INTRODUCTION: Hand transplants unlike solid organ grafts consist of composite tissues with differential antigenicity. Uniquely, in addition to their immunogenicity, some tissues such as skin and bone marrow also have pro-tolerogenic characteristics. The vascularized bone marrow (VBM) component of limb allografts provides the stromal microenvironment for sustained renewal of donor derived hematopoietic stem cell (HSC) production.  We hypothesize that composite tissue allografts (CTA) containing viable VBM offer the inherent potential for establishment of stable mixed chimerism (MC) and robust tolerance.  This study seeks to determine the origin of cells contributing to recipient MC after experimental limb CTA. 

METHODS: Wild type BN or gfp-transgenic SD rats served as bone marrow cell (BMC), vascularized skin/muscle (VSM), vascularized skin/muscle/bone (VSMB) and hind-imb allograft donors.  MHC mismatched LEW rats were recipients. Our regimen to induce CTA tolerance consisted of anti-lymphocyte serum (ALS) (day - 4 and +1) and CsA (day 0 - 7) followed by rapamycin (day 8-21).

RESULTS: 1) In a wild type BN to LEW CTA model, the graft survival times were on an ascending scale: VSM (30, 38, 39 days) < VSMB (69, 74, 90, 104 days) < hind-limb (38, 39, >150, >150, >150, >150 days). 2) Persistent gfp+ multilineage MC were observed in the peripheral blood of gfp+ VSMB and gfp+ hind-limb allograft recipients (2.68-6.66%) throughout follow up until 140 days. 3) In contrast, only 0.5% gfp+ cells were detected at two-weeks post-transplant and disappeared thereafter in recipients receiving gfp+ VSM grafts. 4) The bone marrow from the excised bone graft 140 days post-transplantation contained 39.5% gfp+ donor derived cells. 5) Donor gfp+ cells consisting of >90% CD4+CD8+ cells, were evident in the thymus of VSMB recipients >140-days post-transplantation. 6) Donor gfp+ cells homed into recipient bone marrow at remote sites as well as in contralateral limb.

CONCLUSION: 1) Bone marrow component of hind limb allograft promotes MC and tolerance. 2) Donor HSC home to recipient bone marrow, migrate to thymus and undergo T cell differentiation; 3) Vascular bone component of CTA provides stromal microenvironment for sustained donor derived HSC production, renewal and recipient reconstitution, contributing to MC and tolerance.