Costimulation Blockade, Busulfan, and Bone Marrow Promote Titratable Macrochimerism, Induce Transplantation Tolerance, and Correct Genetic Hemoglobinopathies with Minimal Myelosuppression

Abstract

Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (γ-irradiation, depletion of the peripheral immune system) or administration of “mega” doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-γ-producing T cells re-emerged and skin grafts were rejected at ∼100 days. When applied to a murine β-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.

Establishment of Transplantation Tolerance via Minimal Conditioning in Aged Recipients

“…Bu is typically administered at a dose of 20 mg/kg or higher, with higher levels of chimerism corresponding to dosage increases (6,36,37), as mice are relatively more resistant to Bu than humans. We found that 10 mg/kg Bu was sufficient to deplete LSK progenitors without severely affecting WBM cell numbers.…”

Deletional and regulatory mechanisms coalesce to drive transplantation tolerance through mixed chimerism

“…The contribution of regulation is also time dependent, as it declines over time in protocols leading to progressive peripheral clonal deletion [67]. CD4 + [11,68], CD4 + CD25 + [67], CD4 + CD25 − [60], and CD4 + FoxP3 + [70,71] cell populations have all been found to play a regulatory role in various rodent models of transplantation tolerance [70].…”

“…Regulatory (i.e., nondeletional) mechanisms appear to inhibit donor-specific alloreactivity through a series of incompletely defined, cell contact dependent or cytokine-mediated mechanisms [63,64], taking place in secondary lymphoid organs or in the graft [65,66]. The less drastic the recipient conditioning [11,67,68], the more pronounced becomes the contribution of regulation in addition to deletion. Along these lines, regulation is critical in the NHP setting and in clinical trials (see below) in which permanent mixed chimerism (which is a prerequisite for maintaining continuous central deletion) is not achieved [69].…”

“…Peripheral clonal deletion was identified as a key mechanism tolerizing the unperturbed peripheral T-cell repertoire in such models [3,8,11,56,58]. Mature donor-reactive recipient T cells have been shown to undergo apoptosis soon after BMT under costimulation blockade [8,58].…”

“…The administration of costimulation blockers, such as anti-CD40L mAb and CTLA4Ig, as part of nonmyeloablative conditioning allowed for the first time the successful transplantation of fully allogeneic BM without having to destroy the recipient T-cell repertoire [8]. While some form of (local) irradiation was universally required even when very profound doses of T-cell depleting antibodies (anti-CD4 and anti-CD8 mAb) were given as part of the conditioning [9,10], costimulation blockade allowed the elimination of any irradiation from recipient conditioning if very high doses (mega doses) of allogeneic BM were transplanted [7,11]. Anti-CD40L is sufficiently effective alone (without CTLA4Ig) in strain combinations without minor antigen barriers (e.g., donor B10.A → recipient C57BL/10) [12,13], in particular if CD8 + T cells are depleted [14], but CTLA4Ig is required for an optimal outcome in fully mismatched combinations (MHC plus minor histocompatibility antigen disparities) without T-cell depletion [15].…”