Extended Data Fig. 5: Multi-Switch receptor arrays enhance T cell activity in response to inhibitory ligands.
From: Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors
a,b, Flow cytometry analysis of Zip-sorted dual-CAR BALB/c T cells ± FasBB switch receptor co-cultured for 24 h with BM185-CD19 (E:T = 1:1) or left unstimulated. Data are mean ± SEM (biological replicates) of triplicate wells from n = 3 donor experiments. c, Vector maps for dual-CAR and switch receptor configurations as depicted in the table. d–g, Flow cytometry analysis of Zip-sorted dual-CAR, dual-CAR FasBB, and dual-CAR multi-Switch BALB/c T cells stimulated for 24 h with BM185-CD19 (E:T = 1:2) or left unstimulated. Data are mean ± SEM (biological replicates) of triplicate wells from n = 4 donor experiments. Statistical comparisons were calculated using two-way ANOVA, with Tukey’s test. h, Flow cytometry analysis of BM185-ffluc-Thy1.1-Neo cell lines engineered to express FasL, PD-L1, or CD200. i–k. Sublethally irradiated BALB/c mice were injected with 1:1:1 mixture of BM185-CD19-FasL, BM185-CD20-PD-L1, and BM185-CD20-CD200 and treated with Zip-sorted dual-CAR BALB/c T cells, pre-cultured for 2 days with 1 μM dasatinib. i, Leukaemia BLI, and j, mouse survival, respectively, from one experiment. Survival differences were compared via log-rank test. k, Flow cytometry analysis of inhibitory ligand expression of single-ligand-positive BM185 lines harvested from bone marrow of mice reaching humane endpoints. PD-L1 expression difference was compared via a two-tailed t-test. Data are mean ± SEM (biological replicates). l, Live-cell microscopy (Incucyte) analysis of EGFP-labelled dual-CAR and dual-CAR triple-Switch Zip-sorted BALB/c T cells co-cultured with iRFP713+ BM185 target cell lines as depicted (E:T = 1:1), with repetitive target addition on days 0, 1, and 2. Representative of n = 3 donor experiments. Data are mean ± SEM of triplicate samples. m, Live-cell microscopy (Incucyte) analysis of NFκB-EGFP-reporter labelled dual-CAR and dual-CAR triple-Switch Zip-sorted BALB/c T cells co-cultured with iRFP713+ BM185 target cell lines as depicted at E:T = 1:9. Representative of n = 2 donor experiments. Values represent mean ± SEM of triplicate samples.
