Panel a shows flow cytometry plots showing the surface levels of CD19 on control and Rab5-CARTs at different time points during the CAE assay. The x-axis represents PE-CD19 (FMC63) and the y-axis represents PE-Cy7-CD19. Panel b shows a line graph summarizing the percentage of CD3 positive CD19 positive cells over time for control and Rab5-CARTs. The x-axis represents time points, and the y-axis represents the percentage of CD3 positive CD19 positive cells. Panel c shows flow cytometry plots displaying CD19 levels on the T cell surface at various time points using anti-CD19 (HIB19) and anti-CD19 (FMC63) antibodies. The x-axis represents PE-CD19 (FMC63) and the y-axis represents BV650-CD19 (HIB19). Panel d shows a schematic diagram illustrating the CART fratricide assay. Panel e shows flow cytometry plots showing mCherry-positive tEGFR or Rab5 CARTs 24 hours after co-culture with K.19.GFP cells, measuring surface CD19 and mCherry expression. The x-axis represents mCherry and the y-axis represents PE-Cy7-CD19. Panel f shows flow cytometry plots depicting the population frequencies of mCherry CART and BFP-fused 19BBz-tEGFR CARTs following co-culture for 2 days. The x-axis represents mCherry CART and the y-axis represents BFP CART. Panel g shows a scatter plot summarizing the percentage of mCherry positive cells. The x-axis represents the type of CARTs (tEGFR or Rab5) and the y-axis represents the percentage of mCherry positive cells. Panel h shows a scatter plot summarizing the mean fluorescence intensity (MFI) of the mCherry signal. The x-axis represents the type of CARTs (tEGFR or Rab5) and the y-axis represents the MFI of mCherry. Panel i shows flow cytometry plots assessing apoptosis of mCherry CARTs using SYTOX and Annexin V staining. The x-axis represents APC-Annexin V and the y-axis represents PB-SYTOX. Panel j shows a scatter plot summarizing the percentage of Annexin V positive SYTOX positive cells. The x-axis represents the type of CARTs (tEGFR or Rab5) and the y-axis represents the percentage of Annexin V positive SYTOX positive cells.
Rab5 expression facilitates target antigen clearance and resistance to fratricide. (a) Measurement of CD19 on the surface of control or Rab5-CARTs was measured by flow cytometry at indicated time points during the CAE assay. (b) Summary data of a. Data are representative of three independent experiments with samples from unique healthy donors. (c) Representative flow cytometry plots of CD19 on T cell surface, measured at the indicated CAE assay time points using anti-CD19 (HIB19) and anti-CD19 (FMC63) antibodies. Data are representative of three independent experiments. (d) Schematic representation of the CART fratricide assay used in e–j. mCherry-fused 19BBz-CARTs acquired the CD19 antigen from K.19.GFP tumor cells, triggering fratricide mediated by BFP-fused 19BBz-CARTs. (e) Representative flow cytometry plots of mCherry-positive tEGFR or Rab5 CARTs 24 h after co-culture with K.19.GFP cells, measuring surface CD19 and mCherry expression. (f) Representative flow cytometry plots of mCherry-CART and BFP-fused 19BBz-tEGFR CART population frequencies following co-culture for 2 days. (g and h) Summary of f indicating percentage of mCherry+ cells (g) and MFI of mCherry signal (h). (i and j) Apoptosis of mCherry-CARTs from f was assessed using SYTOX and Annexin V staining. Data in e–j are representative of three 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.