Background: We have developed a reproducible model for induction of tolerance of all components of VCAs across haploidentical MHC barriers in miniature swine. The purpose of this study was to characterize skin-resident leukocytes in tolerant and rejecting VCAs, and to investigate the role of specific sharing of either MHC class I or class II in the outcome of VCA-skin transplantation.

Methods: Using reduced intensity conditioning and hematopoietic cell transplantation (HCT), mixed chimeras were generated across either haploidentical (n=2), MHC class II (n=3) or MHC class I (n=3) mismatched barriers. VCA and host skin were biopsied at serial timepoints. In addition to histological analysis, leukocytes were independently isolated from epidermis and dermis and analyzed by flow cytometry for lineage (CD3, CD4, CD8, MHC Class 2, Langerin) and donor/host hematopoietic origin.

Results: Haploidentical and MHC class II mismatched chimeric animals demonstrated long-term tolerance of VCAs. MHC class I mismatched chimeras experienced acute rejection episodes of the VCA skin. Non-chimeric controls rejected VCAs in 8 days.

In tolerant animals, host-derived T cells were detectable in the VCA dermis, and host Langerhans' cells in epidermis, by two weeks post-transplant. Donor derived T cells were identified in recipient dermis, but entry of donor Langerhans' cells into recipient epidermis took 100 days. Chimerism in VCA, host skin and peripheral blood gradually equilibrated.

In MHC class I mismatched chimeras, a progressive infiltration with host CD8+ T cells, comparable to, albeit more gradual than, that observed in naïve controls, was identified in VCA dermis and epidermis, correlating with histological signs of rejection, and in 2/3 animals eventual loss of the VCA skin.

Conclusions: We have demonstrated that animals tolerant of VCAs experience turnover of skin-resident leukocytes establishing cutaneous mixed chimerism without rejection. The progressive infiltration of recipient CD8+ T cells and ultimate rejection of VCA skin across a full class I mismatch, despite in vitro unresponsiveness to donor, demonstrates that additional factors are required to control local alloreactivity in skin. Current work aims to address the mechanisms involved in these phenomena in detail and to confirm the efficacy of mixed chimerism for induction of VCA tolerance across full MHC barriers.

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