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The Role of Skin-Specific Immunobiology and Major Histocompatibility Complex Sharing in Vascularized Composite Allograft Tolerance
David A. Leonard, MBChB, Kumaran Shanmugarajah, MBBS, Harrison Powell, BS, Alexander Albritton, BS, Christopher Mallard, BS, David H. Sachs, MD, Curtis L. Cetrulo, Jr., MD, Josef M. Kurtz, PhD.
Massachusetts General Hospital, Boston, MA, USA.
Induction of transplant tolerance for vascularized composite allografts (VCAs) would reduce immunosuppression-related morbidity, and avoid skin rejection episodes. Using a mixed chimerism approach in MGH miniature swine, we have developed a reproducible model for inducing tolerance of all components of VCAs across haploidentical MHC barriers. In this study we have utilized this model to study transplantation across a MHC class I mismatch/ class II match, and class II mismatch/class I match and characterized leukocyte turnover in the VCA to identify local factors associated with skin tolerance and rejection.
Using a non-myeloablative protocol (CD3 T-cell depletion, 100 cGy total body irradiation and 45 days of cyclosporine) with hematopoietic stem cell transplantation (HSCT), 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. Biopsies were incubated in Dispase II to separate epidermis from dermis. Dermis was further digested in collagenase D, and epidermis with trypsin to prepare single cell suspensions. For FACS analysis, dermal and epidermal suspensions were analyzed for lineage (CD3, CD4, CD8, MHC Class 2, Langerin) and donor/host hematopoietic origin.
Haploidentical and MHC class II mismatched chimeric animals demonstrated long-term tolerance of VCAs, with no histological evidence of rejection. But MHC class I mismatched chimeras experienced acute rejection episodes of the VCA skin component.
In all animals, host-derived T cells were detectable in the VCA dermis (CD4+ 20-51%, CD8+ 5-48%) at two weeks post-transplant. In the VCA epidermis, recipient-derived Langerhans’ cells were also identified (<15%). Distinct populations of donor derived T cells were also identified in recipient dermis (CD4+ 18-44%, CD8+ 5-28%), but Langerhans’ cells in recipient epidermis remained exclusive of recipient-type.
In haploidentical and MHC class II mismatched recipients, chimerism in VCA, host skin, and peripheral blood equilibrated at later time points (>50 days), with Langerhans’ cell chimerism comparable to that of monocytes in blood. In MHC class I mismatched chimeras however, a steadily increasing proportion of host-derived CD8+ T cells were identified in both VCA dermis and epidermis, correlating with progressive signs of rejection on histology, and in 2/3 animals eventual loss of the VCA skin component , which occurred despite maintenance of peripheral blood chimerism and donor-specific unresponsiveness in vitro.
We have demonstrated that animals tolerant of VCAs across full class II and haploidentical MHC barriers, through a mixed chimerism approach, experience turnover of skin-resident leukocytes within both VCA and recipient skin, to establish cutaneous mixed chimerism without rejection. The progressive infiltration of recipient CD8+ T cells and ultimate rejection of the skin component across a full class I mismatch/class II match, despite in vitro unresponsiveness to donor, demonstrates that additional factors are required to control local alloreactivity in skin. We hypothesize that the presence of a tolerized class II disparity facilitates regulation of skin rejection by linked suppression. Current work aims to address these mechanisms in detail and to confirm the efficacy of mixed chimerism for induction of VCA tolerance across full MHC barriers.
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