LAG-3-Expressing Tumor-Infiltrating T Cells Are Associated with Reduced Disease-Free Survival in Pancreatic Cancer

Abstract

T cells are the predominant immune cell population in the pancreatic tumor microenvironment. High CD8⁺ and Th1-polarized CD4⁺ T cell infiltration is associated with prolonged survival in human pancreatic ductal adenocarcinoma (PDAC). However, the expression pattern of co-stimulatory and inhibitory receptors by PDAC-infiltrating T cells and their prognostic significance are not well defined. In this study, we employed multiplex immunofluorescence to investigate the intratumoral expression of the co-stimulatory receptor inducible T-cell co-stimulator (ICOS), the inhibitory receptors lymphocyte-activation gene 3 (LAG-3), programmed death 1 (PD-1), and V-domain immunoglobulin suppressor of T cell activation (VISTA) by tumor-infiltrating T cells (CD3) in a cohort of 69 patients with resected PDAC. T cells were enriched particularly within the stromal area and were highly heterogeneous across tumors. Further, T cells were associated with prolonged disease-free survival (DFS). However, LAG-3 expression by PDAC-infiltrating T cells was correlated with reduced DFS. Our study highlights the biological importance of LAG-3 expression by tumor-infiltrating T cells. LAG-3⁺ T cells may represent a novel prognostic marker and a particularly attractive target for immunotherapeutic strategies in PDAC.

Keywords: ICOS; LAG-3; PD-1; VISTA; pancreatic cancer; tumor microenvironment; tumor-infiltrating T cells.

Figure 1

Tumor-infiltrating T cells reside within the stromal area in pancreatic cancer. (A) Paraffin-embedded human pancreatic ductal adenocarcinoma (PDAC) specimens were stained for PanCK (cyan), CD3 (yellow), ICOS (red), LAG-3 (orange), PD-1 (purple), and VISTA (green). Representative multiplex immunofluorescence image is shown (200×). Scale bar, 50 μm. (B) Quantification of T cell density in whole PDAC and (C) ductal (red) and stromal (green) tissue areas. Each point represents a single patient (total, n = 69). Dot plots and box-and-whiskers (plus min-max), median. Paired Wilcoxon test. p-values ≤ 0.05 were considered significant. ****, p < 0.0001.

Figure 2

Tumor-infiltrating T cells express co-stimulatory and inhibitory receptors in pancreatic cancer. (A) Density of T cells stained positive for indicated receptor in whole PDAC and (B) ductal (red) and stromal (green) tissue areas. (C) Percentage of T cells positive for indicated receptor in whole PDAC and (D) ductal (red) and stromal (green) tissue areas. Each point represents a single patient (total, n = 69). Dot plots and box-and-whiskers (plus min-max), median. Paired Wilcoxon, and Kruskal–Wallis test and Dunn multiple comparisons test. p-values ≤ 0.05 were considered significant. **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

Figure 3

Intratumoral T cell and PD-1⁺ T cell densities are associated with increased disease-free survival in pancreatic cancer. (A) Overall and (B) disease-free survival of patients with pancreatic cancer, stratified by the median of indicated density. Tick marks indicate censored data. p-values were calculated using a log-rank test. *, p < 0.05.

Figure 4

LAG-3⁺ T cells are associated with poor disease-free survival. (A) Risk of tumor recurrence in pancreatic cancer patients. Hazard ratios and 95% confidence intervals are shown. p-values ≤ 0.05 were considered significant. *, p ≤ 0.05; and **, p < 0.01.