Fig 2: PRDX1 overexpression in primary NK cells and CAR-PD-L1 NK-92MI improves survival and cytotoxicity in oxidative stress. A, Western blotting analysis of PRDX1 in primary NK cells (left) and NK-92MI cell line (right). Primary NK cells (representative donor) were nonstimulated or IL15-stimulated (10 ng/mL, 3 days), and electroporated with in vitro transcribed PRDX1 mRNA or not. NK-92MI cell line was nontransduced (NT) or transduced with control (SFFV-IRES-mRFP) and PRDX1-encoding plasmid (SFFV-PRDX1-IRES-mRFP). B, Representative dot plots (flow cytometry) of control (MOCK) and PRDX1-mRNA electroporated NK cells incubated with GOX. C, Viability of MOCK and PRDX1-mRNA electroporated human primary NK cells in the presence of GOX assessed by flow cytometry (n = 5). Data points show the average from two technical replicates with median (horizontal line) and mean (plus symbol). Statistic: two-way ANOVA (P = 0.0002) with Bonferroni post hoc. D, Migration of mRFP-NK-92MI and NK-92MI-PRDX1 cell lines into an engineered 3D hydrogel scaffold on day 1 (left) or day 7 (right), determined by fluorescence intensity of NK cells normalized to cancer cell fluorescent intensity. Left: n = 18 in both mRFP and PRDX1-mRFP group. Right: n = 9 (mRFP), n = 8 (PRDX1-mRFP). Data show mean and SD. Statistic: unpaired t test. E, Survival of NK-92MI-mRFP and NK-92MI-PRDX1-mRFP cell lines (left, n = 8) or CAR-PDL-1-NK-92MI cells (mRFP) and CAR-PD-L1-PRDX1-NK-92MI (PRDX1-mRFP) cells (right, n = 6) in the presence of GOX (20 hours) assessed with flow cytometry. Data show median (horizontal line) and mean (plus symbol) with range. Dots represent the average from two technical replicates. Statistic: two-way ANOVA (P < 0.0001) with Bonferroni post hoc. Results of Bonferroni post hoc were marked on the graph. F, IVIS imaging of the bioluminescence signal of NK-92MI-mRFP-LUC or PRDX1-mRFP-LUC cells administered intratumorally into 4T1 tumors in NSG mice. Data show median (horizontal line) and mean (plus symbol) with range. Points represent bioluminescence signal from each mouse. The experiment was performed twice. All data from two independent experiments were pooled (n = 9). Statistic: two-way ANOVA (P < 0.0001) with Bonferroni post hoc. Bonferroni post hoc results were marked on the graph. Intratumoral administration scheme was created with BioRender.com. G, RTCA cytotoxicity of CAR-PD-L1-NK-92MI and CAR-PD-L1-PRDX1-NK-92MI cells toward PD-L1+MDA-MB-231 cells in different GOX concentrations (E:T ratio 1:1). Representative results from one experiment. Data shown as averages ± SD from two technical replicates. H, Summary RTCA cytotoxicity data (n = 6) at 19 hours after effectors' addition. Data shown as median (horizontal line) and mean (plus symbol) with range. Points represent the average of two technical replicates. Statistic: two-way ANOVA (P < 0.001) with Bonferroni post hoc. Significant differences of Bonferroni post hoc were marked on the graph.

Fig 3: Influence of NK cell priming with IL15 on the natural cytotoxicity and the expression of select antioxidant enzymes. A, Representative RTCA cytotoxicity of nonstimulated (left) and IL15-stimulated (right) NK cells toward K562 targets in the presence of increasing concentrations of GOX (E:T ratios: 1:1, 2:1, 5:1). Data show averages ± SD from two technical replicates. B, Summary of RTCA cytotoxicity experiments (n = 5) at 19 hours after effectors' addition. Left: nonstimulated; right: IL15-stimulated NK cells. Data show median with range. Dots represent average of two technical replicates. C, scRNA-seq analysis of PRDX1 in different NK cell subsets stimulated with IL15. The scRNA-seq data were processed using Seurat 3. Samples within each subset were merged, and the dot plot shows for each gene the average expression level in the subsets as well as the percentage of cells within the subsets that expressed the gene. Visualization was generated using the DotPlot function within the Seurat toolkit. D, Western blot analysis of PRDX1 in nonstimulated and IL15-stimulated (10 ng/mL) NK cells. Left: representative blot from one donor; right: densitometry analysis of PRDX1/ß-actin ratio (n = 7 donors). Data shown as median and range. Dots represent individual donors. Statistic: Wilcoxon test. E, qRT-PCR analysis of PRDX1 in nonstimulated and IL15-stimulated NK cells (10 ng/mL, 24 hours). Data show median and range. (n = 8 donors). Dots represent the average of two technical replicates. Statistic: Wilcoxon test. F, qRT-PCR analysis of PRDX1 after mTOR inhibition with Torin 1 (1 µmol/L) in nonstimulated and IL15-stimulated NK cells (10 ng/mL). Data show median and range (n = 7 donors). Dots represent the average from two technical replicates. Statistic: Wilcoxon test, in comparison with control nonstimulated group. G, qRT-PCR analysis of PRDX1 in nonstimulated NK cells (CON), after IL15 stimulation (10 ng/mL, 48 hours; IL15A) and IL15 stimulation (48 hours) followed by cytokine withdrawal (24 hours) (IL15W). Data show median and range (n = 13 donors). Dots represent the average from two technical replicates. Statistic: Wilcoxon test.