Figure 4.

Gamma irradiation and LEEI result in DNA damage and an accumulation in G2/M phases of CD123-directed CAR-NK-92 cells. (A) Alkaline comet assays were performed with LEE- and gamma-irradiated versus non-irradiated CD123-directed CAR-NK-92 cells 2 h (left) or 24 h (right) after treatment to determine irradiation-induced DNA strand breaks. Values are depicted as means ± SEM (n = 6 independent experiments, each carried out in technical triplicates, minimum number of 100 cell nuclei analyzed per sample). Statistical significance is symbolized by asterisks (ns for p > 0.05, * for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001, Kruskal-Wallis test adjusted for multiple comparisons by Dunn’s test). (B) CD123-CAR-NK-92 cells (n = 8) were fixed and stained with propidium iodide (PI). Representative histograms of non-irradiated (control, left), LEE-irradiated (middle), and gamma-irradiated (right) cells are shown 2 h (d0, top), 2 days (d2, middle), and 6 days (d6, bottom) after treatment. Gating indicates cells defined as Sub-G1- (1), G1- (2), S- (3), G2/M phase (4) or polyploid (5). (C) Quantification of PI-staining of CD123-directed CAR-NK-92 (n = 8). Values are indicated as means ± SEM. (D) Light microscopy of non-irradiated (control, top), LEE-irradiated (center), and gamma-irradiated (bottom) CD123-directed CAR-NK-92 cells on day 10 post-irradiation. Arrows show enlarged cells. (E) Preliminary results of gene expression levels of gamma-irradiated (left) and LEE-irradiated (right) CD123-directed CAR-NK-92 cells both compared to non-irradiated cells 2 h after treatment. Heat maps of normalized, variance-stabilized and per gene standardized expression values of three technical replicates show up- (red) or down-regulated (blue) genes. The total number of significantly differentially regulated genes is indicated below (FDR < 0.01).