Single-Cell Transcriptomic Mapping of PD-L1/TLR4 Remodeling Informs Topical Immunoprevention Timing in Skin Carcinogenesis

Cutaneous squamous cell carcinoma (cSCC) represents a growing public health burden, with incidence projected to increase 23-29% over the coming decade. Topical immunoprevention strategies targeting the PD-L1/PD-1 and TLR4 axes have demonstrated preclinical efficacy, yet optimal intervention timing in humans remains undefined. To address this gap, single-cell RNA sequencing was performed on matched sun-protected (SP), sun-damaged (SD), and actinic keratosis (AK) biopsies from the same individuals, along with independent cSCC cases. Immune checkpoint and innate inflammatory signals were detectable as early as SD skin, prior to histologically confirmed dysplasia. Monotonically increasing expression of CD274 (PD-L1), CTLA4, PDCD1, CD27, and STAT1, alongside progressive TLR4-MYD88 innate immune signaling, was revealed through pseudobulk data analysis, with earliest upregulation at the SD stage. Fuzzy c-means trajectory clustering identified cell-typespecific programs across dendritic cells, macrophages, T cells, fibroblasts, endothelial cells, and keratinocytes. Dendritic cells shifted from early inflammatory antigen-presenting programs toward late PD-L1/IFN-regulatory states; macrophages showed monotonically increasing TLR4-associated myeloid activation; and T cells defined a "hot but exhausted" microenvironment in established cSCC. These findings identify SD and AK as biologically active stages for topical immunoprevention and provide a cellular roadmap for PD-L1/PD-1 and TLR4 blockade strategies.