ATR inhibition in combination with hypofractionated radiotherapy is superior to ATM inhibition with regard to ex vivo CD8 + T cell activation particularly for HPV-negative head and neck cancer cells

• Induction of CD8 + T cell proliferation in co-culture with HNSCC cells is dependent on the HPV status and on radiotherapy. • HLA-DR expression on cytotoxic T cells is highest after treating HPV-negative tumor cells with irradiation and ATRi. • ATMi treatment of tumor cells reduces IFN-γ secretion by CD8 + T cells. • PD-1/PD-L1 axis is not involved in regulation of CD8 + T cells activity after contact with treated HPV-neg HSC4 tumor cells. • 3D co-cultures predominantly confirm T cell stimulatory effects of ATRi. Even in the area of immune therapies , multimodal treatment of head and neck squamous cell carcinoma (HNSCC) is a big challenge. Small molecule kinase inhibitors (smKI), that target the DNA damage repair (DDR) system, have been described to alter, in addition to their radio-sensitizing effects, the immune phenotype of HNSCC. Thereby, the ATM and ATR kinases are key mediators of the DDR. However, the consequences these changes of the tumor cell phenotype have on immune cells are still poorly understood. Consequently, we here investigated how single and combination treatment of HNSCC cells with an ATM (ATMi) or an ATR inhibitor (ATRi) in combination with hypo-fractionated radiotherapy (RT) differently affects the activation of human cytotoxic CD8 + T cells regarding proliferation, activation marker surface expression and cytokine secretion. Human papillomavirus (HPV)-negative (Cal-33, HSC4) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC tumor cells were treated with hypo-fractionated RT of 2 x 5 Gray (Gy) in combination with AZD0156 (ATMi) or VE-822 (ATRi) and co-cultured with human CD8 + T cells for 96 h. T cell activation was quantified by CFSE-signal detected proliferation rates, activation marker expression on surfaces of the T cells, and by secretion of interferon gamma (IFN-γ) in the supernatants of the co-cultures via ELISA. In 2D co-cultivation settings RT-, HPV-status- and inhibitor-dependent influences on T cell activation were observed. Thereby, a stronger activation of T cells could be achieved after co-cultivation with ATRi-treated HNSCC tumor cells compared to ATMi. However, the addition of immune checkpoint inhibitors did not lead to enhanced T cell activation. In 3D co-cultures, a shift from RT-induced to a more inhibitor-induced effect on human T cells was visible and confirmed the superiority of ATRi, especially in combination with additional RT. The combination of RT + ATRi resulted in increased T cell activation compared to the combined treatment with ATMi, respectively. This suggests an advantage of ATRi in comparison to ATMi in combination with RT for induction of beneficial anti-tumor immune responses in HNSCC.