m909 CAR T cells do not inhibit CD34⁺ HSC colony formation or eliminate FRβ-low healthy monocytes in vitro. (A) After healthy adult human bone marrow CD34⁺ HSCs were isolated, they were stained for FRβ expression using m909-IgG (black) or human IgG isotype control (gray). One representative donor is shown. (B) Isolated CD34⁺ HSCs were cocultured with CAR T cells at an E:T ratio of 1:1 for 4 hours. Wells were diluted in methylcellulose and cultured for 14 days. Total colonies were counted and scored for CFU-GEMM, CFU-GM, CFU-G, CFU-M, and BFU-E. There were no significant differences between total or lineage-specific colonies for any of the treated groups compared to untreated CD34⁺ HSCs. Bar graphs represent mean + standard deviation for n = 2 wells per condition. Results are representative of 4 independent experiments and 3 normal bone marrow donors. (C) Low surface expression of FRβ on normal human monocytes detected by flow cytometry using m909-IgG (black) or human IgG isotype control (gray). One representative of 7 normal donors is shown. (D) CD14⁺ human monocytes were cocultured with indicated engineered T cells at E:T ratios of 3:1 and 1:3 for 4 hours, after which the total number of live CD3⁻, CD14⁺ monocytes per well was quantified by bead-based flow cytometry. Data incorporates results using 3 different CAR T-cell donors and 4 different monocyte donors as target cells. BFU-E, erythroid blast forming unit; G, granulocyte; GEMM, granulocyte/erythrocyte/monocyte/megakaryocyte; GM, granulocyte/monocyte; M, monocyte.