HLA-DR-negative human myeloid cells predominate in hSCF Tg NSG recipient BM. (A) Flow cytometric contour plots demonstrating FSC and SSC characteristics of 6 hSCF Tg NSG recipient BM (S1-1, S1-2, S9-1, S4-1, S12-2, and S2-1) and 3 non-Tg NSG recipient BM (N1-1, N1-2, and N9-1). Polymorphonuclear myeloid cells (red asterisks) are present at high frequencies in hSCF Tg NSG recipient BM. (B) Flow cytometry contour plots demonstrating hCD33 and HLA-DR expression in the same recipients as shown in panel A. Consistent with their FSC and SSC characteristics, hSCF Tg NSG recipient BM contained a prominent CD33⁺HLA-DR⁻ granulocyte population (red asterisks; N1-1, killed at 21 weeks; N1-2, killed at 16 weeks; N9-1, killed at 20 weeks; S1-1, killed at 23 weeks; S1-2, killed at 20 weeks; S9-1, killed at 16 weeks; S4-1, killed at 13 weeks; S12-2, killed at 8 weeks; and S2-1, killed at 16 weeks). (C) Representative flow cytometric scatter plots of hSCF Tg NSG recipient BM demonstrating the identification of human c-Kit⁺CD203c⁺ mast cells within the hCD33⁺ fraction and HLA-DR⁻SSC^high granulocytes and HLA-DR⁺SSC^low APCs within the c-Kit⁻CD203c⁻ fraction (S4-1, killed at 13 weeks; and S2-1, killed at 16 weeks). (D) Frequencies of human c-Kit⁺CD203c⁺ mast cells, CD33⁺HLA-DR⁻ granulocytes, and CD33⁺HLA-DR⁺ APCs within the total hCD45⁺hCD33⁺ myeloid cell population in the BM of hSCF Tg and non-Tg NSG recipients. Numbers of cells in the granulocyte/neutrophil fraction were significantly higher in hSCF Tg NSG recipient BM (hSCF Tg n = 20, non-Tg n = 12, P = .0001). (E) CD33⁺HLA-DR⁻ cells from hSCF Tg and non-Tg NSG recipient BM were FACS-purified and examined by MGG staining. In 9 of 13 hSCF Tg recipients (S4-1 and S12-2 shown as representative), immature myeloid cells composed the majority of cells in this fraction. In 4 of 13 hSCF Tg recipients (S2-1 shown as representative) and 4 of 5 non-Tg NSG recipients (N12-1 shown as representative), mature neutrophils (band and segmented forms) were observed (N12-1, killed at 8 weeks; S2-1, killed at 16 weeks; S4-1, killed at 13 weeks; and S12-2, killed at 8 weeks). (F-G) Global transcriptional profiles of FACS-purified CD33⁺cKit⁻CD203⁻HLA-DR⁻ granulocytes and CD33⁺c-Kit⁻CD203c⁻HLA-DR⁺CD14⁺ monocytes derived from hSCF Tg NSG and non-Tg NSG recipient BM as well as human CD16⁺ neutrophils and CD14⁺ monocytes were compared. (F) Unsupervised clustering for each group is shown. (G) The expression heatmap demonstrates genes that are significantly under- and over-represented in each population.