Chimeric antigen receptor–induced BCL11B suppression propagates NK-like cell development
Marcel Maluski, Arnab Ghosh, Jessica Herbst, Vanessa Scholl, Rolf Baumann, Jochen Huehn, Robert Geffers, Johann Meyer, Holger Maul, Britta Eiz-Vesper, Andreas Krueger, Axel Schambach, Marcel R.M. van den Brink, Martin G. Sauer
Marcel Maluski, Arnab Ghosh, Jessica Herbst, Vanessa Scholl, Rolf Baumann, Jochen Huehn, Robert Geffers, Johann Meyer, Holger Maul, Britta Eiz-Vesper, Andreas Krueger, Axel Schambach, Marcel R.M. van den Brink, Martin G. Sauer
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Research Article Immunology

Chimeric antigen receptor–induced BCL11B suppression propagates NK-like cell development

Abstract

The transcription factor B cell CLL/lymphoma 11B (BCL11B) is indispensable for T lineage development of lymphoid progenitors. Here, we show that chimeric antigen receptor (CAR) expression during early phases of ex vivo generation of lymphoid progenitors suppressed BCL11B, leading to suppression of T cell–associated gene expression and acquisition of NK cell–like properties. Upon adoptive transfer into hematopoietic stem cell transplant recipients, CAR-expressing lymphoid progenitors differentiated into CAR-induced killer (CARiK) cells that mediated potent antigen-directed antileukemic activity even across MHC barriers. CD28 and active immunoreceptor tyrosine–based activation motifs were critical for a functional CARiK phenotype. These results give important insights into differentiation of murine and human lymphoid progenitors driven by synthetic CAR transgene expression and encourage further evaluation of ex vivo–generated CARiK cells for targeted immunotherapy.

Authors

Marcel Maluski, Arnab Ghosh, Jessica Herbst, Vanessa Scholl, Rolf Baumann, Jochen Huehn, Robert Geffers, Johann Meyer, Holger Maul, Britta Eiz-Vesper, Andreas Krueger, Axel Schambach, Marcel R.M. van den Brink, Martin G. Sauer

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Figure 7

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

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CAR-induced developmental shifting from T cell to NK cell–like different...
(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.