Vascularized composite allotransplantation (VCA) would likely be more widely performed if maintenance immunosuppression was not essential for graft acceptance. Accordingly, we used murine models of heterotopic and orthotopic limb transplantation (BALB/c->C57BL/6) to assess the potential for peri-transplant therapy to promote long-term VCA survival.

Injection of CD40L mAb (MR-1) and donor splenocyte transfusion (5×10⁶ cells), plus 30 days of rapamycin (RPM, 2 mg/kg/d, Alzet pumps) induced long-term VCA survival (>100 days, p<0.01). Likewise, CTLA4Ig (500 [micro]g, i.p., on days 0, 2 and 4) plus RPM also induced long-term orthotopic VCA survival (>100 days, p<0.01). The success of either protocol required the presence of a bone-associated, radiation-sensitive component since removal of the long-bone or pre-transplant donor irradiation (800 cGy) prevented long-term allograft survival. Efficacy also required a T or B cell component since allograft rejection occurred when using Rag1-/- donors; and long-term allograft acceptance could not be restored by infusion of donor bone marrow cells peripherally at the time of engraftment (p<0.05). Analysis of donor bone marrow showed ~40% of CD4+ T cells were Foxp3+ Treg cells, constituting the largest population of Tregs within the immune system. Use of a CXCR4 inhibitor (AMD3100) to mobilize donor bone marrow cells pre-transplant abrogated the efficacy of either protocol (p<0.01), as did use of limbs from mice with conditional deletion of CXCR4 within Foxp3+ Tregs. Lastly, donor Foxp3+ T-regulatory (Treg) depletion, by diphtheria toxin administration to DEREG donor mice whose Foxp3+ Treg cells expressed the diphtheria toxin receptor, restored rejection with either protocol, whereas without Treg depletion long-term survival was associated with an active tri-lineage bone marrow.

Long-term VCA survival is possible across a full MHC disparity using costimulation blockade-based approaches. Surprisingly, the efficacy of costimulation blockade in these models depends on the presence of a population of radiation-sensitive, CXCR4+ Foxp3+ Tregs resident within donor bone marrow. The mechanisms by which these cells promote VCA survival post-transplant, including migration of donor Tregs to recipient lymphoid tissues, and the interactions of recipient lymphoid cells with donor bone marrow cells resident within long-bones, are under investigation.

CITATION INFORMATION: Wang L., Wang Z., Han R., Ge G., Levin L., Levine M., Hancock W. Donor Bone Marrow CXCR4+ Foxp3+ Treg Cells Are Essential for Costimulation Blockade-Induced Long-Term Survival of Murine Limb Transplants Am J Transplant. 2017;17 (suppl 3).

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