*Purpose: Recent studies have yielded conflicting results on the capacity of oocyte-encoded mtDNA polymorphisms to stimulate rejection . Therefore, there are still questions as to whether or not genetic differences in oocyte mtDNA can encode minor histocompatibility antigens (MiHAg) that stimulate rejection of tissue transplants from SCNT clones that are otherwise identical to their nuclear donors. The purpose of this research is to investigate clinical and pathologic signs of rejection following transplantation of skin grafts containing allogeneic mitochondria to hosts with identical nuclear genomes.

*Methods: We transplanted 15 C57BI/6J females with two orthotopic tail skin grafts from B6.PWD-mt females that differ only by mtDNA from the PWD strain. Grafts were scored twice weekly until 78 days post-transplant or until the time of rejection that was indicated by the loss of epidermal scale pattern, pigment, and hair on both allografts. Ten recipients that had rejected one or both grafts were re-grafted with second-set B6.PWD-mt grafts. These recipients were euthanized at the time of rejection, and tissue and blood were collected for analyses.

*Results: The majority of recipients rejected either both or one of the allografts within 78d (the median survival time is underlined): 58d, 58d, 58d, 58d, 64d, 64d, 78d, 78d, >78 (one graft rejected 64d), >78d (one graft rejected 78d), >78d (one graft rejected 78d), >78d (one graft rejected 78d), and >78 (one graft rejected 78d). Two recipients did not reject either allograft by 78d. Ten recipients that had either rejected both allografts or one allograft received two second-set B6.PWD-mt skin grafts. Eight/10 recipients were euthanized at 13-16 days post-grafting when they were in the process of rejecting their allografts. PCR amplification using DNA from rejecting B6.PWD-mt grafts and healed-in syngeneic grafts revealed reduced mtDNA copy numbers (based on relative amplification of Cytb and Cox3 sequences) and increased frequencies of mtDNA deletions in rejecting allografts relative to syngeneic grafts harvested at the same post-transplantation time point.

*Conclusions: In comparison to the range of median survival times of other known MiHAgs in mice, the median survival time of 78d & the chronic rejection process suggest that a polymorphism(s) that distinguishes PWD mtDNA from B6 mtDNA defines only a weak MiHAg. The accelerated rejection of second-set B6.PWD-mt grafts indicates the action of adaptive immune responses.

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