Fig. 5: The effect of anti-CD123 CAR T cells on normal hematopoietic progenitor cell development.

a CFU-GM (HD BMMC only n = 12; NTD Tc + HD BMMC n = 9; anti-CD123 CAR Tc + HD BMMC n = 12), b CFU-GEMM (HD BMMC only n = 9; NTD Tc + HD BMMC n = 6; anti-CD123 CAR Tc + HD BMMC n = 12), and c BFU-E (HD BMMC only n = 9; NTD Tc + HD BMMC n = 6; anti-CD123 CAR Tc + HD BMMC n = 11) colonies were scored using an inverted microscope following a 14-day culture in methocult supplemented with hSCF, IL-3, EPO, G-CSF, and GM-CSF. CD34⁺ HD BM mononuclear cells (BMMC) were cultured alone, with NTD T cells, or anti-CD123 CAR T cells at an E:T ratio of 1:1 for 6 h before the cell suspension was transferred and plated in methocult. Colony numbers are represented per 1000 plated cells. Data was pooled from three different primary HD samples, each plated in at least duplicates. d Flow cytometry-based analysis depicting the total absolute cell numbers of B-lymphoid (CD45⁺CD34⁻CD19⁺), Monocyte (CD45⁺CD34⁻CD14⁺CD16⁺CD33⁺/CD123⁺), Myeloid (CD45⁺CD34⁻CD13⁺CD33⁺/CD123⁺), and plasmacytoid dendritic cell (pDC) (CD45⁺CD34⁻CD4⁺CD45RA⁺CD11c⁻MHC-II⁺CD304⁺CD123⁺) graft in the PB of HD CD34⁺ cord blood (CBMC) engrafted NSG mice pre- (day 0) and post-infusion (day 7 and day 16) with 5 × 10⁶ NTD Tc (n = 4) or anti-CD123 CAR Tc (n = 2). e Flow cytometry-based analysis depicting the absolute cell numbers (cells/µL) of B-lymphoid, monocyte, myeloid, and pDCs in the spleen and BM of HD CD34⁺ CBMC NSG engrafted mice 16 days following infusion with 5 × 10⁶ anti- NTD Tc (n = 4) or anti-CD123 CAR Tc (n = 2). f Flow cytometry-based analysis depicting the absolute cell numbers (cells/µL) of hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), and granulocytic macrophage progenitors (GMPs) in the BM of HD CD34⁺ CBMC NSG engrafted mice 16 days following infusion with 5 × 10⁶ NTD Tc (n = 4) or anti-CD123 CAR Tc (n = 2). Data were pooled from two independent experiments (d–f) and all graphed data are represented as mean ± SEM. p-values were calculated using two-sided one-way ANOVA (Tukey’s test or Kruskal–Wallis test with Dunn’s multiple comparison) (a–c) or two-sided unpaired student’s t-test (Mann–Whitney) (d–f).