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The Effect of CXCR4 Inhibition on Hindlimb Allograft Survival
Joani M. Christensen, BA1, Lehao W. Wu, MD1, Georg Furtmuller, MD1, Saami Khalifian, BS1, Michael Kimelman, MD2, Angelo Leto Barone, MD1, Nance Yuan, MD1, Justin M. Sacks, MD1, W P Andrew Lee, MD1, Gerald Brandacher, MD1, Damon S. Cooney, MD, PhD1.
1Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2Innsbruck Medical University, Innsbruck, Austria.
Wider application of vascularized composite allotransplantation (VCA) is hindered by need for chronic immunosuppression post-transplant Evidence suggests that autologous, bone marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) may promote induction of central and peripheral immunologic tolerance. Recipients of VCAs possess two sources of HSPCs, donor and recipient bone marrow, but the role of these cells in the immune response to a VCA is yet to be explored. Antagonism of the CXCR4 axis has been shown to mobilize bone marrow HSPCs to the periphery. Our objective was to investigate the effect of CXCR4 receptor antagonists AMD3100 and plerixafor on the ability to mobilize stem cells, promote peripheral chimerism, and prolong graft survival in a rodent hindlimb transplantation model.
Lewis rats received orthotopic hindlimb transplants from Brown Norway (BN) donors. Treatments consisted of tacrolimus (0.5mg/kg) for 21 days, AMD3100 (5mg/kg) POD 0,1,2,3,7; or plerixafor (1mg/kg) POD 0,1,2,3,7. Groups included 1) tacrolimus; 2) tacrolimus + AMD3100; 3) tacrolimus + plerixafor; 4) AMD3100; 5) plerixafor. Mobilization of peripheral blood mononuclear cells was assessed by performing a colony forming unit (CFU) assay on day 7 and day 21 using peripheral blood mononuclear cells of rats (n=3) receiving the regimens above. Peripheral hematopoietic chimerism was detected using flow cytometry staining PBMCs with antibodies for RT1Ac, MHC class 1 marker for BN, on POD 7, 21, 28, and at the time of rejection. Clinical rejection was assessed according to Banff criteria.
CFU assays from day 7 peripheral blood revealed an average of 54.3 CFU in the AMD3100 group, 48.0 in the tacrolimus + AMD3100, 43.0 in the plerixafor group, and 49.8 in the tacrolimus + plerixafor group, all significantly elevated compared to control, 22.7 (all p<0.05). MHC-I chimerism was detectable in all experimental animals. Rats receiving combination treatment with tacrolimus + AMD3100 had average peripheral lymphoid chimerism of 4.1%, higher than that in treatment with tacrolimus alone, 0.84% (p=0. 23) on POD 7. No significant difference in chimerism levels was found at any other time point. Average rejection-free graft survival with conventional tacrolimus was 35 days, while it was 37.0 for those receiving tacrolimus + AMD3100, and 36.0 for those receiving tacrolimus + plerixafor. Average rejection free graft survival after AMD3100 treatment alone was 11.0 days, and 12.5 after plerixafor alone.
The results suggest that treatment with AMD3100 or plerixafor, alone or in combination with conventional tacrolimus therapy elevates the number of circulating HSPCs as expected. Additionally this treatment led to increases in circulating donor-derived cells in animals following hindlimb transplant. Despite the ability to increase circulating stem cells and peripheral chimerism these treatments did not significantly prolong graft survival. Further studies will explore this disconnect between chimerism and graft survival as well as explore whether variations in the dosing may be more successful in prolonging graft survival.
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