BackgroundThe majority of patients with recurrent or metastasized head and neck squamous cell carcinoma (HNSCC) do not benefit from immune checkpoint blockade (ICB) while several patients experience severe and persistent immune-mediated side effects. Therefore, predictive biomarkers are urgently needed to allow for a personalized treatment. In this study, we investigated DNA methylation of the immune checkpoint geneCTLA4with regard to its predictive value.MethodsWe analyzedCTLA4promoter methylation in tumors of HNSCC patients (N= 29) treated with ICB at the University Medical Center Bonn with regard to response to ICB and progression-free survival. We further analyzed a second cohort (N= 138) of patients that did not receive ICB with regard toCTLA4promoter methylation, CTLA-4 protein expression, and immune cell infiltrates. Finally, we tested inducibility of CTLA-4 protein expression in HNSCC cells using the DNA methyltransferase inhibitor decitabine.ResultsLowerCTLA4promoter methylation correlated with response to ICB and prolonged progression-free survival. We could show that not only tumor infiltrating immune cells, but also HNSCC cells harbor cytoplasmic and nuclear CTLA-4 expression.CTLA4promoter methylation inversely correlated with infiltrates of CD3+, CD4+, CD8+, and CD45+immune cells.CTLA4methylation did not correlate with protein expression in tumors, however, decitabine treatment led to decreasedCTLA4methylation and an induction ofCTLA4mRNA and CTLA-4 protein expression in HNSCC cell lines.ConclusionsOur results indicate thatCTLA4DNA hypomethylation is a predictive biomarker for response to ICB in HNSCC. Our study warrants further analyses of the predictive value ofCTLA4DNA methylation in clinical trials of anti-PD-1 and/or anti-CTLA-4 immunotherapy in HNSCC.