Extended Data Fig. 6: Coexpression of switch receptors attenuates inhibitory receptor upregulation, ROS production, and endoplasmic reticulum stress response activation.
From: Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors
a, MAGIC110 imputed gene expression violin plots of inhibitory receptor and transcription-factor genes for dual-CAR ± multi-Switch T cells related to Fig. 5. Differentially expressed genes were compared via Wilcoxon test prior to imputation. b, Flow cytometry analysis of dual-CAR ± multi-Switch T cells 24 h following stimulation with BM185-CD19. Representative of n = 2 donor experiments. c, Dual-CAR ± multi-Switch T cells were stained with CM-H2DCFDA to measure total ROS two days following the last TCR stimulation. Left, representative flow cytometry plots from one of two donors. Right, bar graphs depicting mean CM-H2DCFDA values from triplicate wells from n = 2 donors. Replicate values were compared via one-way ANOVA, with Tukey’s test. Data are mean ± SEM. d, ER stress score gene set (GOBP_RESPONSE_TO_ENDOPLASMIC_RETICULUM_STRESS) comparison for dual-CAR ± multi-Switch T cells related to Fig. 5. Single-cell ER stress score values were compared via one-way ANOVA, with Tukey’s test. e, STRING115 analysis combining functional and physical protein interaction networks corresponding to the union of GO:0034976 ER stress response genes significantly upregulated (adjusted P < 0.05) in CD4+ or CD8+ dual-CAR T cells compared with dual-Switch and triple-Switch T cells. Gene nodes without connecting edges were excluded. Thickness of edges indicates strength of data support. f, Heat map depicting gene scaled expression of selected ER stress response genes in the IRE1α and ATF6 ER stress pathways. g, Full GSEA pathway illustrations for Fig. 5g.
