Figure 8.

Hyperthermia in combination with radiotherapy affects the expression of inhibitory immune checkpoint molecules (PD-L1, PD-L2, and HVEM) on MDA-MB-231 breast cancer cells. MDA-MB-231 cells were irradiated 2 times with 5 Gy (RT) or treated with HT of different temperatures (39 °C, 41 °C, 44 °C) and a combination of both, either HT followed by RT (HT (39 °C, 41 °C, 44 °C) +RT) or vice versa (RT + HT (39 °C, 41 °C, 44 °C)). The time interval between HT and RT was less than 2 h. The expression of ICMs ((a–d): PD-L1, (e–h): PD-L2 and (i–l): HVEM) was analyzed by multicolor flow cytometry. The mean fluorescence intensity (ΔMFI) was calculated by subtracting the fluorescence intensity of unstained samples from stained samples. Mean ± SD are presented from at least five independent experiments. Statistical significance is calculated by using Kruskal–Wallis tests with Dunn’s correction by comparing the ΔMFI of cells after the treatment to untreated control of the corresponding timepoint, and Mann–Whitney U tests to compare the ΔMFI of different sequences of HT and RT. * (p < 0.1), ** (p < 0.01). RT alone was compared with combinational treatments (HT + RT and RT + HT); # (p < 0.1).