaling pathway, enhancing the uptake of glucose, glutamine, and fatty acids, inducing metabolic reprogramming to promote rapid ATP production, ultimately enhancing T cell effector functions, including cytokine production and the secretion of cytotoxic molecules such as granzyme B, thereby improving their ability to kill tumor cells.

Figure 5: NaCl enhances membrane hyperpolarization through Na⁺/K⁺-ATPase.

Finally, they tested the high cytotoxicity of NaCl-treated T cells through in vitro CAR-T and in vivo mouse experiments. They analyzed T cells in the tumor microenvironment of mice and humans and found that the presence of NaCl was associated with enhanced T cell cytotoxicity.

Figure 6: NaCl licenses CD8⁺ T cells for killing of tumor cells in vitro and in vivo.

Conclusion

In summary, the authors of this article found that the high NaCl concentration in the tumor microenvironment significantly impacts cellular metabolism and enhances the effector functions of human CD8⁺ T cells through Na⁺/K⁺-ATPase-dependent molecular mechanisms, improving T cell anti-tumor cytotoxicity both in vitro and in vivo. Furthermore, NaCl helps enhance the cytotoxicity of T cell adoptive therapy, providing a novel therapeutic strategy for tumor immunotherapy and diseases reliant on T cell toxicity. However, the article still has some limitations, including the following two aspects:

Overall, this article deepens our understanding of the regulation of CD8⁺ T cells and reveals a new mechanism for modulating T cell activation.

Original Link

DOI: 10.1038/s41590-024-01918-6

References

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