Salt Can Boost Antitumor Responses of T Cells

Sodium in tumor microenvironments found to heighten T-cell activation, suggesting new cancer treatment avenues

In a recent study published in Nature Immunology, researchers investigated the direct influence of sodium (Na+) ions on the cytotoxic cluster of differentiation 8 (CD8)-expressing T cells and hence on antitumor cytotoxicity.

Background

The metabolic status of cytotoxic T cells or lymphocytes controls antitumor immunity. These cells are sensitive to the tumor microenvironment (TME). The TME can inhibit the anticancer immune responses by reducing T cell invasion, decreasing T cellular maintenance, and lowering effector activities. Studies indicate that extracellular ions such as potassium (K+) may influence T-cell functions.

K+ ions are abundant within the necrotic TME, inhibiting T-cell receptor (TCR)-driven effector functions while increasing stemness and multipotency. Elevated sodium ion concentrations stimulate T helper 17 (Th17) differentiation and increase self-regulatory cytokine expression. However, the effects of sodium ion concentrations on cytotoxic T-cell-mediated antitumor immunity are unclear.

About the study

The researchers investigated the effect of sodium ion concentrations on cytotoxic T-cell activity and antitumor immunological responses.

Inductively coupled plasma optical emission spectrometry (ICP-OES) assessed K+ and Na+ concentrations in breast cancer and adjacent tissues. Researchers investigated the transcriptome imprint of sodium chloride exposure on cytotoxic T cells. They found that high NaCl treatment dramatically increased differentially expressed genes (DEGs), resulting in the sodium-chloride signature. They next investigated the enrichment of this signature in tumor tissues vs. healthy tissues. They also investigated transcriptome NaCl signature enrichment in cytotoxic T cells.

The researchers investigated the influence of NaCl on the transcriptome of human CD45RA-ve cytotoxic T cells expanded with CD3 and CD28 monoclonal antibodies (mAbs) in vitro. They analyzed transcriptome alterations using single-cell ribonucleic acid sequencing (scRNA-seq). They investigated the mammalian target of rapamycin (mTOR) signaling induction by TCR crosslinking of CD45RA−ve cytotoxic T cells through ribosomal subunit S6 phosphorylation under high and low NaCl circumstances.

The researchers investigated the molecular pathways by which increasing extracellular sodium-chloride concentrations can convert T cell receptor engagement in cytotoxic T lymphocytes into greater activation of T cells. They expected higher extracellular sodium chloride concentrations to enhance electromotive forces for Ca2+ entry following TCR-induced calcium (ORAI) channel activation. They investigated NaCl-induced relative hyperpolarization of membrane potential (or Vm).

The researchers created antigen-specific cytotoxic memory T lymphocytes by nucleofecting a melanoma-associated antigen recognized by T cells (MART-1)-specific TCR. They designed T lymphocytes that expressed second-generation chimeric antigen receptors (CAR) with 4-1BB-derived costimulatory domains that identified Panc02-mROR1 pancreatic cancer cells. They also investigated the effect of NaCl on CAR T lymphocytes bearing a CD28-derived costimulatory domain. Using a pancreatic cancer mouse model and the PancOva pancreatic cancer cell line, they tested whether NaCl increased T cell cytotoxicity during adoptive T cell therapy in vivo. Principal component analysis (PCA) and uniform manifold approximation and projection (UMAP) yielded the results.

Results

The study discovered elevated NaCl levels in breast cancer tissues, which resulted in transcriptional alterations in immune cells. Most tumor entities from the Cancer Genome Atlas (TCGA) have a transcriptomic sodium footprint compared to healthy tissues. The findings indicated that sodium is a relevant component of the tumor microenvironment. NaCl increased cytotoxic T-cell activation and activity, which improved metabolic fitness. Thus, NaCl enhances tumor cell death in vitro and in vivo. Elevated levels of genes like mTOR, tumor necrosis factor (TNF), interleukin-2 (IL-2), programmed cell death 1 (PD-1), mitogen-activated protein kinase (MAPK), interferon regulatory factor 4 (IRF4), and hypoxia-inducible factor 1 subunit alpha (HIF1A) corroborated these findings. 

NaCl-induced alterations in cytotoxic T lymphocytes are associated with sodium-induced increased Na+/K+-adenosine triphosphatase (ATPase) activities and membrane hyperpolarization, which amplifies electromotive forces for TCR-induced downstream TCR signaling and Ca2+ influx. The adoptive transfer of cytotoxic T lymphocytes (CTLs) activated under high NaCl conditions effectively decreased tumor growth in a mouse pancreatic cancer model.

NaCl increased anticancer activity in the native T lymphocyte repertoire, CAR T lymphocytes, and transgenic ones. Under high NaCl circumstances, cytotoxic memory T cells increased the nuclear factor of activated T cell 5 (NFAT5) expression, resulting in prolonged survival in pancreatic cancer patients. Similarly, increased ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) gene expression was associated with pancreatic cancer survival in patients with Na+/K+-ATPase regulation. Researchers consequently argue that NaCl positively regulates acute antitumor immunological responses that may require adjustment to train therapeutic T lymphocytes like the CAR T variety, ex vivo.

Conclusion

The study showed that NaCl in the tumor microenvironment improves T-cell metabolic fitness and cytotoxicity. The mechanism of NaCl-induced T-cell hyperactivation includes internal Na+ translocation, increased Na+/K+-ATPase activity, membrane hyperpolarization, and higher electromotive forces for Ca2+ entry generated by TCR. Treatment strategies based on this mechanism may help cancer patients. Further clinical trials must evaluate NaCl's short- and long-term effects on antitumor immunity.