Fig. 4.

Macrophages are not (re-)polarized upon the uptake of HPV-positive or HPV-negative HNSCC cells including their supernatants, but CD206 is downregulated on M2 macrophages. For the analysis of the (re-)polarization of the macrophages after the addition of irradiated (2 × 5 Gy) and mock-treated (0 Gy) HPV-negative (cal33; a–d) and HPV-positive (UD2; e–h) HNSCC cells, after 24 h macrophages were stained with the viability stain Zombie NIR and the antibodies for CD11b, HLA-DR, CD80, CD163, and CD206. Macrophages without the addition of any tumor cells are referred to as “w/o,” and used as control groups. Background correction for the respective marker expression was performed by subtracting the median fluorescence intensity of the Zombie NIR and CD11b staining from the complete staining. The fold change in expression of the markers HLA-DR (a, e), CD80 (b, f), CD163 (c, g), and CD206 (d, h) is presented in relation to the “w/o” control. Statistical analysis was performed with the Kruskal–Wallis test and Dunn’s correction for multiple testing comparing “w/o,” “0 Gy,” and “2 × 5 Gy” with each other within each phenotype. Significance is indicated as: * p < 0.05