Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Intrathymic In Vivo Two-photon Microscopy Reveals The Interaction Of Donor Bone Marrow-derived Dendritic Cells And Host Thymocytes In A Model Of VCA Central Tolerance
Wensheng Zhang, M.D., Ph.D, Yang Yang, M.D., Ph.D, Yang Li, M.D., Ph.D., Kia M. Washington, M.D., Vijay S. Gorantla, M.D., Xin Xiao Zheng, M.D., Mario G. Solari, M.D..
University of Pittsburgh, Pittsburgh, PA, USA.

PURPOSE: We have demonstrated that vascularized composite allografts (VCA) containing viable vascularized bone morrow offer the inherent potential for establishment of stable mixed chimerism (MC) and robust tolerance. Studies also showed that dendritic cells (DC) contribute critically to central tolerance-induction by elimination of developing thymocytes. In this study, we track donor bone marrow-derived DCs (BM-DCs) in a mouse VCA model, and visualized their dynamic communication with the recipient thymocytes in the chimeric thymus environment related to VCA tolerance induction.
METHODS: Syngeneic or allogeneic hind-limb transplantation was performed from B6.CD11c-YFP or BALB/c.CD11c-DTR/GFP transgenic donor mice, whose DCs expresses enhanced fluorescent protein, to wild-type B6 recipient mice. MHC-mismatched recipients were treated with the combined immunosuppressive regimen of rapamycin, CTLA4/Fc and anti-CD40L mAb. In a control group, diphtheria toxin (DT) was administered after transplantation for deletion of DCs from BALB/c.CD11c-DTR/GFP donors. 90 days after transplantation, the long-term surviving chimeric B6 recipients were sacrificed as donors, and B6 nude mice served as recipients of the secondary thymus transplantation. Nude mice were gamma-irradiated and reconstituted with BM from B6.DsRed.MST transgenic mice 3 weeks before thymus transplantation under the renal capsule. After thymus acceptance, the kidney containing the transplanted thymus was exposed for in vivo imaging by two-photon microscopy. The MC and DCs profiles were also studied by flow cytometry and immunofluorescence analysis.
RESULTS: 1) Long-term survival of hind-limb allografts was achieved in the BALB/c to B6 strain combination under comstimulation blockade plus rapamycin treatment. 2) Donor derived DCs were detected in peripheral blood of the recipient from 2 weeks post limb transplantation and over a sustained period of time. 3) Donor BM-DCs presented in the thymus of VCA recipients for an extended period as determined by flow cytometry and immunofluorescence analysis. 4) The functional interaction between donor BM-DCs and the new developing thymocytes were visualized in vivo in the transplanted chimeric thymus from living mice that lack an endogenous thymus.
CONCLUSION: Donor BM-DCs migrate to the host thymus and are implicated in the clonal deletion process by presenting donor-derived antigens. This intravital imaging study provides further insights into the activities of DCs in VCA tolerance induction.


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