AMD3100 Enhances Engraftment Figure 8. Schematic diagram of AMD3100’s mechanisms of action. Post transplant administration of AMD3100 likely has 3 effects. 1. AMD3100 mobilizes residual recipient’s radioresistant HSCs (indicated as red circle), thus freeing up more niches for healthy donor HSCs (indicated as purple circles) to engraft. This is supported by Fig. 5. 2. AMD3100 relieves the inhibitory effect of SDF-1 on primitive HSCs, allowing for engrafted HSCs to proliferate. 3. AMD3100 mobilizes engrafted donor HSCs and mobilized HSCs are more active and more proliferative. Effects 2 and 3 are supported by Fig. 6. Recipient HSCs after irradiation likely have survival disadvantage compared to donor HSCs. The end result is selective engraftment and expansion of donor hematopoietic cells. doi:10.1371/journal.pone.0011316.g008 benzoyl-TN14003). Abraham et al recently reported enhanced recovery of marrow after transplantation with the CXCR4 antagonist 4F-benzoyl-TN14003[49]. The overall effects of the blockage of CXCR4/SDF-1 with AMD3100 on hematopoiesis are outlined in Fig. 8. Additionally, AMD3100 could affect molecular components of niche microen- vironment such as cytokines, chemokines, neurotransmitters, and oxidative status that are crucial to the homing, proliferation and mobilization of HSCs. Our results indicate that AMD3100 promotes recovery of all donor cell lineages, including myeloid cells, T and B lymphocytes, erythrocytes and platelets. The data suggest that AMD3100 enhances engraftment of multipotent primitive HSCs. Furthermore, as shown in Fig. 2 and 4, the total blood counts and marrow KLS stem cells in AMD3100-treated mice at day +65 were significantly higher than those in PBS- injected control mice. Moreover, using secondary transplant model, we found that prolonged post transplant administration of AMD3100 did not adversely affect the long-term engraftment of primitive HSCs (Fig. 7). Our current study provides important insights into the roles of SDF-1/CXCR4 interaction in transplant-related mortality and in hematopoiesis following HCT. Our study suggests that blocking SDF-1/CXCR4 interaction with CXCR4 antagonist has many important ramifications not only in clinical HSC transplantation, but also potentially in other clinical settings that involve exaggerated inflammatory responses. Since AMD3100 is FDA approved for mobilization of HSCs, the translational potential of this agent is significant. Its use to enhance engraftment and decrease the toxicity would be especially important in clinical setting such as cord blood transplantation where engraftment remains a problem. AMD3100 can also be used routinely in any myeloablative allogeneic transplantation to ensure robust donor cell engraftment. Our studies indicate that post transplant administration of AMD3100 is a new approach for enhancing hematological recovery and reducing transplant-related mortality in HCT. Methods Animals Eight to twelve week old C57BL/6, CD45.2, Thy1.2 (H2b, termed ‘‘CD45.2 mice’’) mice were purchased from the Jackson Laboratories (Bar Harbor, ME). C57BL/Ka, CD45.1, Thy1.1 mice (H2b, termed ‘‘CD45.1 mice’’) were bred and maintained in our specific pathogen-free animal facility. Studies were performed in accordance with Duke University Institutional Animal Care and Use Committee-approved procedures. Antibodies and reagents AMD3100 used in our first 2 sets of in vivo experiments was purchased from Sigma (St Louis, MO). AMD3100 (Plerixafor, MozobilTM, Genzyme Corp.) provided by Genzyme Corp under a research agreement was used in our third set of in vivo experiment and in all other studies, including stem cell homing studies, cytokine studies and in vivo proliferation experiments. Antibodies for phenotypic analyses included: R-phycoerythrin (PE)-conjugat- ed anti-CD45.1 (clone A20); FITC- conjugated anti-CD45.2 (clone 104); APC-Cy7-labeled anti-CD45.2; APC-conjugated anti- CD3 (clone 145-2C11); APC-Cy7-labeled anti mouse CD4 (L3T4, clone GK1.5); PE-Cy7 labeled anti mouse CD8a (Ly-2, clone 53– 6.7); Biotin conjugated anti mouse Gr-1 (clone RB6-8C5), and APC conjugated anti mouse CD117 (c-kit, clone 2B8). All of these antibodies were from BD Pharmingen (San Diego, CA). Tricolor- conjugated anti mouse CD45R (B220) was from Caltag (South San Francisco, CA); and PE-Cy7 conjugated anti mouse Sca-1 (clone D7) was from eBiosciences (San Diego, CA). Antibodies used for purification of hematopoietic stem cells are noted below. Murine hematopoietic stem cell transplantation Donor mouse c-Kit+Thy1.1lowLin2/lowSca-1+ hematopietic stem cells (KTLS cells) were obtained from CD45.1 mice as PLoS ONE | www.plosone.org 11 June 2010 | Volume 5 | Issue 6 | e11316