Figure 1. im1928z1-CAR expression in HSPCs cells prevents T cell, but favors NK-like cell development of lymphoid progenitors in vitro and in vivo.

(A) The lentiviral control and the murine CD19 CAR construct: iTom (inducible dTomato reporter gene only) and im1928z1 (inducible murine CD19 CAR, CD28 costimulation, 1 functional ITAM containing CD3ζ domain) linked to an IRES dTomato cassette. LTR, long terminal repeats; T11, Dox-inducible promotor; scFv, single chain variable fragment; TM, transmembrane domain; IRES, internal ribosome entry site; PRE, woodchuck hepatitis virus posttranscriptional regulatory element. (B) Representative data showing im1928z1 expression on in vitro–generated lymphoid progenitors. (C) Representative FACS plots of NK1.1 and CD3 expression on in vitro–generated im1928z1-engineered lymphoid progenitors (left), NK1.1⁺ population within CD25⁺CD44⁺ lymphoid progenitors (middle), and NK1.1⁺ expression on iTom and im1928z1-transduced lymphoid progenitors before cotransplantation (right) (n = 3 independent cultures were pooled). (D) Irradiated B6 recipients were reconstituted with 3 × 10⁶ B6 TCD-BM and cotransplanted with either 8 × 10⁶ im1928z1-engineered lymphoid progenitors or iTom‑engineered lymphoid progenitors. (E) Thymic sections were imaged for Tom⁺ cells. Scale bars: 50 μm; Original magnification, × 20. Single cells from harvested thymi were analyzed by FACS for Tom⁺ progeny of cotransplanted lymphoid progenitors (n = 3 mice, respectively). (F) Lymphoid progenitor–derived progeny in the BM on day 14 (top). Numbers of NK1.1⁺ cells within the Tom⁺ population are depicted (bottom) (n = 3 mice per group). (G) Numbers of NK1.1⁺ and (H) frequencies of CD4⁺, CD8⁺, and CD3⁺TCRβ⁺ progeny within the Tom⁺ gate in BM and spleens on day 28 (im1928z1, n = 5; iTom, n = 4). Results from 1 of 2 independent experiments are shown. Statistics was performed using Student’s t test (2 tailed). Data are shown as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.