ACK2 treatment plus LD-IR allows long-term engraftment of syngeneic wild-type bone marrow cells and gene-modified donor HSCs. (A) Posttransplantation donor chimerism of F1 C57/BoyJ mice conditioned with LD-IR (300 cGy) or ACK2 + LD-IR prior to transplantation with 1 × 106 freshly isolated C57 marrow cells. Recipients received 2 mg ACK2 or saline on day 1 and LD-IR on day 4, followed by transplantation on day 7. Peripheral blood was sampled at different times posttransplantation. Solid squares, ACK2 + LD-IR cohort; gray diamonds, LD-IR cohort. Donor cell chimerism at 24 weeks in ACK2 + LD-IR cohort was 79.3 ± 8.3% versus 15.3 ± 4.6% in LD-IR cohort (**P < .01, n = 4). (B) Posttransplant donor chimerism of F1 C57/BoyJ mice conditioned with saline, ACK2 alone, LD-IR (300 cGy), or ACK2 + LD-IR prior to transplantation with 20 × 106 (saline or ACK2) or 1 × 106 (LD-IR or ACK2 + IR) freshly isolated C57 marrow cells. PB leukocyte donor chimerism was determined 24 weeks posttransplantation. Bars represent the mean donor cell CD45.2 chimerism. **P < .01 (n = 4 in each group). (C) Secondarily transplanted donor HSCs from ACK2-treated mice give rise to long-term engraftment in secondary recipients. Marrow from 2 primary recipients in each cohort shown in Figure 2A was harvested at 24 weeks posttransplantation and for each primary recipient, 1 × 106 cells were transplanted into each of 4 lethally irradiated F1 C57/BoyJ mice. Donor chimerism in primary recipients was 3.4% and 5.2% for the 300 cGy mice and 71.2% and 69.3% for the ACK2 + LD-IR mice. Secondary recipients were analyzed for PB leukocyte donor chimerism 18 weeks after transplantation (mean ± standard deviation is shown). **P < .01 compared with 300 cGy cohort. One of 2 independent experiments is shown. (D) Conditioning with ACK2 + LD-IR prior to transplantation of lentivirus-tranduced cells in murine X-CGD. Lineage-negative BM cells from C57 X-CGD mice were purified using a MACS Separation System, then transduced in vitro with vesicular stomatitis virus–glycoprotein–pseudotyped CL20i4r-EF1a-gp91-OPT lentiviral vector overnight in the presence of interleukin-6 and SCF. Recipient F1C57/BoyJ X-CGD mice were treated with LD-IR (300 cGy) alone or ACK2 + LD-IR as described in panel A prior to transplantation with 5 × 105 transduced X-CGD lin- BM cells. n = 4 in each group. PB leukocyte donor chimerism and neutrophil NADPH oxidase activity was assayed at various times after transplantation. Different colored symbols represent individual mice. *P < .02 compared with LD-IR cohort. One of 2 independent experiments is shown.
