Normal HSPCs are not recognized by Siglec-6 CAR T cells. (A) Flow cytometric analysis of cell surface expression of different CAR target antigens on CD34⁺CD38^– hematopoietic stem cells (HSCs) (left) and CD34⁺CD38⁺ hematopoietic progenitor cells (HPCs) (right) from 5 healthy donors. Bar diagrams show NMFI ± standard deviation (SD). Two-way analysis of variance (ANOVA) *P < .05; **P < .01;***P < .001. (B) Quantification of Siglec-6 expression on HSPCs by dSTORM super-resolution microscopy. U937 cells and Siglec-6–negative U937 knockout (KO) cells were used for comparison. Each data point represents a cell. (C) Real-time qPCR was used to assess Siglec-6 mRNA transcripts in CD34⁺CD38^– HSCs and CD34⁺CD38⁺ HPCs. Data are normalized to MOLM-13 Siglec-6 mRNA transcripts. AML blasts from patient 24 were included in the analysis as a positive control (supplemental Figure 6). (D) Flow cytometric analysis of Siglec-6 expression on granulocyte colony-stimulating factor–mobilized CD34⁺CD38^– HSCs and CD34⁺CD38⁺ HPCs from peripheral blood of 5 healthy donors. Inset numbers indicate the NMFI. (E) Left: percentage of live (7-AAD negative) HSCs after 24-hour coincubation with CD8⁺ Siglec-6_BBz CAR, CD123_BBz CAR, or UTD T cells. The assay was performed in triplicate wells with 5000 target cells per well. Counting beads were used to quantify the number of residual live HSPCs at the end of co-culture. Data are from 3 independent experiments. Right: colony formation assay was performed with residual live HSPCs after 24 hours of co-incubation with CD8⁺ Siglec-6_BBz CAR, CD123_BBz CAR, or UTD T cells. Graphs show the absolute number of colonies (mean ± SD) per 55-mm plate as determined by microscopy on day 14 from 3 independent experiments. (F) Flow cytometric analysis of Siglec-6 expression on healthy donor peripheral blood mononuclear cells. Siglec-6 expression by B cells (CD45⁺CD19⁺), myeloid cells (CD45⁺CD33⁺), T cells (CD45⁺CD3⁺CD56^–), NK cells (CD45⁺CD56⁺CD3^–), and NK T cells (CD45⁺CD3⁺CD56⁺) in 7 healthy donors. Siglec-6 expression by Siglec-6–positive (U937, TF-1, MV4;11, and MOLM-13) and Siglec-6–negative (K562, Kasumi-1) cell lines are plotted for reference. (G) Flow cytometric analysis of Siglec-6 expression on healthy B cells, CD33⁺ myeloid cells, neutrophils (CD33⁺CD15⁺CD16⁺), and basophils (CD33⁺CD123⁺HLA-DR^–). *P < .05; **P < .01; ***P < .001; ****P < .0001, Student t test. BFU-E, burst-forming unit erythroid; CFU-G, colony-forming unit granulocyte; CFU-M, colony-forming unit macrophage; GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte; GM, granulocyte-macrophage.