Figure 3.
Anti-CD117 mAb (ACK2) combined with AZA enhances HSC depletion and delays HSC recovery in vivo. (A) Treatment schema for panels B-F. C57BL/6 mice were injected intravenously with a single dose of ACK2 or 2B8 at 500 µg 5 days before the start of treatment with AZA. AZA was administered intraperitoneally at 5 mg/kg once per day for 5 days. Mice were euthanized at 6, 10, or 20 days after the first dose of AZA; spleens and BM from both legs and spine were harvested and analyzed by flow cytometry for HSC depletion and depletion of mature myeloid and lymphoid cells. (B) Hematoxylin and eosin staining of a BM section of 1 femur at day 6 from a mouse treated with ACK2 only, AZA only, or ACK2-AZA. (C) Absolute cell counts of the different HSPC compartments in the BM of untreated controls and ACK2-AZA–treated mice on days 6, 10, and 20 after the start of AZA treatment as measured by flow cytometry. (D) Comparison of the absolute cell counts of different HSPC compartments in the BM of mice treated with AZA only or ACK2-AZA at baseline and at 6, 10, and 20 days after start of AZA. (E) Comparison of the absolute cell counts of LT-HSCs in the BM of mice treated with 2B8-AZA vs ACK2-AZA at baseline and on days 10 and 20 after the start of AZA treatment. (F) Comparison of the absolute cell counts of ST-HSCs in the BM of mice treated with 2B8-AZA vs ACK2-AZA at baseline and on days 10 and 20 after the start of AZA treatment. For panels C-F, data for each experimental group were pooled from 2 independent experiments; n = 8-9 mice per group per time point. (G) Treatment schema for panels H-I. C57BL/6 mice were injected intravenously with a single dose of ACK2 500 µg 5 days before the start of treatment with AZA (administered intraperitoneally at a dose of 2.5 mg/kg per day for 3 days). Mice were euthanized 4 days after the first dose of AZA, and BM from both legs was harvested and analyzed by flow cytometry for annexin V and propidium iodide (PI) staining. (H) Representative flow cytometry plots of annexin V and PI staining (gated from Lin–Sca1+c-Kit+) in untreated controls and mice treated with ACK2, AZA 2.5 mg/kg, or ACK2-AZA. (I) Top: annexin V+; bottom: annexin V+/PI+; stained cells are shown as a percentage of LSK cells in the different treatment groups compared with untreated controls (n = 3-5 mice). Data are expressed as mean ± SD. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, nonsignificant.

Anti-CD117 mAb (ACK2) combined with AZA enhances HSC depletion and delays HSC recovery in vivo. (A) Treatment schema for panels B-F. C57BL/6 mice were injected intravenously with a single dose of ACK2 or 2B8 at 500 µg 5 days before the start of treatment with AZA. AZA was administered intraperitoneally at 5 mg/kg once per day for 5 days. Mice were euthanized at 6, 10, or 20 days after the first dose of AZA; spleens and BM from both legs and spine were harvested and analyzed by flow cytometry for HSC depletion and depletion of mature myeloid and lymphoid cells. (B) Hematoxylin and eosin staining of a BM section of 1 femur at day 6 from a mouse treated with ACK2 only, AZA only, or ACK2-AZA. (C) Absolute cell counts of the different HSPC compartments in the BM of untreated controls and ACK2-AZA–treated mice on days 6, 10, and 20 after the start of AZA treatment as measured by flow cytometry. (D) Comparison of the absolute cell counts of different HSPC compartments in the BM of mice treated with AZA only or ACK2-AZA at baseline and at 6, 10, and 20 days after start of AZA. (E) Comparison of the absolute cell counts of LT-HSCs in the BM of mice treated with 2B8-AZA vs ACK2-AZA at baseline and on days 10 and 20 after the start of AZA treatment. (F) Comparison of the absolute cell counts of ST-HSCs in the BM of mice treated with 2B8-AZA vs ACK2-AZA at baseline and on days 10 and 20 after the start of AZA treatment. For panels C-F, data for each experimental group were pooled from 2 independent experiments; n = 8-9 mice per group per time point. (G) Treatment schema for panels H-I. C57BL/6 mice were injected intravenously with a single dose of ACK2 500 µg 5 days before the start of treatment with AZA (administered intraperitoneally at a dose of 2.5 mg/kg per day for 3 days). Mice were euthanized 4 days after the first dose of AZA, and BM from both legs was harvested and analyzed by flow cytometry for annexin V and propidium iodide (PI) staining. (H) Representative flow cytometry plots of annexin V and PI staining (gated from Lin–Sca1+c-Kit+) in untreated controls and mice treated with ACK2, AZA 2.5 mg/kg, or ACK2-AZA. (I) Top: annexin V+; bottom: annexin V+/PI+; stained cells are shown as a percentage of LSK cells in the different treatment groups compared with untreated controls (n = 3-5 mice). Data are expressed as mean ± SD. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, nonsignificant.

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