Long-Term Survival of Intestinal Allografts
Tolerance-inducing strategies that infuse donor bone marrow cells in conjunction with costimulation blockade have not been applied to intestinal transplantation. Intestines from BALB/c mice were transplanted into C57BL/6 recipients treated with anti-CD40L mAb, CTLA4-Ig, donor bone marrow, and busulfan. The majority of mice transplanted after completion of this regimen developed hematopoietic macrochimerism, although the degree of chimerism varied widely between recipients, and experienced long-term allograft survival. T cells from these mice demonstrated donor-specific hyporesponsiveness in vitro. However, T cells from chimeric mice proliferated to donor alloantigen in vivo. Furthermore, chimeric mice bearing intestinal allografts were capable of rejecting subsequently placed donor-strain skin grafts. These data suggest that although long-term allograft survival occurs in the absence of acute or chronic rejection, recipient mice are not completely unresponsive to donor alloantigens. When intestinal transplantation was performed at the time of initial bone marrow infusion (initiation of the chimerism protocol), most recipients failed to develop chimerism and promptly rejected the intestinal allograft. Although this is the most effective protocol that we have tested using this stringent model of transplantation, our observations suggest that modifications will be necessary before it can be reliably applied to the transplantation of highly immunogeneic organs like the intestine.

Intestinal transplantation is now recognized as definitive therapy for selected patients with intestinal failure. Despite the increased number of intestinal transplants performed, the outcome of intestinal transplantation remains inferior to that associated with the transplantation of other organs. This is mainly because of the greater immunogenicity of intestinal allografts and the associated increased frequency and severity of rejection. In addition to the problem of rejection itself, the relatively greater amount of immunosuppression required to prevent or control rejection following intestinal transplantation results in significantly increased rates of infection and post transplant lymphoproliferative disease (PTLD) relative to other transplanted organs.

The broader application of intestinal transplantation awaits the development of more effective and less toxic immunosuppressive regimens. In this regard, immunosuppressive strategies that promote donor-specific tolerance may offer particular benefits to patients undergoing intestinal transplantation. We have previously shown that agents including anti-CD4 mAb, anti-CD40L mAb, anti-LFA1, anti-B7.1 and anti-B7.2 mAb, and CTLA4Ig that promote tolerance and/or long-term survival of allografts in other transplant models fail to produce the same effect in the murine model of intestinal transplantation (K. A. Newell, unpublished observations). Of the currently available approaches to tolerance induction, combined donor bone marrow and organ transplantation affords the advantage of inducing a robust tolerance to allografts in a number of experimental models. It was first recognized that hematopoietic chimerism was associated with donor-specific tolerance five decades ago. In 1955 Main and Prehn demonstrated that donor-specific tolerance could be acquired by infusing bone marrow cells into lethally irradiated adults[12]]. Concerns about infectious complications in fully allogeneic chimeras prompted investigators to design strategies for inducing mixed allogeneic chimerism. Subsequently protocols were designed that replaced the need for lethal irradiation with conditioning regimens that utilized anti-T-cell antibodies, low-dose TBI, and thymic irradiation or costimulatory blockade combined with infusion of very large doses of donor bone marrow (200 × 10 BM cells/mouse). However, persisting concerns about the long-term consequences of irradiating transplant recipients and the difficulty of obtaining the number of bone marrow cells required for the 'mega-dose' protocols may limit the clinical application of these approaches. Recently we have reported a regimen that completely replaces radiation as a conditioning agent with the alkylating agent busulfan. Busulfan preferentially depletes early hematopoietic stem cells without significantly affecting the number of mature circulating leukocytes in murine transplant models. Mice treated with busulfan, anti-CD40L mAb, CTLA4-Ig, and infused with two doses of donor bone marrow developed donor-specific tolerance as indicated by the indefinite survival of primary skin and heart allografts and acceptance of subsequently placed donor, but not third party skin grafts.

The aim of the current study was to determine the effect of this tolerance-inducing strategy on the outcome of intestinal transplants in mice. The results of our studies demonstrate that the infusion of donor bone marrow together with busulfan and costimulation blockade induces hematopoietic chimerism and promotes the long-term survival of intestinal allografts transplanted into mice that have completed the treatment regimen. This long-term survival is associated with donor-specific hyporesponsiveness in vitro and deletion of donor-reactive T cells in vivo. Interestingly, mice bearing long-term surviving intestinal allografts displayed significant prolongation of subsequently placed donor-strain skin grafts but were not tolerant by the strictest definition in that most donor-strain skin grafts were eventually rejected. Finally, unlike results obtained using skin and heart transplant models, most intestinal allografts placed on the initial day that the tolerizing regimen was begun were promptly rejected. These results provide a cautionary note and demonstrate that in its current form this approach to tolerance induction may not be clinically applicable for highly immunogenic organ allografts.