Panel a shows a schematic of continuous antigen exposure. Panel b features a scatter plot with axes labeled PC1 and PC2, with units in percent. The plot shows two distinct clusters of samples, indicating transcriptomic differences driven by tumor engagement. Panel c displays a volcano plot of gene expression changes in 19BBz-tEGFR CARTs, with significantly upregulated genes highlighted in green, downregulated genes in purple, and unchanged genes in grey. The axes are labeled Significance (Log10) and Fold Change (Log2). Panel d presents a dot plot. The axes are labeled Term and NES, with the color gradient indicating the negative log10(P Value). Panel e includes a histogram. The axis is labeled pHrodo Dextran Red. Panel f shows confocal microscopy images of 19BBz-tEGFR CARTs assessing endocytic activity by AM1-44 uptake in the absence or presence of K.19.GFP, with different colors indicating GFP, CD45, nuclei, and AM1-44. Panel g features a scatter plot quantifying the integrated density of AM1-44 dye in T cells, with the axis labeled IntDen of AM1-44. Panel h displays a Western blot analysis of Rab protein expression in CARTs before and after treatment by tumor cells, with axes labeled with protein names and days.
Continuous tumor interactions lead to downregulation of endocytic activity in CARTs. (a) Schematic of CAE assay. (b) Principal component analysis (PCA) of bulk RNA-seq data from 19BBz-tEGFR CARTs cultured in the presence (CAE samples) or absence (rested samples, D9) of K.19.GFP tumor cells. Each dot represents an individual donor sample. Separation along PC1 and PC2 reflect transcriptomic differences driven by tumor engagement. (c) Volcano plots of bulk RNA-seq data displaying gene expression changes in 19BBz-tEGFR CARTs cultured in the presence or absence of K.19.GFP tumor cells. Significantly (P value <0.05) upregulated genes (>twofold), downregulated genes (<-twofold), and unchanged genes are highlighted in green, purple, and grey, respectively. (d) Normalized enrichment score (NES) for biological pathways obtained post GO analysis, comparing 19BBz-tEGFR CARTs cultured with or without K.19.GFP tumor cells. Data in b–d are representative of four samples from different donors. (e) Endocytic activity of rested T cells or T cells activated with anti-CD3/CD28 beads for 24 h was measured by uptake of pHrodo Red dextran using flow cytometry. Data are representative of three independent experiments with samples from independent healthy donors. (f) Endocytic activity of 19BBz-tEGFR CARTs was assessed by AM1-44 uptake in the absence or presence of K.19.GFP and visualized by confocal microscopy. GFP (green), endogenous CD45 (blue), nuclei (red), and AM1-44 (yellow) are shown. (g) Integrated density (IntDen) of AM1-44 dye in T cells shown in f was quantified using ImageJ. Data in f and g are representative of two independent experiments with samples from independent healthy donors. (h) Western blot analysis of Rab protein expression in CARTs before they were treated by tumor cells. Primary human T cells were activated on day 1, which induced Rab protein expression. Following anti-CD3/CD28 beads removal on day 3, Rab protein levels were downregulated. Data are representative of three independent experiments with samples from independent healthy donors. Error bars show mean ± SEM. Statistical comparisons were made using unpaired t tests. ****P < 0.0001. Source data are available for this figure: SourceData FS1.