Background
The development of VCA tolerance protocols would obviate the need for chronic immunosuppression and allow widespread use of this highly promising reconstructive modality. However, achieving robust tolerance of the skin component of VCAs has proven particularly challenging in pre-clinical large animal models. Our laboratory has recently reported acceptance of VCAs (>600 d), including the skin component, across a haploidentical MHC barrier in a miniature swine model using a mixed chimerism approach. In this study, we investigated whether sharing of MHC class I and/or class II between donor and recipient influences skin tolerance.
Methods
Miniature swine recipients (n = 5) underwent a non-myeloablative protocol and haematopoietic stem cell transplantation (HSCT), generating mixed chimeras across either a MHC class I (n = 2) or MHC class II mismatch (n = 3). Fasciocutaneous VCAs were transplanted at the time of HSCT. Flow cytometry was used to assess chimerism in the peripheral blood and tissues. Immune responsiveness was assessed using mixed lymphocyte reaction and cell-mediated lymphocytotoxicity assays. Serial biopsies of VCA skin were performed and digested into epidermis and dermis, to allow assessment of cellular infiltrate and rejection under flow cytometry.
Results
All animals displayed stable chimerism in lymphoid (50–70%), myeloid (40–80%) and granulocyte (40–80%) lineages. All MHC class II-mismatched chimeras remained tolerant of VCAs while off immunosuppression (>400 d), without clinical or histological features of rejection. In contrast, MHC class I-mismatched animals experienced rejection of the epidermal component of the VCAs following tapering of immunosuppression, characterized by recipient type CD8+ T lymphocytes infiltrating into the epidermis. Chimerism levels remained stable despite skin rejection. All animals demonstrated systemic donor-specific non-responsiveness in vitro, including after rejection episodes.
Conclusions
Our data demonstrate that sharing of MHC class I antigens between donors and recipients has a beneficial effect on VCA skin tolerance using mixed chimerism. These data suggest that local regulation of immune tolerance is critical in long-term acceptance of all VCA components. Finally, these results may help elucidate undetermined mechanisms underlying VCA skin tolerance and identify clinically relevant strategies to induce tolerance of VCAs.