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. 2024 Feb 27;12(2):e008155. doi: 10.1136/jitc-2023-008155

Figure 4.

Figure 4

CAR-NK cells retain ICAM-1-independent activity and cytokine production in long-term killing assays. (A–C) MDA-MB-453 ICAM-1 KO, NT and WT control cells were labeled with CytoLightRed and allowed to adhere to 96-well plates. NK-92/5.28.z CAR-NK cells or haNK cells in combination with trastuzumab were added at an E:T ratio of 1:1 in medium supplemented with CytoToxGreen viability dye, and cytotoxicity over time was analyzed using the IncuCyte live-cell imaging system. Where indicated, NK cells were treated with LFA-1 blocking antibody or control IgG1 prior to co-culture. (A) Microscopic images showing the co-cultures after 50 hours, with MDA-MB-453 cells labeled in red. Overlapping red and green signals (yellow) indicate dead MDA-MB-453 cells. (B, C) Graphs depicting cytotoxicity at the indicated time points calculated as the percentage of dead (double-positive) cancer cells out of total (red) cancer cells. Data were pooled from at least three independent experiments. Mean values±SEM are shown. Statistical analysis was performed using two-way analysis of variance. (D) Flow cytometric analysis of LFA-1 expression on NK-92, haNK and NK-92/5.28.z cells. Filled gray areas indicate isotype controls. Representative data out of at least three independent experiments are shown. (E, F) haNK cells combined with trastuzumab (E) or NK-92/5.28.z cells (F) were co-cultured with MDA-MB-453 ICAM-1 KO or WT cells at an E:T ratio of 1:1 for 6 hours in duplicates. Where indicated, NK cells were treated with LFA-1 blocking antibody or control IgG prior to co-culture. Supernatants from duplicate samples were pooled, and cytokine concentrations were measured using the ProcartaPlex Luminex system. Mean values±SEM calculated from three independent experiments are shown. CAR, chimeric antigen receptor; E:T, effector to target; haNK, high-affinity FcγRIIIa-modified NK-92 cells; IFN, interferon; KO, knockout; NK, natural killer; NT, non-targeting; MIP-1α, macrophage inflammatory protein 1-alpha; TNF-α, tumor necrosis factor alpha; WT, wild type.