Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Detailed Characterization and Functional Analysis of Skin-Resident Leukocytes from Vascularized Composite Allografts in Tolerant Miniature Swine Mixed Chimeras
Harrison Powell, BS, David A. Leonard, MBChB, MRCS, Alexander Albritton, BS, Christopher Mallard, BS, Curtis L. Cetrulo, Jr., MD, FACS, Josef M. Kurtz, PhD.
Massachusetts General Hospital, Boston, MA, USA.

PURPOSE:
We have previously reported induction of vascularized composite allograft (VCA) tolerance across major histocompatibility (MHC) barriers by hematopoietic stem cell transplantation (HSCT) in MGH Miniature Swine, with stable mixed chimerism permitting indefinite immunosuppression-free survival of all components of the VCAs. While previous studies have primarily focused on peripheral blood leukocytes for analysis, the characterization of both lineage and function of the resident skin leukocyte populations remain to be investigated.
METHODS:
Two animals underwent haplomatched HSCT and VCA. VCA and host skin were biopsied for FACS and mixed lymphocyte reaction (MLR) analysis. Biopsies were first incubated in Dispase II to separate epidermis from dermis, following which the tissue was further digested to produce single cell suspensions. Dermis was digested in collagenase D, while epidermis was digested with trypsin. For FACS analysis, dermal and epidermal cell suspensions were analyzed for lineage markers (CD3, CD4, CD8, g/d T cells, MHC Class 2, Langerin) and donor/host hematopoietic origin. For MLR, Langerhans cells were FACS sorted and plated in culture as stimulators to peripheral blood leukocyte responders.
RESULTS:
Two weeks post-VCA and HSCT, host skin dermis contained distinct populations of donor derived T cells (CD4+ 18-30%, CD8+ 5-10%, and g/d 8-10%) cells, but no donor-derived Langerhans cells were detected within the epidermis. In contrast, the VCA contained a significant population of host-derived leukocytes; host derived T cells (CD4+ 20-30%, CD8+ 5-6%, and g/d 20-60%) cells were found within VCA dermal tissue. In the VCA epidermis, the majority of Langerhans cells remained of donor origin, but a small population (5-15%) of host derived Langerhans cells was detected. At later time points (150 days post-VCA/HSCT), skin chimerism levels reflected peripheral blood chimerism. The majority of leukocytes from the dermis were donor-derived in both host and VCA skin. While the majority of T cells in peripheral blood were donor-derived, a small but significant number of host T cells were found in the host and VCA dermis. Interestingly, at >150 days, 30-40% of the Langerhans cells in host skin epidermis remained host derived. This suggests that while donor derived Langerhans cells migrate into host epidermis, the turnover is slower compared to other cell lineages.
Langerhans cells, of both donor and host origin, stimulated proliferative responses in naive responders of host- and donor type as well as third party peripheral blood lymphocytes in MLRs. However, no proliferation was observed in responders from the chimeric VCA recipient, demonstrating tolerance of both host- and donor-derived lymphocytes to host- and donor-derived Langerhans cells in these animals. Furthermore, the ability to stimulate non-tolerant host and donor responders suggests that the Langerhans cells isolated were not suppressive in vitro, while their in vivo suppressive capability remains to be investigated.
CONCLUSION:
Given the difficulty in achieving tolerance to skin in the transplant setting, these studies will contribute significantly to our understanding of the mechanisms of skin-specific immunobiology. Understanding the kinetics of cellular infiltrate and resident leukocyte turnover will enhance our understanding of the balance of rejection and tolerance in VCA transplantation research.


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