How do you measure a single cell's metabolism?

An immune cell's ability to act depends on how it generates energy. But studying this in human tumors has been hard, because standard methods for measuring metabolism require a large, pure cell population.

"What arrives is a mixture; there isn't much of it, and the cells you most want to study are often the rarest ones. So, most of what we know about immune cell metabolism comes from mouse tissue and cells grown in dishes," says first author Sujeetha A. Rajakumar, Ph.D., a former research associate in the Department of Otolaryngology–Head and Neck Surgery at Johns Hopkins Medicine.

The team measured each cell's metabolic machinery alongside the surface markers that identify it, reading a single cell's identity, functional state and metabolic profile at once. They applied the method to tumor-infiltrating immune cells including MAIT cells, CD8+ T cells and innate lymphoid cells.

Tissue came from patients in two phase 2 trials at Johns Hopkins Medicine in which immunotherapy was given before surgery: the anti-PD-1 antibody nivolumab, alone or combined with an antibody against interleukin-8, a signaling protein that directs immune cells toward inflammation. Samples were taken before treatment and again four weeks later at surgery.

What did the researchers find?

Three findings stood out:

• A population of T cells within the tumors proved metabolically fitter and less exhausted than neighboring cells.
• The more exhausted cells showed elevated levels of the enzyme glucose-6-phosphate dehydrogenase (G6PD), marking a metabolic pathway those cells rely on — a possible point of intervention.
• A subset of innate lymphoid cells carried a metabolic profile associated with immune suppression, suggesting they may obstruct checkpoint therapy.

Did blocking the pathway work?

To test the G6PD finding, the researchers used cultures of tumor-infiltrating CD8+ T cells from a separate group of untreated patients. They sorted the cells by expression of CD39, a protein that can contribute to immunosuppression. Then they treated the cells with a G6PD inhibitor, an anti-PD-1 antibody, both, or neither.

The combination produced the largest gene expression changes in the most exhausted cells and increased their secretion of CD27, a marker of T-cell activation.

"When we blocked this metabolic pathway alongside checkpoint inhibitor treatment, the cells we expected to be least reachable changed the most and became reactivated," Rajakumar says. "That tells us the pathway is worth pursuing."

What are the limits?

The results come from patient tissue and lab dishes, not from tests in living systems. "These findings need confirmation in larger patient groups before anything can be built on them," Alphonse says. Next steps include reproducing the findings in independent patient cohorts and testing whether interfering with this metabolic step has any effect in a living organism.