We recently developed a murine protocol for the induction of allogeneic mixed chimerism and tolerance employing nonmyeloablative total body irradiation (TBI), standard-dose bone marrow transplantation (BMT), and costimulation blockade (cobl) with an anti-CD154 monoclonal antibody (mAb) plus CTLA4Ig. We now evaluated whether a short course (1 month) of immunosuppressive drugs, which would be ethically required in the clinical setting of organ transplantation to prevent graft loss in case tolerance is not achieved, interferes with tolerance induced with this regimen. Our results show that calcineurin inhibitors (cyclosporin A [CyA] or tacrolimus [FK]) inhibit development of long-term chimerism and abrogate tolerance induction in this model. Rapamycin (rapa), methylprednisolone (MP), FTY720, and mycophenolate mofetil (MMF), in contrast, have no negative effect on chimerism or tolerance development. Peripheral deletion of donor-reactive T cells, which usually occurs in the weeks following BMT in this model, is blocked by CyA and FK, but not by the other drugs tested. Furthermore, we found that the additional use of compatible immunosuppressive drugs (rapa plus MMF plus MP) allows the dose of TBI to be reduced, so that mixed chimerism and donor skin-graft acceptance can be achieved with 1 Gy using clinically feasible cell numbers. Thus, this protocol of BMT with costimulation blockade can be safely combined with a clinically tested immunosuppressive regimen to permit success with a lower dose of irradiation. These results should facilitate clinical application of this tolerance strategy.

Peripheral and central clonal deletion are important tolerance mechanisms in models using bone marrow transplantation (BMT) with costimulation blockade (CB). However, since tolerance can be found before peripheral deletion is complete and since elimination of recipient CD4⁺ cells at the time of BMT prevents tolerance induction, we investigated the potential roles of regulation and anergy in such a murine model. We found that transient elimination of CD25⁺ cells or neutralization of IL2 immediately after BMT and CB prevented the induction of skin graft tolerance. Cotransfer into SCID mice of CD4⁺ cells taken from chimeras early after BMT, together with naïve recipient-type CD4⁺ cells significantly prolonged donor skin graft survival. In contrast, cotransfer of CD4⁺ cells harvested from chimeras late after BMT did not prolong donor skin graft survival. Besides, depletion of CD25⁺ cells in established chimeras several months post-BMT did not break tolerance. In vivo administration of recombinant IL2 inhibited chimerism and tolerance neither early nor late post-BMT, arguing against a decisive role for classical anergy. Thus, CD4 cell-mediated regulation contributes significantly to tolerance induction early after BMT, but appears to have no critical role in the maintenance of tolerance.

Induction of mixed chimerism and tolerance usually requires cytoreduction or transplantation of high numbers of bone marrow cells (BMC). However, such protocols have only a suboptimal success rate and, more importantly, equivalent numbers of BMC cannot be routinely obtained in the clinical setting. The authors therefore evaluated whether a short-course of immunosuppression (IS) given in addition to co-stimulation blockade would facilitate chimerism induction and allow reduction of the minimally required number of BMC without cytoreduction. B6 mice received 200, 100, or 50×106 unseparated BMC from Balb/c donors plus an anti-CD40L monoclonal antibody (mAb) and CTLA4Ig (without irradiation or cytotoxic drugs). Some groups were treated additionally with IS (rapamycin, methylprednisolone, and mycophenolate mofetil for 4 weeks after bone marrow transplantation), donor-specific transfusion (DST), or anti-OX40L mAb, as indicated. IS led to long-term multilineage chimerism in 9 of 10 mice receiving 200×106 BMC (without IS, 1 of 4; P<0.05), in all mice (n=10) receiving 100×106 (without IS, 6 of 9; P<0.05), and notably in 9 of 10 mice treated with 50×106 BMC (without IS, 4 of 10; P<0.05). With transient IS, donor skin grafts were accepted longer than 170 days in 9 of 10 mice receiving 200×106 (without IS, 0 of 5 mice; P<0.05), all mice receiving 100×106 (without IS, 6 of 9; P<0.05), and 6 of 11 mice receiving 50×106 BMC (without IS, 4 of 10). The use of DST or anti-OX40L mAb had no beneficial effect. Transient IS significantly improves rates of chimerism and donor skin graft survival, and allows lasting mixed chimerism after transplantation of only 50×106 BMC. Thus, IS might help in the further development of noncytoreductive chimerism protocols.

The transplantation of donor hematopoietic stem cells has been used successfully in numerous experimental settings to induce donor-specific tolerance. After appropriate host conditioning, hematopoietic stem-cell transplantation leads to a lasting state of donor macrochimerism that is associated with a robust form of tolerance. One of the key factors in the success of this approach is its reliance on intrathymic clonal deletion to ensure lifelong tolerization of newly developing T cells. Evidence for ongoing central deletion comes from studies following superantigen-reactive T cells and from experiments using mice transgenic for an alloreactive T-cell receptor. In protocols inducing tolerance through macrochimerism, the preexisting mature T-cell repertoire is controlled by either globally destroying all T cells before the hematopoietic cell transplantation or, in more recent models, by tolerizing it through co-stimulation blockade. The peripheral mechanisms induced by hematopoietic stem-cell transplantation and co-stimulation blockade include both extrathymic clonal deletion and the nondeletional mechanisms anergy, suppression, or both. In addition to these immunologic hurdles, a physiologic engraftment barrier has to be surmounted for the successful induction of mixed chimerism. This can be achieved by cytoreductive host treatment or by the infusion of high numbers of donor hematopoietic cells. A detailed delineation of the mechanisms responsible for tolerance induction after hematopoietic stem-cell transplantation is expected to help in the translation of these experimental protocols to clinical organ transplantation.

Bone marrow transplantation (BMT) under costimulation blockade induces mixed chimerism and tolerance in rodent models. Recent data, predominantly from in vitro studies, suggest that in addition to blocking the CD28 costimulation pathway CTLA4Ig also acts through upregulating the tryptophan-catabolizing enzyme indoleamine-2,3-dioxygenase (IDO). Here we demonstrate that even though CTLA4Ig is critically required for the induction of chimerism and tolerance in a murine model of nonmyeloablative BMT, IDO activity is not. No significant differences were detectable in the kynurenine to tryptophan ratios (indicative of IDO activity) in sera of BMT recipients treated with CTLA4Ig (tolerant group) versus BMT recipients treated without CTLA4Ig (nontolerant group) versus naïve controls. In vivo inhibition of IDO immediately after BMT with CTLA4Ig or several months thereafter did not block achievement of chimerism and tolerance. Thus, IDO does not play a critical role in the induction or maintenance of chimerism and tolerance in a CTLA4Ig-based BMT model.

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