Inhibition of T-cell activation by costimulatory blockade has demonstrated significant potential in both solid organ transplantion[11
] and nerve allotransplantation[18
] models. While solid organ transplants requires a lifetime of aggressive immunosuppression, nerve allotransplantation only requires temporary systemic immunosuppression. Under sufficient immunosuppression, donor SCs assist migrating host SCs as axonal regeneration occurs across the nerve allograft[21
]. Once adequate host SC migration has occurred and the target end-organ has been reinnervated, immunosuppression can be withdrawn[21
]. Blockade of these costimulatory signals has proven effective at decreasing the required dose of other more traditional immunosuppressive agents, achieving temporary allotransplantation tolerance and preventing T-cell clonal expansion and maturation[25
Anti-CD40L mAb (MR-1) has been studied extensively, and prolonged graft survival and tolerance induction in the murine cardiac model have been demonstrated [31
]. CTLA4-Ig causes arrest of T cells in the G1 phase of the cell cycle by inhibiting T cell activation through a reduction in IL-2 production and IL-2 receptor expression[33
], and it has been shown to produce long term cardiac allograft survival in mouse and rat models[35
]. ICOS is a structurally analogous costimulatory molecule that is not constitutively expressed on T cells but is induced following T cell activation[37
]. ICOS blockade has been shown to prolong liver allograft survival in a murine model[40
]. Recent work in our lab has demonstrated that combination therapy with multiple costimulatory blocking agents (MR-1, CTLA-4-Ig, and Anti-ICOSL), produces neural regeneration not significantly different from isograft controls[41
In this study we investigated the role of the direct and indirect pathways in the immune hyporesponsiveness produced by costimulation-blocking antibodies. We found that the redundancy of the immune system facilitates nerve rejection in the absence of a normal direct or indirect pathway. The significance of the direct pathway has long been understood, and there is now better appreciation for an important role for indirect recognition[12
]. Both pathways are known to play a role in allograft rejection[42
], but their relative roles in differing settings are incompletely understood. Immunological factors such as antigen presentation may vary with the cellular composition of the allograft and may contribute to differential tissue resistance to immunomodulation. The parenchymal component constitutes a much smaller proportion of the entire peripheral nerve allograft than an organ or composite tissue allograft. The immune response is directed at the cellular components, which in the nerve allograft include SCs, endothelial cells, and perivascular macrophages. In the untreated control groups, the strength of the rejection response based only on the indirect pathway was just as strong as when both pathways were intact. However when the indirect pathway was eliminated leaving only the direct pathway intact, total axonal regeneration was improved and was very close to reaching statistical significance. Indirect antigen presentation therefore seems to play a greater role in the nerve allograft rejection than expected. The non-vascularized nature and relative lack of professional APC’s on the nerve allograft may explain a weaker direct component. Professsional APC’s include dendritic cells, macrophages and other mononuclear phagocytes and B lymphocytes, while non-professional APC’s include epithelial and mesenchymal cells. The role of non-professional APC’s in antigen presentation remains unclear, and it is unlikely that they play a major part in most T cell responses[24
]. The nerve allograft is more homogenous in tissue composition and is quantitatively smaller than an organ allograft which also contains sizeable blood vessels for surgical revascularization. Microvascular endothelial cells may also present antigen and may be significant during allograft rejection. Non-vascularized nerve grafts contain microvessels in the epi- and perineurium, but they are miniscule and not directly exposed to host blood initially. As such, the nerve allograft is more analogous to a non-vascularized skin allograft except that donor skin also contains Langerhan cells which, as dendritic cells, are professional APC’s. Because SC’s are only facultative APC’s, the relative lack of donor professional APC’s may account for a strong indirect component of the host response to the nerve allograft.
The use of transgenic mice that lack class II MHC molecules allowed us to eliminate either the direct or indirect pathways of antigen recognition to evaluate their relative roles with and without treatment with costimulation blockade[11
]. In this model, triple costimulatory blockade provides a permissive environment for neural regeneration in the nerve allograft. This effect was not achieved when only direct allorecognition was functional and was further enhanced when only the indirect pathway was available. As demonstrated in the solid organ literature, the presence of the indirect pathway appears necessary for costimulatory blockade induced immune hyporesponsiveness[11
]. Similarly, we found that the capacity to mount an indirect response is necessary to achieve costimulatory blockade-mediated neural regeneration. The direct pathway of allorecognition appears to be more resistant to costimulation blockade because it is less dependent on CD40 and CD28 costimulation.
One potential limitation of this study involves the relative number of circulating CD4+ T cells in MHC deficient mice. MHC
−/− mice are known to have lower numbers of CD4+ cells with an increased number of CD8+ T cells[45
], which may impair the immune response or impart a selective resistance to costimulatory blockade. The threshold level of required CD4+ T cells to generate an adequate allograft rejection response is not precisely known. In order to account for these findings we performed an additional control experiment utilizing the adoptive transfer of wild-type CD4+ T cells into MHC
−/− recipients receiving costimulatory blockade (Group 9). Our results corroborate similar experiments done with cardiac transplantation[11
], and we found that costimulatory blockade-induced immune hyporesponsiveness in the absence of an indirect pathway is not due to an abnormal level of CD4+ T cells. MHC deficient mice that received wild-type CD4+ T cells with costimulatory blockade still failed to have any meaningful neural regeneration. Our results suggest, therefore, that the indirect pathway is necessary for costimulatory blockade induced immune hyporesponsiveness.