Extended Data Fig. 3: OR7A10 ORF engineering enhances multiple effector functions of human primary CAR-PBNK cells, validated with multiple independent donors.
From: OR7A10 GPCR engineering boosts CAR-NK therapy against solid tumours
a, Left, Flow cytometry of population in CAR-PBNK cells derived from Donor 0005 (same representative donor in Fig. 2). Right, RT-qPCR for overexpression of OR7A10 in CAR-PBNK cells from Donor 0005 (same representative donor in Fig. 2). b, Flow cytometry of population (left) and surface flag-CAR expression (right) in CAR-PBNK cells derived from Donor 7014. c, Co-culture assays of PBNK, CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells with HT29-GL, H1299-PL, K562-GL and MCF-PL cancer cells with indicated E: T ratios at 6 h (n = 3). d, Flow cytometry of population and surface flag-CAR expression in CAR-PBNK cells derived from Donor 1. e, RT-qPCR for overexpression of OR7A10 in CAR-PBNK cells (n = 3). f, Co-culture assays of CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells with K562-GL and HT29-GL cells with indicated E: T ratios at different time points (n = 3). g, Flow cytometry of population and surface flag-CAR expression in CAR-PBNK cells derived from Donor 2. h, RT-qPCR for overexpression of OR7A10 in CAR-PBNK cells (n = 3). i, Co-culture assays of CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells with K562-GL and HT29-GL cells with indicated E: T ratios at different time points (n = 3). j, Flow cytometry of population and surface flag-CAR expression in CAR-PBNK cells derived from Donor 3. k, Co-culture assays of CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells with K562-GL and HT29-GL cells with indicated E: T ratios at different time points (n = 3). l, Flow cytometry of population and surface flag-CAR expression in CAR-PBNK cells derived from Donor 4. m, Co-culture assays of CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells with K562-GL and HT29-GL cells with indicated E: T ratios at different time points (n = 3). n, Flow cytometry of cell trace dye dilution and cell count of PBNK, CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells after 3 days of culture, starting from 1 million cells per group. o, Flow cytometry of chemokine receptor CXCR2 surface expression of PBNK, CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells upon HT29 stimulation at E: T = 1: 1 for 24 h. p-r, Flow cytometry of activation marker CD69 and CD25 and costimulatory receptor 4-1BB surface expression of PBNK, CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells upon HT29 stimulation at E: T = 1: 1 for 6 h. Data represent technical quadruplicates from one representative donor out of 2 different human donors. s, Schematic representation of repeated challenge and cytolysis after the third round of stimulation. The E: T ratio was maintained at 1: 1 across three rounds of stimulation. Data represent technical triplicates from one representative donor out of 3 different human donors. t-v, Flow cytometry of exhaustion markers Tim-3, Lag-3, NKG2A, and PD-1 expression in PBNK, CAR-PBNK;OR7A10(STOP) and CAR-PBNK;OR7A10 cells at 24 h following the third round of stimulation with HT29 cells. Data represent technical triplicates from one representative donor out of 3 different human donors. Note: in all bar blots, data are shown as mean ± SEM. The statistical significance levels are indicated in the plots by Two-way ANOVA with Sidak post-hoc analysis and FDR-correction to p values (c, f, i, k, and m) or unpaired t test (a, e, h, n, o, p, q, r, s, t, u, and v). Statistical tests are two-sided unless otherwise noted. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Exact p-values and detailed statistics are provided in the Source Data Excel file. The schematic in s was created using BioRender (https://biorender.com).
