The study, published in Cancer Immunology Research, a journal of the American Association for Cancer Research, tackles a question that immunologists have rarely been able to address directly in human tissue: does the way a T cell generates and uses energy determine whether it mounts an effective attack or slides into exhaustion? Exhaustion is a state in which a cell, activated too long and too continuously, stops responding to the threats it was designed to destroy. Checkpoint immunotherapy, such as antibodies against the PD-1 receptor, releases one of the brakes holding those cells back. For some patients the effect is dramatic; for many others it fails, and the reasons have remained stubbornly unclear.

Metabolism has long seemed a plausible place to look for answers. An immune cell’s capacity to act — to proliferate, to produce inflammatory signals, to kill tumor cells — is constrained by how it generates energy and building blocks. But probing that relationship in human tumors has been technically formidable. The standard laboratory method for measuring cellular metabolism requires a large, pure population of a single cell type, something that is nearly impossible to obtain from a biopsy. As first author Sujeetha A. Rajakumar, Ph.D., a former research associate in the Department of Otolaryngology–Head and Neck Surgery at Johns Hopkins Medicine, explains, what arrives from the clinic is a mixture, there is not much of it, and the cells researchers most want to study are often the rarest. Most of what the field knows about immune cell metabolism has therefore come from mouse tissue and cells grown in dishes, systems that do not always faithfully reflect what happens inside a patient.

The Johns Hopkins team circumvented this bottleneck by measuring each cell’s metabolic machinery alongside the surface markers that identify it. This approach allowed them to read three things from a single cell simultaneously: which cell type it is, what functional state it is in, and what metabolic profile it uses. They applied the method to several populations of tumor-infiltrating immune cells, including mucosal-associated invariant T (MAIT) cells, conventional CD8+ T cells, and innate lymphoid cells (ILCs). Because the technique works on scarce and mixed samples, it opened a window onto metabolic states that had previously been invisible in human tumor tissue.

The tumor samples came from patients enrolled in two phase II clinical trials at Johns Hopkins Medicine in which immunotherapy was administered before surgery. Participants received either the anti-PD-1 antibody nivolumab alone or nivolumab combined with an antibody against interleukin-8, a signaling protein that recruits immune cells to sites of inflammation and infection. Tissue was collected before immunotherapy began and again four weeks later at the time of surgery, giving the investigators a rare opportunity to observe how the immune and metabolic states of individual cells changed within the same patients over the course of treatment.

Three findings stood out. First, the team identified a population of T cells within these tumors that proved to be metabolically fitter and less exhausted than their neighbors, suggesting that not all tumor-infiltrating lymphocytes are equally depleted and that metabolic state tracks closely with function. Second, the more exhausted cells showed elevated levels of the enzyme glucose-6-phosphate dehydrogenase, or G6PD, the rate-limiting enzyme of a metabolic pathway those cells appear to rely on — making it a possible point of intervention. Third, a subset of innate lymphoid cells carried a metabolic profile associated with immune suppression, raising the possibility that these cells may actively pose an obstacle to checkpoint therapy rather than merely failing to help it.

To test whether the exhausted cells truly depend on the G6PD pathway, the researchers turned to laboratory cultures of tumor-infiltrating CD8+ T cells from a separate group of untreated patients. They sorted the cells according to their expression of CD39, a protein that can contribute to immunosuppression by limiting the activation of T cells. The cultures were then treated with a G6PD inhibitor, with an anti-PD-1 antibody, with both, or with neither. The combination treatment produced the largest gene expression changes in the most exhausted cells and increased their secretion of CD27, a marker of T-cell activation. In other words, the cells that seemed least reachable by conventional immunotherapy were the ones that changed the most when the metabolic pathway was blocked alongside checkpoint blockade.

Senior author Martin Alphonse, Ph.D., an assistant professor of dermatology at the Johns Hopkins University School of Medicine, frames the work around a deceptively simple question: take a piece of head and neck tumor tissue and you will find immune cells inside it whose sole purpose is to recognize something abnormal and destroy it — so what has happened to them? The answer emerging from this study is that part of what has happened is metabolic. Exhausted cells appear to have rewired their energy production in a way that can be mapped, and at least one node of that rewiring can be targeted in the laboratory to partially restore function. Rajakumar notes that when the pathway was blocked alongside checkpoint inhibitor treatment, the cells expected to be least reachable changed the most and became reactivated — evidence, she says, that the pathway is worth pursuing.

The clinical context makes the work consequential. Head and neck squamous cell carcinoma, which arises from the cells lining the mouth, throat and voicebox, is the seventh most common cancer worldwide, with roughly 890,000 new cases each year. With checkpoint inhibitors helping fewer than a quarter of these patients, any strategy that could expand the fraction of responders would address a major unmet need. Growing evidence has suggested that metabolic reprogramming influences whether a T cell mounts an effective response or becomes exhausted, but this study is among the relatively few to examine that link directly in human tumor tissue rather than in animal models or cell lines, and to do so across multiple immune cell types at single-cell resolution.

The researchers and their collaborators are careful to stress what the findings are and what they are not. Blocking G6PD in laboratory cultures reactivated exhausted cells, but no patient in the trials received a G6PD inhibitor, and the approach should not be considered a treatment itself. Alphonse emphasizes that these findings need confirmation in larger patient groups before anything can be built on them. The next steps, he says, are to reproduce the results in independent cohorts and to test whether interfering with this metabolic step has any effect in a living system, not just in a dish. Additional co-authors are Namya Nanda, Chloe Kim, Dustin Dikeman, Tanguy Y. Seiwert, Zubair Khan and Sewon Kang, all affiliated with Johns Hopkins Medicine. Study co-author Carole Fakhry, M.D., M.P.H., senior associate dean for clinical affairs at Johns Hopkins, underscores that such interdisciplinary, collaborative studies are critical to understanding how to make therapies more effective in the future, leveraging the translational capabilities of clinical trials to generate insights that laboratory work alone cannot provide. The work was supported by the Johns Hopkins Department of Dermatology, a Career Development Award from the Dermatology Foundation, the Department of Otolaryngology–Head and Neck Surgery, and the Bloomberg~Kimmel Institute for Cancer Immunotherapy. Alphonse and Rajakumar are inventors on two provisional patent applications related to the work, assigned to The Johns Hopkins University, which manages these relationships under its conflict-of-interest policies.

Subject of Research: Immunometabolic profiling of exhausted tumor-infiltrating T cells in head and neck squamous cell carcinoma

Article Title: Johns Hopkins investigators identify a metabolic weak point in exhausted immune cells in head and neck cancer

Article References: Johns Hopkins investigators identify a metabolic weak point in exhausted immune cells in head and neck cancer. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: head and neck cancer, T cell exhaustion, immunotherapy, checkpoint inhibitors, G6PD, tumor metabolism, CD8+ T cells, innate lymphoid cells, MAIT cells, nivolumab, single-cell profiling, Cancer Immunology Research