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. Author manuscript; available in PMC: 2024 Oct 1.
Published in final edited form as: Nat Biotechnol. 2023 Feb 2;41(10):1434–1445. doi: 10.1038/s41587-022-01650-2

Figure 2: CAR.PDZ NK cells exhibit enhanced avidity and calcium flux upon cancer cell recognition.

Figure 2:

(a) Single cell assessment of at least NK cell avidity to EphA2-positive A549 tumor cells; at least 100 cells for each independent experiment was analyzed. UN; n=3 CAR Δ; n=4, CAR; n=3, CAR.PDZ; n=4 donors,mean±SEM shown.

(b) Normalized fold change of CAR NK cell binding compared to untransduced NK cells from (a). Arrowed line indicates the point of statistical difference at CAR.PDZ vs. CAR Δ and vs CAR at 548pN which continued to 1000pN. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR; mean±SEM shown.

(c) Avidity score of CAR NK cells determined by plateau of One-Phase Decay analysis from (a). One-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR. mean±SEM shown.

(d) Same experimental conditions as (a) except utilizing EphA2 deleted A549 cells.

(e) Same normalized fold change as described in (c) utilizing EphA2 deleted A549 cells.

(f) Representative single cell calcium flux analysis of NK cells interacting with A549 tumor cells over indicated time points. Tumor cells labelled in green, NK cells in red, and calcium flux seen as a yellow burst. White bar indicates 10 microns.

(g) Fold Change Calcium flux quantification of NK cells with A549 tumor cells. UN; n= 28, CAR Δ; n=35, CAR; n=44, CAR.PDZ; n=48. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR q<0.001 *** at minute 1; 1st peak AUC analysis with unpaired Brown-Forsythe and Welch’s ANOVA. mean±SEM shown of one donor.

(h) Fold Change Calcium flux quantification of NK cells with DIPG007 tumor cells. CAR Δ; n=30, CAR; n=19, CAR.PDZmut: n=26, CAR.PDZ n=43. Two-Way ANOVA was used to determine statistical significance with Two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli to correct for FDR q<0.001 *** at minute 6; 1st peak AUC analysis with unpaired Brown-Forsythe and Welch’s ANOVA. mean±SEM shown of one donor.