HBD3 Induced PD-L1 Expression in HNSCC
Objectives: Human β-defensin 3 (HBD3) is present in high concentrations in the oral cavity of individuals with head and neck squamous cell carcinomas (HNSCC). The objectives of this study were i) to determine if HBD3 contributes to HNSCC pathogenesis by inducing programmed death-ligand 1 (PD-L1) expression on HNSCC cell lines and ii) to identify putative pathways involved in HBD3-induced PD-L1 expression.
Methods: HNSCC cell lines SCC4, SCC15, SCC19, SCC25, and SCC99 (5.0 × 104 viable cells) were incubated without and with 0.6 µM IFNg, a well-known PD-L1 inducer used as a positive control, and 0.2, 2.0, or 20.0 µM HBD3 for 24 hours. Cells were stained with anti-human APC-CD274 and Live/Dead Fixable Green Dead Cell Stain. Unstained cells and cells stained with an isotype antibody were included as controls. Cell suspensions were then examined using an LSR II Violet Flow Cytometer. Flow cytometric data was analyzed using FlowJo software. A one-way ANOVA followed by the Tukey’s HSD test was used to determine differences (p < 0.05). An integrated cancer cell network was used to predict signaling pathways for HBD3-induced PD-L1 expression.
Results: Untreated HNSCC cell lines contained lower levels of PD-L1 on their surfaces. Treatment with 0.6 µM IFNg increased the number of cells expressing PD-L1. Treatment with 20.0 µM HBD3 also increased the number of cells expressing PD-L1, and these results not significantly different than IFNg-induced PD-L1 expression (p > 0.05). HBD3-induced PD-L1 expression may signal via CCR6, Gi, LYN, PI3K, PIP3, AKT, MTOR, S6K PD-L1, sharing intermediates with IFNg starting at PI3K.
Conclusions: HBD3 increases the number of cells expressing PD-L1, a finding that suggests HBD3 may play an immunosuppressive role in the pathogenesis of HNSCC.