Panel a shows a histogram with the x-axis representing the fluorescence intensity, and the y-axis represents the normalized count. Panel b displays confocal microscopy images comparing endocytosis activity between tEGFR-CARTs and Rab5-CARTs, with GFP (green), CD45 (blue), nuclei (red), and AM1-44 (yellow) shown. Panel c presents flow cytometry plots of K.19.GFP tumor cell killing by different CARTs during the CAE assay, with the x-axis representing PE-CY7-CD3 and the y-axis representing GFP-K.19. Panel d is a bar graph summarizing the percentage of GFP-K.19 positive cells at different CAE time points for various CARTs. Panel e shows flow cytometry plots of K.19.GFP tumor cells mixed with non-transduced T cells, tEGFR-CARTs, Rab5-CARTs, or Rab5-S35N-CARTs at indicated time points. Panel f is a line graph summarizing the percentage of remaining K.19.GFP tumor cells during the CAE assay, with the x-axis representing time points and the y-axis representing the percentage of GFP-K.19 positive cells. Panel g is a line graph showing the persistence of CD3 positive T cells during the CAE assay, with the x-axis representing time points and the y-axis representing the percentage of CD3 positive T cells.
Forced Rab5 expression restores endocytic activity and CART function. (a) pHrodo Red dextran endocytosis in 19BBz CARTs expressing tEGFR, Rab5, Rab4, or Rab11 from third round CAE assay, measured by flow cytometry. Data are representative of three independent experiments with samples from unique healthy donors. (b) Comparison of endocytosis activity between tEGFR-CARTs and Rab5-CARTs by tracking AM1-44 uptake in the presence of K.19.GFP using confocal microscopy. GFP (green), CD45 (blue), nuclei (red), and AM1-44 (yellow) are shown. Data are representative of two independent experiments. (c and d) Representative flow cytometry plots of K.19.GFP tumor cell killing by 19BBz-tEGFR, -Rab5, -Rab4, or -Rab11 CARTs in CAE assay (c). Summary data of K.19.GFP survival during CAE assay time points shown in c. Each dot represents an independent experiment using a unique donor (d). Data are representative of three independent experiments with samples from unique healthy donors. (e) Representative flow cytometry plots of K.19.GFP tumor cells after being mixed with NTDs, tEGFR-CARTs, Rab5-CARTs, or Rab5-S35N-CARTs at indicated time points in the CAE assay. (f and g) Summary of K.19.GFP tumor cells remaining (f) and CD3+ T cell persistence (g) during the indicated CAE assay, as shown in e. Experiments shown in e–g 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 < 0.001; ns, not significant.