PD-1 and LAG-3 Double-Positive T Cells Predict Better Gastric Cancer Survival

Gastric cancer remains one of the world’s most lethal malignancies, and oncologists have long struggled to explain why two patients with seemingly identical tumors can experience dramatically different outcomes. A new study published in Medical Oncology offers a striking piece of that puzzle, showing that a specific subset of immune cells inside the tumor—CD8-positive T cells that simultaneously carry two immune checkpoint molecules, PD-1 and LAG-3—is associated with significantly improved survival. The finding upends the conventional assumption that checkpoint expression simply marks dysfunctional, exhausted cells and instead points to a paradox: the very markers of potential T cell fatigue may, in combination, flag a population of tumor-fighting lymphocytes at the peak of its anti-cancer activity.

The research team, led by investigators at Zhongshan Hospital of Fudan University in Shanghai, analyzed four independent gastric cancer cohorts totaling more than 1,100 patients. The largest dataset came from their own institution, a cohort of 298 patients known as ZSGC, complemented by 371 cases from The Cancer Genome Atlas (TCGA), 45 patients from an immune checkpoint blockade (ICB) treatment cohort, and 433 patients from the Yonsei cohort, a publicly available Korean dataset. By triangulating across these populations, the researchers sought to ensure that their observations were not artifacts of a single institution, staining protocol, or patient population.

Quantifying these cells required two complementary strategies. In the ZSGC cohort, the team used multiplex immunofluorescence staining to directly visualize PD-1-positive LAG-3-positive CD8-positive T cells within tumor tissue, distinguishing true intratumoral infiltration from cells lingering in the adjacent stroma or normal mucosa. For the transcriptomic cohorts, they scored bulk gene-expression profiles against a gene signature derived from this double-positive population, allowing them to infer infiltration levels in specimens that could not be re-stained. Survival outcomes were then assessed using Kaplan-Meier curves and multivariate Cox regression models that adjusted for established clinical variables such as stage, grade, and treatment.

The central result was remarkably consistent. Patients whose tumors harbored high levels of PD-1-positive LAG-3-positive CD8-positive T cells survived significantly longer than those with low infiltration, a relationship that held in both the ZSGC and TCGA cohorts. Even more telling was the control experiment: T cells expressing only PD-1 or only LAG-3 showed no such survival association. It was the co-expression of both checkpoint receptors on the same cytotoxic T cell that carried the prognostic signal, suggesting that the double-positive phenotype represents a biologically distinct state rather than a simple sum of its parts.

This specificity matters because PD-1 and LAG-3 have historically been lumped together as hallmarks of T cell exhaustion, a gradual loss of function that occurs when T cells are chronically stimulated, as in persistent infections or long-standing tumors. Exhausted T cells lose their ability to proliferate and kill, and in many cancer types their abundance correlates with poor outcomes. But immunologists have increasingly recognized that checkpoint expression is not synonymous with exhaustion. PD-1, in particular, is also expressed transiently on freshly activated T cells, and recent work across several malignancies has suggested that PD-1-positive tumor-infiltrating lymphocytes often represent the tumor-reactive fraction of the immune infiltrate—the cells most likely to recognize cancer antigens.

To determine what these double-positive cells were actually doing, the researchers turned to flow cytometry on freshly resected gastric cancer tissue. The cells exhibited an activated phenotype, characterized by elevated levels of CD137, a co-stimulatory receptor that marks T cells receiving antigen-driven stimulation. They also produced abundant interferon-gamma and perforin, signature molecules of cytotoxic effector function, and expressed CXCL13, a chemokine increasingly associated with tumor-reactive T cells that coordinate organized immune responses within tumors. Importantly, these cells retained expression of TCF7, a transcription factor associated with stem-like and progenitor states, while showing lower PD-1 levels than fully exhausted cells.

Taken together, this molecular profile paints a picture of what immunologists call a pre-exhausted state: T cells that have encountered tumor antigen, are actively engaged in the anti-cancer fight, and retain the proliferative and self-renewing capacity to sustain that fight. They sit at a biological crossroads. With the right support—such as immune checkpoint blockade—they can be rescued and expanded into potent effectors. Without intervention, or under continued immunosuppressive pressure, they may progress to terminal exhaustion. This interpretation also explains why their presence predicts better survival: their abundance signals that the immune system has recognized the tumor and is actively responding to it.

The clinical implications extend beyond prognosis. In the ICB cohort of 45 gastric cancer patients treated with pembrolizumab, an anti-PD-1 antibody, higher infiltration of the double-positive cells was associated with a higher rate of response to therapy. This is biologically coherent: checkpoint inhibitors work by releasing the brakes on pre-exhausted T cells, and tumors rich in such cells provide more targets for the drug to act upon. If validated in larger prospective studies, measuring PD-1-positive LAG-3-positive CD8-positive T cell infiltration could help clinicians identify which gastric cancer patients are most likely to benefit from immunotherapy, complementing existing biomarkers such as PD-L1 expression, microsatellite instability, and Epstein-Barr virus status.

Intriguingly, the study also found that high infiltration correlated with advanced tumor stage, poor differentiation, microsatellite instability, and EBV-positive molecular subtypes. This association is less contradictory than it appears. Microsatellite instability and EBV infection are known to generate abundant neoantigens and viral antigens, respectively, providing the immune system with more targets and thereby recruiting more tumor-reactive T cells. The double-positive infiltrate may thus be the immune signature of antigen-rich tumors—and may partially explain why these molecular subtypes respond particularly well to checkpoint blockade.

Computational analyses of the broader tumor microenvironment reinforced the theme. Tumors with high double-positive infiltration displayed an immune-active landscape, with gene-expression patterns indicating coordinated activity of cytotoxic lymphocytes, antigen-presenting machinery, and inflammatory signaling rather than the immunosuppressive, fibrotic architecture that characterizes immune-cold tumors. The authors suggest that these cells could serve as both independent prognostic biomarkers and candidate predictive biomarkers for immunotherapy stratification in gastric cancer, though they caution that larger, prospective validation will be needed before the metric enters routine pathology practice. With LAG-3 inhibitors such as relatlimab already approved in combination with PD-1 blockade in other malignancies and under investigation in gastroesophageal cancers, a simple staining strategy that identifies patients whose tumors are populated by pre-exhausted, tumor-reactive lymphocytes could meaningfully sharpen the selection of patients for these increasingly important therapies.

Subject of Research: Prognostic and functional characterization of intratumoral PD-1-positive LAG-3-positive CD8-positive T cells in gastric cancer

Article Title: Intratumoral PD-1+LAG-3+CD8+ T cells are associated with improved prognosis in gastric cancer

Article References: Liu, D., Xue, Z., Yu, K., Sun, Z., Ma, G., Zhao, J., Li, H., Sun, Y., Wang, Z., & Wang, X. (2026). Intratumoral PD-1+LAG-3+CD8+ T cells are associated with improved prognosis in gastric cancer. Medical Oncology, 43(10), Article 285. https://doi.org/10.1007/s12032-026-03384-6

Image Credits: AI Generated

DOI: 10.1007/s12032-026-03384-6

Keywords: gastric cancer, PD-1, LAG-3, CD8 T cells, T cell exhaustion, tumor microenvironment, immune checkpoint blockade, pembrolizumab, biomarkers, prognosis, tumor immunology, immunotherapy