Transcriptomic Profiling of anti-CD276/NKG2DLs CAR-T Cells Stimulated by A549 Cells

1. we performed a bulk RNA-seq analysis after 24-hour co-culture with A549 cells. Compared with untransduced T cells, CAR-T cells presented broad transcriptomic differences. Pathway enrichment analysis of the differentially expressed genes (DEGs) revealed significantly upregulated cytokine-receptor cross-talk pathway, as well as TNF, JAK-STAT, NF-?B, and IL-17 signaling pathways in CAR-T cells compared with that in UTD. All these pathways are critical for T cell activation, proliferation, differentiation, and effector functions. 2. We also carried out continuous antigen exposure (CAE) wherein CAR-T cells were induced into an exhaustion state. We observed substantial transcriptomic differences and thousands of DEGs among distinct CAR-T cell groups and the UTD group. To better understand the specific advantages of Bicephali CAR-T cells over two single-targeting CAR-T cells, we employed Venn diagrams to visualize the DEGs among the three groups and revealed 346 unique DEGs of multi-targeting Bicephali CAR-T cells. We further subjected these 346 DEGs to Gene Ontology (GO) pathway enrichment analysis and found that positive regulation of T cell function and oxidative stress were among the top enriched pathways. Overall design: CAR-T cells were incubated with A549 tumor cells for 24h at a 1:1 E/T ratio, or incubated with A549 cells (E:T = 1:2) for five cycles (CAE). Then the cell suspensions were harvested, and the CAR-T cells were sorted using biotin-conjugated anti-BCMA mAbs (Cat# 357514, BioLegend) and anti-biotin magnetic beads (Cat# 480016, BioLegend). Total RNA was then extracted using RNA Extraction Reagent (Cat# R401-01, Vazyme) following the manufacturer's instructions. RNA quality assessment and transcriptome sequencing were conducted by Oebiotech (Shanghai, China).