Fig. 1. The CAR construct impacts the differentiation of CAR-iPSCs into T-cell lineages.

a, Schematic presentation of the CAR constructs 19z, 1928z, 19bbz, 1928bbz and w/oED28z CAR (w/oED28z lacks the extracellular scFv domain of 1928z CAR). Each construct was inserted at the indicated internal promotor(s) of the lentiviral vector pCS. b, Comparison of surface antigen profiles of immature iCAR-T cells from various kinds of iCARs. Haematopoietic progenitor cells derived from iPSCs were transduced with CARs (see a) and differentiated into T-cell lineages. To compensate for well-to-well variation, we used hKO1-negative cells in the same well as internal controls. Differentiation efficiencies were calculated as follows: CD5CD7 DP = (percentage of CD5CD7 DP cells in hKO1-positive cells)/(percentage of CD5CD7 DP cells in hKO1-negative cells); CD4CD8 DP = (percentage of CD4CD8 DP cells in hKO1-positive cells)/(percentage of CD4CD8 DP cells in hKO1-negative cells). Each dot represents biological replicates (n = 6). One-way ANOVA with Dunnett’s multiple comparisons test. c, Haematopoietic progenitor cells derived from inducible 1928z or 1928bbz CAR-transduced T-iPSCs were divided into two groups and subsequently differentiated on FcDLL4 for 2 days in the presence (2 μg ml⁻¹) or absence of doxycycline. Representative FCM data representing the phosphorylation of CD3ζ in the two groups are shown. d, Representative FCM data comparing surface antigen expression of CD4 and CD8β on immature iCAR-T cells from 1928z-transduced and 1928bbz-transduced iPSCs. NTD, not transduced.