A) ACK2 is cleared from serum of RAG2−/−γc−/− mice seven days after injection. Serum of mice receiving 500μg ACK2 was analyzed every two days for ACK2 antibody by staining c-kit⁺ mast cells (31). B) ACK2 administration leads to depletion of BM HSCs. The number of KLS CD135⁻ CD150⁺ HSCs in both femurs and tibia of ACK2-treated and control mice was determined. Mean values +/− SEM are shown (n = 3 for each time point); ** indicates p-value < 0.001. C) ACK2, but not 2B8 treatment, depletes functional HSCs from BM. 200,000 unfractionated bone marrow cells from RAG2^−/−γc^−/− mice treated with 500μg ACK2 or 2B8 nine days earlier were transplanted into wild type irradiated recipients alongside 200,000 untreated competitor wild type bone marrow cells. Mean values +/− SEM are shown (n = 5–8); ** indicates p-value <0.01. D) ACK2 treatment does not directly cause HSC mobilization to the spleen. Entire splenocyte populations from mice treated with 500μg ACK2 nine days earlier were transplanted alongside 200,000 competitor bone marrow cells from wild type mice. Mean values +/− SEM are shown (n = 3–9); ** indicates p-value <0.001. E) ACK2 inhibits SCF mediated HSC proliferation. HSC were isolated from wild type mice, plated at 10 cells/well cultured in the presence of SCF or TPO and ACK2 or 2B8. Proliferation was observed by light microscopy. ** indicates p-value <0.05 as compared to ACK2 treated samples.

A) ACK2 treatment increases available HSC niche space. In two separate experiments, RAG2−/−γc−/− mice were treated with ACK2 and transplanted nine days later with varying doses of HSCs (CD45.1). Donor granulocyte chimerism was measured as above 24 weeks after transplantation for the first experiment, and 4 weeks for the second experiment. Mean values +/− SEM are shown. B) Flow cytometry profiles of mice transplanted with 35,000 HSCs. Chimerism of CD3⁻B220⁻Mac1^high side scatter^high peripheral blood granulocytes is shown.

Peripheral blood of transplanted unconditioned RAG2−/−γc−/− mice was analyzed 16 weeks after HSC transplantation for GFP⁺ granulocytes. The bottom panel represents an expanded view of results from transplantation of 10–1000 HSCs. In the bottom right panel, mice were co-transplanted with CD45.1 1000 HSCs and 100,000 GFP⁺ KLS CD34⁺ cells. Mean values +/− SEM are shown (n = 4–5 for each dose); ** indicates p-value <0.05 relative to the chimerism arising from the 10 HSC-transplanted group. The dashed line represents the theoretical HSC chimerism if engraftment were to increase linearly with transplanted cell dose from the observed chimerism at the 50-HSC dose group.

A) ACK2 conditioning leads to higher donor myeloid chimerism. Donor granulocyte chimerism was measured following transplantation of 5000 HSCs in RAG2−/− mice conditioned with ACK2 seven days prior to transplant and compared to that of unconditioned mice. Mean values +/− SEM are shown (n = 4); ** indicates p-value <0.01. B) HSC transplantation of ACK2-treated animals leads to lymphocyte reconstitution. Splenic donor-derived B and T-cells from ACK2-treated and unconditioned RAG2^−/− mice were enumerated 39 weeks after transplantation with wild type HSCs. Mean values +/− SEM are shown (n = 3–5); ** indicates p-value <0.01. C) Granulocyte chimerism accurately measures BM HSC chimerism. Peripheral blood granulocyte (Ter119⁻CD3⁻B220⁻Mac-1^highside scatter^high) chimerism at 37 weeks post-transplantation was correlated with HSC (c-kit⁺ lineage⁻ Sca-1⁺ CD34⁻CD150⁺) chimerism in the BM at 39 weeks post-transplantation upon sacrifice. Solid line illustrates linear regression with 95% confidence interval shaded in gray. Dashed line represents theoretical values if donor granulocyte chimerism were identical to donor HSC chimerism. D) Secondarily transplanted donor HSCs from ACK2 treated give rise to long term multilineage engraftment. Peripheral blood chimerism of B cells (B), T cells (T), and granulocytes (G) are shown 16 weeks post-secondary transplant for 2 independent experiments. Mean values +/− SEM are shown (n = 7–8 in each experiment).