Integrative transcriptomic and single-cell analyses identify KLRD1 enrichment in exhausted CD8⁺ T cells in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) is characterized by complex interactions between tumor cells and the immune microenvironment. Here, we performed integrated transcriptomic and single-cell analyses to dissect immune-related molecular features and identify key regulators of CD8⁺ T cell exhaustion in cSCC. Differential expression analysis of GSE42677 identified 2,149 genes significantly dysregulated in cSCC compared with normal skin, with enrichment in immune- and inflammation-related pathways. Immune cell infiltration profiling revealed distinct patterns between tumor and normal tissues, and weighted gene co-expression network analysis (WGCNA) highlighted modules negatively correlated with activated CD8⁺ T cells and other immune populations. Protein–protein interaction network analysis and hierarchical clustering defined two molecular subtypes of cSCC with divergent immune landscapes. Single-cell RNA sequencing analysis further revealed expansion of exhausted CD8⁺ T (CD8_Exh) cells in tumors. Subpopulation-specific pathway enrichment and hub gene analysis demonstrated distinct functional programs across CD8⁺ T cell subsets, including antigen processing, apoptosis, MAPK and NF-κB signaling, and cytokine interactions. Pseudotime and trajectory analyses using CytoTRACE, Monocle3, and Slingshot identified differentiation trajectories from naive to exhausted and effector CD8⁺ T cells, highlighting dynamic expression of key genes such as CCL5, CD3D, CTSD, KLRD1, and PTPN6 in the CD8_Exh subset. Spearman correlation with pseudotime indicated a strong positive association for KLRD1, suggesting its role in driving CD8⁺ T cell exhaustion. Experimental validation using quantitative PCR and immunofluorescence confirmed elevated expression of KLRD1 in cSCC tissues. Collectively, these results provide a comprehensive landscape of immune regulation in cSCC and identify KLRD1 as a candidate marker of exhausted CD8⁺ T cells, offering a potential target for immunotherapeutic strategies.