Integrative multi-omics reveal a CD4⁺ T cell–derived six-gene signature linking the immune–stromal ecosystem to prognosis and immunotherapy selection in pancreatic cancer

BACKGROUND: Pancreatic cancer (PC) exhibits dismal outcomes and an immune-excluded microenvironment that blunts immunotherapy. Robust, immune-anchored biomarkers are needed to stratify risk and inform treatment design. METHODS: We integrated The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) dataset and validated findings in Gene Expression Omnibus (GEO) dataset (GSE57495). Immune infiltration, survival modeling, pathway activity, tumor microenvironment, and immunotherapy responses were analyzed. Single-cell RNA sequencing (GSE212966) informed CD4⁺ T-cell differentially expressed genes (DEGs), pseudotime, and intercellular communication (CellChat). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in H6C7, CAPAN-1, and PANC-1 cell lines were performed. RESULTS: Among 28 immune subsets, CD4⁺ T-cell populations most strongly associated with overall survival (OS). A six-gene signature (KLF3, EZR, SMDT1, JPT1, ISG15, MT1X) derived from 111 CD4⁺ T-cell DEGs successfully stratified OS with time-dependent AUCs ranging from 0.637 to 0.838 across the training and validation cohorts. The high-risk group showed significantly poorer OS, higher stromal scores, broad checkpoint upregulation, and greater immune exclusion (P < 0.05), indicating an immune-stromal ecosystem favoring tolerance. Single-cell RNA sequencing localized model genes to fibroblasts, ductal cells, and CD4⁺ T cells subsets. CellChat revealed globally intensified crosstalk in PC and strengthened CD4⁺ T-cell interactions with myeloid and stromal compartments. Western blot and qRT-PCR results confirmed up-expression of all model gene except SMDT1 in PC cell lines. CONCLUSIONS: CD4⁺ T-cell subsets serve as central determinant of the immune landscape and clinical outcome in PC. The CD4⁺ T cell-anchored six-gene signature enables risk stratification, links immune contexture to stromal ecology, and motivates biomarker-guided trials and rational combinations that alleviate immunosuppression while targeting metabolic-stress pathways.