Cold preservation of nerve allografts has evolved into a clinically applicable practice, decreasing nerve antigenicity and reducing required doses of systemic immunosuppression with only 7 days of allograft pretreatment. We know that after only 2 weeks of cold preservation, SCs decrease their expression of MHC Class II molecules,2
and it has been demonstrated in numerous studies that SCs behave as facultative APCs, representing the primary target of host allograft rejection.9,10,13,15,18,24,26
Although prolonged cold preservation has proven useful in a short gap model, the viable SCs that are required for regeneration across longer distances are ultimately lost with progressive cold preservation.5,6,14,23
Despite the inherent limitations of cold preservation, it remains a useful adjunct in nerve allotransplantation and when used with immunosuppression allows for the use of cadaveric nerves as a source of graft material.
In this study we investigated the effect of cold nerve allograft preservation on the unmodified rejection response and antigen presentation. The importance of direct recognition in acute allograft rejection is well established, but a significant role for indirect recognition is now understood.3,26
However, the relative contribution of each pathway and the role of T-cell subsets in mediating rejection remains unclear. These factors may vary with the cellular makeup of the allograft and may contribute to differential tissue resistance to immunomodulation. The parenchymal component of a peripheral nerve allograft is much smaller than an organ allograft and the immune response is directed at cellular components including SCs, endothelial cells, and perivascular macrophages. The use of transgenic mice lacking 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 cold allograft preservation.
In the unmodified rejection response groups without cold preservation, the strength of the rejection response when the direct pathway was not functional was just as strong as when both pathways were intact. However, when the indirect pathway was eliminated, total axonal regeneration was much improved and very close to reaching statistical significance. The indirect pathway would therefore appear to play a greater role in the unmodified rejection response to the nerve allograft than expected. Perhaps the nonvascularized nature and a relative lack of APCs on the nerve allograft could account for a weaker direct component. Professional APCs include dendritic cells, macrophages and other mononuclear phagocytes, and B-lymphocytes, whereas nonprofessional APCs include epithelial and mesenchymal cells. The physiological significance of nonprofessional APCs in antigen presentation remains unclear, and it is unlikely that they play a major role in the initiation of most T-cell responses.1
The nerve allograft is much more homogeneous in tissue type and quantitatively smaller than a vascularized organ allograft, which also contains sizable blood vessels for vascular anastomoses. In humans, microvascular endothelial cells may also present antigens and may be particularly important during allograft rejection. Nonvascularized nerve grafts do contain microvessels in the epi- and perineurium, but they are extremely small and not directly exposed to host blood initially. As such, they are more like a nonvascularized skin allograft, except that donor skin also contains Langerhans cells, which like dendritic cells are professional APCs. Because SCs are only facultative APCs, the relatively smaller quantity of donor professional APCs may provide an explanation for a strong indirect component to the host response in the nerve allograft model.
As we have previously shown, longer periods of cold nerve allograft preservation resulted in significant attenuation of the host immune response and improvement in nerve regeneration.6
Based on our current knowledge of the effects of cold preservation, we expected to see a greater degree of nerve regeneration when the indirect pathway was not functional, leaving only an intact direct pathway that would be diminished by the decrease in donor Class II MHC expression during cold preservation. If direct antigen recognition were eliminated leaving only a functional indirect pathway, progressive periods of cold preservation would be expected to have little effect and regeneration should remain poor. However our findings demonstrated the opposite trend. When the indirect pathway was eliminated and therefore only direct recognition was intact, treatment with 1 week of cold allograft preservation in these recipients showed no improvement in regeneration compared with no allograft pretreatment. Although 4 weeks of cold preservation did show some improvement, it did not quite reach statistical significance. On the other hand, when the direct pathway was not functional and therefore only indirect recognition was intact, 4 weeks of cold preservation improved regeneration significantly, even though the improvement after 1 week of cold preservation was modest and not statistically significant. We have previously shown that 1 week of cold allograft preservation decreases donor Class II MHC expression,2
and that SC viability is maintained for up to 3 weeks of cold preservation.4,5
However, up to 7 weeks of preservation is required before all donor allograft antigenicity is diminished,6
indicating the degeneration and loss of donor peptides capable of eliciting a host immune response. It would follow, then, that after 4 weeks of cold preservation in this model, most of the donor cells are no longer viable and there has also been significant loss of antigenic donor peptides. Therefore, indirect recognition by host APCs of donor antigens is decreased by a diminishing pool of allograft peptides. Direct recognition by donor APCs is also decreased by the loss of donor cell viability, but likely to a lesser degree because of the smaller contribution by the direct pathway in the unmodified nerve allograft rejection response as discussed above.