Strategic exposure to donor antigens prior to transplantation can be an effective way for inducting donor-specific tolerance in allogeneic recipients. We have recently shown that pre-transplant infusion of donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces indefinite islet allograft survival in a full MHC-mismatched model without the need for any immunosuppression. Mechanisms of allograft protection by this strategy remain elusive. In this study, we show that the infused donor ECDI-SPs differentially target T cells with indirect versus direct allo-specificities. To target indirect allo-specific T cells, ECDI-SPs induce up-regulation of negative, but not positive, co-stimulatory molecules on recipient splenic CD11c+ DCs phagocytosing the injected ECDI-SPs. Indirect allo-specific T cells activated by such CD11c+ DCs undergo robust initial proliferation followed by rapid clonal depletion. The remaining T cells are sequestered in the spleen without homing to the graft site or the graft draining lymph node. In contrast, direct allo-specific T cells interacting with intact donor ECDI-SPs not yet phagocytosed undergo limited proliferation and are subsequently anergized. Furthermore, CD4+CD25+Foxp3+ T cells are induced in lymphoid organs and at the graft site by ECDI-SPs. We conclude that donor ECDI-SPs infusions target host allogeneic responses via a multitude of mechanisms including clonal depletion, anergy and immunoregulation, which act in a synergistic fashion to induce robust transplant tolerance. This simple form of negative vaccination has significant potential for clinical translation in human transplantation.

CD4+ regulatory T cells play a critical role in tolerance induction in transplantation. CD8+ suppressor T cells have also been shown to control alloimmune responses in pre-clinical and clinical models. However, the exact nature of the CD8+ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen-specific CD8+Foxp3+ T cells can be induced and significantly expanded by stimulating naive CD8+ T cells with donor dendritic cells in the presence of IL-2, TGF-β1, and retinoic acid. These CD8+Foxp3+ T cells express enhanced levels of CTLA-4, CCR4 and CD103, inhibit the up-regulation of co-stimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor-specific and contact-depended manner. Importantly, upon adoptive transfer, the induced CD8+Foxp3+ T cells protect full MHC-mismatched skin allografts. In vivo, the CD8+Foxp3+ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4+Foxp3+ T cells and concurrently suppress effector T cell expansion. We conclude that donor-specific CD8+Foxp3+ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.

We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid derived suppressor cells (MDSCs) in the graft protection provided by ECDI-SPs is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b+ cells in the spleen that phenotypically resemble monocytic-like (CD11b+Ly6CHI) and granulocytic-like (CD11b+Gr1HI) MDSCs. Both populations suppress T cell proliferation in vitro, and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b+Gr1HI cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ dependent mechanisms. This form of preemptive donor apoptotic cell infusions has significant potential for the therapeutic manipulation of MDSCs for transplant tolerance induction.

No abstract

Recent studies have shown significantly increased expression of matrix metalloproteinases (MMP) and disintegrin-type metalloproteinases (ADAM) during allograft rejection. In this regard, our previous studies have demonstrated contrasting roles for MMP-2 and MMP-9 during allograft rejection: MMP-2-deficiency enhanced allograft survival while MMP-9-deficiency decreased allograft survival. The aim of this study was to determine the effect of broad-spectrum MMP/ADAM inhibition on the pathogenesis of allograft rejection. Toward this, heterotopic BALB/c cardiac allografts were transplanted into C57BL/6 recipients treated with MMP/ADAM inhibitors, GM6001 or doxycycline. Systemic MMP/ADAM inhibition significantly enhanced allograft survival. Functioning allografts recovered from MMP/ADAM inhibitor-treated recipients showed lower cellular infiltration and tissue remodeling than rejected allografts recovered from control recipients. In addition, decreased chemotaxis of CD4+ and CD8+ T cells, B cells and macrophages was observed in vitro in the presence of MMP/ADAM inhibitors. Enhanced T-cell alloreactivity was also observed ex vivo in MMP/ADAM inhibitor-treated recipients and in vitro in the presence of MMP/ADAM inhibitors. These observations were associated with enhanced cytokine, chemokine and growth factor production. These results indicate that MMPs and ADAMs play a critical role in the pathogenesis of allograft rejection and may represent novel therapeutic targets for the treatment and/or prevention of this disease.