Immune checkpoint expression was compared between paired peripheral blood lymphocyte and tumor infiltrating lymphocyte subsets of the same HNSCC patients (n = 7). (A) All analyzed T cell subsets isolated from the tumor site revealed significantly higher PD-1 expression compared to circulating T cells. (B),(C) Similarly, GITR and OX40 expression was significantly upregulated on TIL subsets, both displaying the largest difference on the regulatory T cell (Treg) population (p < 0.0001). (D) B- and T-lymphocyte attenuator (BTLA) expression on tumor-infiltrating T cells was decreased compared to circulating T cells in all presented subpopulations. (E) On CD8+ T cells and CD39+ Treg isolated from the tumor an increase in CD137 expression could be measured, whereas the population of CD4+ T cells did not show a significant difference. p-values < 0.05 were considered to be significant with (*), p-values < 0.01 (**), p-values < 0.001 (***), and p-values < 0.0001 (****), p-values > 0.05 (ns).