ACK2 synergizes with AZA and permits engraftment of congenic HSCs in immunocompetent mice. (A) Schematic of congenic transplantations. C57BL/6 (H-2^b, Thy1.1, CD45.1) mice were donors for C57BL/6 (H-2^b, Thy1.1, CD45.1/CD45.2) recipients. ACK2 at 500 µg was injected intravenously 10 days before cell infusion, and AZA was administrated on days −5 through −1 at a dose of 2.5 or 5 mg/kg per day. Recipients received 20 × 10⁶ whole BM cells (WBM) or 5 × 10⁴ FACS-sorted LSK cells on day 0 via retro-orbital injection. PB chimerism was assessed by flow cytometry using the CD45 allelic marker to distinguish between donor and recipient live total cells, myeloid cells (Gr1⁺Mac1⁺), B cells (CD19⁺CD3^–), and T cells (CD19^–CD3⁺). (B) Higher levels of sustained multilineage donor engraftment were observed in the ACK2-AZA group compared with single-agent ACK2 or single-agent AZA groups after transplantation of 20 × 10⁶ WBM cells (n = 3-6 mice per group). (C) ACK2-AZA at 5 mg/kg per day enables sustained multilineage engraftment of 5 × 10⁴ FACS donor congenic LSK cells (n = 6-7 mice per group). Data were pooled from 2 independent experiments and represent mean ± SD.