Bone Marrow Component of Hind Limb Allograft Promotes Marrow-Derived Hematopoietic Stem Cell Engraftment, Chimerism and Tolerance

INTRODUCTION: Although, hand allograft contains large amount of most antigenic skin component, current immunosuppressant regimens for composite tissue allograft (CTA), which does not fundamentally different from that used for other organ transplantation, have allowed more than 95% 1-year survival in the current compliant hand transplant patients, suggesting unique intrinsic mechanisms make CTA, as a complex unit, more pro-tolerogenic than its most antigenic component: skin. One unique feature that distinguishes many CTA from other organ transplants is the presence of the hematopoietic microenvironment as a component. Bone marrow has long been appreciated to possess immunomodulatory properties. Experimental studies showed that a long-lasting chimerism in rat hind limb recipients treated with conventional immunosuppressant. We hypothesize that the vascular bone marrow in CTA provides stromal environment for sustained donor derived hematopoietic stem cell (HSC) production, homing and renewal, provide an opportunity to achieve chimerism and CTA tolerance using clinically more acceptable preparative regiments. METHODS: We utilized wild type BN or gfp-transgenic SD rats as bone marrow cells (BMC), vascularized skin/muscle (VSM), vascularized skin/muscle/bone (VSMB) and hind limb allograft donors and MHC mismatched LEW rats as recipients to determine the role of donor bone component in establishing mixed chimerism and inducing tolerance in CTA recipients. To induce CTA tolerance we developed a novel tolerizing strategy by tailoring the dosing and timing of immunosuppressive drugs to induce CTA tolerance, which consists of anti-lymphocyte serum (ALS) (day -4 and +1) and CsA (day 0-7) followed by RMP (day 8-21). RESULTS: 1) Persistent gfp+ multilineage donor cells were observed in the peripheral blood of gfp+ VSMB and gfp+ hind limb allograft recipients (2.68-6.66%) through out follow up 100 days. 2) The simultaneous gfp+ BMC and gfp+ hind limb transplantation significantly increase gfp+ multilineage donor cells in the peripheral blood (9.7-15%). 3) Interestingly, in the recipients receiving gfp+ donor BMCI gfp+ cells were homing to both recipient's and donor's bone marrow (3-6%) respectively. 4) In contrast, only 0.5% gfp+ donor cells were detected at two weeks post-transplant and disappeared thereafter in recipients receiving gfp+ VSM grafts. 5) In a BN to LEW CTA model using a novel tolerizing regiment the graft survival time were on an ascending order: VSM (30, 38, 39 days) < VSM+BMCI (42, 61, 62, 87 days) < VSMB (69, 74, 90, 104 days) < hind-limb (38, 39, >150, >150, > 150, >150 days). CONCLUSION: 1) vascular bone marrow of CTA contributes to the establishment of mixed chimerism in CTA recipients; 2) bone marrow infusion contributes to the establishment of mixed chimerism in CTA recipients; 3) Infused bone marrow cells home to bone marrow of both recipient and donor graft; 4) simultaneous BMC and hind limb transplantation promote HSC engraftment and chimerism; 5) vascular bone component and BMCI promote chimerism and CTA tolerance.