Skip to main content
. 2019 Oct 22;129(12):5108–5122. doi: 10.1172/JCI126350

Figure 7. CAR-induced developmental shifting from T cell to NK cell–like differentiation translates to humans.

Figure 7

(A) Representation of the lentiviral human CD19 CAR constructs with either CD28 costimulatory and CD3ζ signaling domain (h1928z3) or without signaling domains (h19delta). An IRES dTomato reporter cassette was used. (B–F) Human CD34+ CB-derived HSPCs were engineered with respective CAR constructs and consecutively differentiated on OP9-DL1 stromal cells. FACS analyses were performed within the Tom+ gate on day 21 of coculture. For stimulation, h1928z3 lymphoid progenitors were cocultured with irradiated hCD19+ Daudi cells at a 1:10 ratio from day 4 onwards. Results from 1 of 2 experiments are shown. (B) Expression of the CAR constructs on differentiating human HSPCs analyzed by protein L staining. (C) CD7+CD5+ engineered human lymphoid progenitor cells were evaluated for CD5 and CD1a expression. Numbers represent percentages in the respective gates. (D) Histograms represent NOTCH1 expression on engineered early hematopoietic human progenitors. (E) CAR-modified HSPCs were analyzed for CD161 and CD56 expression. (F) Human CAR-engineered lymphoid progenitors were evaluated for TCRB rearrangement by PCR analysis of genomic DNA on day 18 of culture. Human PBMCs and nontransduced and h19delta-modified progenitors were used as controls. (G) Hierarchical clustering of the 500 most differentially expressed (P < 0.05) transcripts across lymphoid progenitors expressing the h1928z3 CAR that had been either stimulated with hCD19 or not; h19delta CAR served as signaling-deficient control. (H) Heatmap showing the relative expression of exemplary transcripts that related to either T cell or NK cell development. Data are normalized according to expression in each row. (G and H) Experiments were performed once. h1928z3, h1928z3 + hCD19 (n = 3); h19delta (n = 4). (I) qPCR analysis of BCL11B expression in nontransduced or h1928z3-expressing progenitors stimulated with hCD19. Data show mean of triplicates and upper and lower limit from 1 experiment performed.