Fig 2: Co-incubation of treated HPV-positive and HPV-negative HNSCC tumor cells had no significant effect on the expression of activation markers of hmDCs. 24 h after the last treatment, the cells were harvested and co-cultured with hmDCs. 48 h after the co-incubation the hmDCs were analyzed using flow cytometry. A: Presenting the gating strategy. B-E: For the hmDCs, which were co-cultured with the HPV-negative cell lines Cal33 and HSC-4, as well as HPV-positive cell lines UD-Scc-2 and UM-Scc-47, the mean fluorescence intensity (ΔMFI) of CD70 (B), CD80 (C), CD83 (D) and CD86 (E) is shown as stacked bars showing the mean with ±SD. A Mann-Whitney test was calculated to compare hmDCs without maturation cocktail and hmDCs with maturation cocktail. Furthermore, a Kruskal-Wallis test with Dunn's correction for multiple testing was calculated to compare the different treatment approaches versus hmDCs without maturation cocktail. *p < 0.05, **p < 0.01, ***p < 0.001; n≥5.

Fig 3: Flow cytometric analysis of immune checkpoint surface marker on HNSCC after ATMi or ATRi w/o 2x5Gy RT. Combining RT with ATRi leads to decreased expression of PD-L2 in HPV-positive cell lines UM-SCC-47 and UD-SCC-2. 48 hours after the last treatment, the HNSCC cells were harvested and the expression of the immunosuppressive ICMs PD-L1 (A), PD-L2 (B), HVEM (C), OX40-L (D) and CD70 (E) was examined by flow cytometry. ICM expression is presented as ΔMFI (delta of mean fluorescence intensity of stained samples – background fluorescence) (A–E). Combined treatment with RT and ATRi had no significant impact on the expression of PD-L1 compared to RT alone, except for the Cal-33 cell line, where it resulted in upregulation. However, it led to decreased expression of PD-L2 (B) in both HPV-positive cell lines and decreased expression of HVEM (C) in one HPV-positive cell line. The impact of the treatment approaches on the expression of OX40-L (D) and CD70 (E) showed cell line-specificity, regardless of the HPV status. A one-tailed Mann-Whitney U test was performed to compare the different treatment approaches within one cell line: *p ≤ 0.05, n=4.

Fig 4: RNAseq analysis of treated Cal-33. Modulation of specific transcripts based on a RNAseq analysis of treated HPV-negative Cal-33 cells (RT vs. RT+ATMi vs. RT+ATRi). Cells were treated according to the standard scheme in this study (see Figure 1 ). Immune-modulating cell surface marker such as CD70 (A), TNFRSF9 known as CD137-L (B), TNFRSF8 known as CD30 (C) and LGALS9 known as Galectin-9 (D) were significantly downregulated by the combination of RT and ATMi AZD0156. Further, soluble factors such as IDO1 (E) and immune-related cytokines such as IL-24 (F), IL-1b (G), IL-1a (H) and IL-6 (I) were also downregulated by the combination of RT + ATMi. Data representing 4 independent replicates. All transcripts were declared as significant when log-(foldchange) > ± 1 was determined and the adjusted p-value was ≤ 0.050. All transcripts were declared as significant (*) when log-(foldchange) > ±1 was determined and the adjusted p-value was ≤0.050.

Fig 5: Long-term culture induced CAR-T cell exhaustion, excessive activation and reduced cytotoxicity. (A) Representative flow cytometric profile showing the expression of CCR7 and CD45RA on CAR-T cells. (B) Proportions of Tscm (CCR7+CD45RA+), Tcm (CCR7+CD45RA-), Tem (CCR7-CD45RA-), and Teff (CCR7-CD45RA+) subsets in anti-CD70 CAR-T cells as measured by flow cytometry. (C) Representative flow cytometric profile showing the expression level of CD62L on CAR-T cells. (D) Histogram plot showing the percentage of CD62L+ CAR-T cells. (E, F) Mean fluorescence intensity (MFI) of CD25 (E) and CD69 (F) expression on CAR-T cells. (G) Levels of TNF-α and IFN-γ released by CAR-T cells at different time points, measured by ELISA. (H–J) MFI of exhaustion markers PD-1 (H), TIM-3 (I), and LAG-3 (J) in anti-CD70 CAR-T cells. (K–M) Cytotoxicity of anti-CD70 CAR-T cells against 786-0 cells after co-culture at the indicated effector-to-target (E:T) ratios for 24 hours (K), 48 hours (L), and 72 hours (M). Data were expressed as mean ± SD from at least 3 independent donors. Statistical significance was determined by t-test (D–F, H–J) and two-way ANOVA (G, K–M). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.