Panel a: A series of flow cytometry plots show the presence of K.19 tumor cells (GFP positive) and 19BBz-tEGFR CARTs (CD3 positive) at different time points during the CAE assay. The x-axis represents PE-CD3 and the y-axis represents GFP-K.19. The plots indicate the percentage of GFP positive and CD3 positive cells at each time point. Panel b: A Venn diagram displays the blue circle represents downregulated genes, and the yellow circle represents endocytic genes, with 54 genes overlapping. Panel c: A heatmap shows the expression profiles of endocytosis-related genes in samples from four healthy donors. The x-axis represents individual donor samples, and the y-axis lists the genes. Colors range from green (low expression) to red (high expression). Panel d: A histogram compares the endocytic activity of CARTs on day 10 post T cell stimulation (D10) and after a second-round CAE assay (CAE). The x-axis represents pHrodo Dextran Red intensity, and the y-axis represents the normalized count. Panel e: A scatter plot shows the mean fluorescence intensity (MFI) of pHrodo Red dextran uptake in CARTs on day 10 and after the CAE assay. The x-axis represents the groups (D10 and CAE), and the y-axis represents MFI. Panel f: Flow cytometry plots and a histogram compare the endocytic activity of tumor-infiltrating 19BBz-tEGFR and MESOBBz-tEGFR CARTs. The flow cytometry plots show AF647-MSLN protein expression, and the histogram shows pHrodo Dextran Red intensity. Panel g: A scatter plot shows the MFI of pHrodo Red dextran uptake in 19BBz and MESOBBz CARTs. The x-axis represents the CART types, and the y-axis represents MFI. Panel h: A series of histograms show the intracellular Rab5 expression level in 19BBz-tEGFR CARTs during the CAE assay. The x-axis represents PE-Rab5c intensity, and the y-axis represents the normalized count.
Continuous tumor interactions lead to downregulation of endocytic activity in CARTs. (a) Flow cytometry of the CAE assay at indicated time points, used to analyze the presence of K.19 tumor cells (GFP+) and 19BBz-tEGFR CARTs (CD3+). Primary human T cells were activated and transduced with 19BBz-CAR. After 9 days of culture, 1 × 106 CAR-positive T cells were co-cultured with 2 × 106 K562 cells expressing CD19 and GFP (K.19.GFP). Every 2 days, additional 2 × 106 K.19.GFP were added to the co-culture assay. Data are representative of three independent experiments with samples from unique healthy donors. (b) Venn diagram showing the overlap between downregulated genes and genes associated with endocytic processes. Bulk RNA-seq identified over 2,000 genes downregulated in 19BBz-tEGFR CARTs after tumor engagement, of which 54 were annotated as endocytosis-related. (c) Heatmap showing bulk RNA-seq expression profiles of endocytosis-related genes in b, excluding four genes with low read counts. Each column represents an individual donor sample (n = 4 healthy donors). (d and e) Endocytic activity of CARTs obtained on day 10 after T cell stimulation or after a second-round CAE assay was measured by uptake of pHrodo Red dextran at 37°C for 20 min, analyzed by flow cytometry. Data are representative of three independent experiments with samples from unique healthy donors. (f and g) pHrodo Red dextran endocytosis was assessed in tumor-infiltrating CARTs. 19BBz-tEGFR and MESOBBz-tEGFR CARTs were mixed at a 1:1 ratio and infused into K.19.GFP tumor-bearing mice 7 days after tumor implantation. 14 days after CART infusion, tumor-infiltrating CARTs were isolated and incubated with pHrodo Red dextran at 37°C for 20 min. Endocytic activity was compared between CD19-responsive 19BBz-tEGFR and CD19-nonresponsive MESOBBz-tEGFR CARTs. Data are representative of two independent experiments, n = 5 mice for each group. (h) Intracellular Rab5 expression level in 19BBz-tEGFR CARTs, measured during the CAE assay by flow cytometry; data are representative of two independent experiments with samples from unique healthy donors. Error bars show mean ± SEM. Statistical comparisons were made using unpaired t tests. *P < 0.05 and **P < 0.01.