Characterization And Functional Analysis Of Skin Dendritic Cell Subpopulations In Vascularized Composite Allotransplantation
WENSHENG ZHANG, M.D., Ph.D.1, Chiaki Komatsu, M.D.1, Jiaqing Wu, M.D., Ph.D.1, Firuz Feturi, M.D.2, Jeff Walsh, PhD3, Jingjing Li, M.D., Ph.D.1, Lin He, M.D., Ph.D.1, Maxine Miller, M.D.1, Alicia Mathers, Ph.D.4, Angus Thomson, Ph.D.3, Vijay Gorantla, M.D., Ph.D.5, Kia Washington, M.D.1, Mario Solari, M.D.1.
1Dept. Plastic Surgery, University of Pittsburgh, Pittsburgh, PA, USA, 2Dept. Pharmaceutical Science, University of Pittsburgh, Pittsburgh, PA, USA, 3Dept. Surgery, University of Pittsburgh, Pittsburgh, PA, USA, 4Dept. Dermatology, University of Pittsburgh, Pittsburgh, PA, USA, 5Wake Forest Institute for Regenerative Medicine, Wake Forest Baptist Medical Center, Winston Salem, NC, USA.
PURPOSE: Skin dendritic cells (DC) are believed to play an important role in both the initiation and regulation of skin alloimmunity. We have developed a technique for the isolation and characterization of distinct skin DC subsets and have demonstrated the dynamics and the associated immune response profiles in vascularized composite allotransplantation (VCA). Resident skin DC can migrate out of allogeneic skin into the recipient's skin and draining lymph nodes, and may exert regulatory functions after VCA. Thus, isolating migratory skin DC from limb transplants and examining their capacity to effect T cell immune responses is part of our ongoing effort. In the present study, we assessed the function of skin-resident and skin-migrated DC subsets on regulating the T-cell immune response and characterized their changes in VCA under topical FK506 immunosuppression. METHODS: 1) To evaluate the impact of topical immunosuppressive treatment on the skin DC in VCA, LEW rats received hindlimb transplants from BN rats and were treated with topical FK506 (0.5mg 0.1% FK506 ointment, applied once daily). Skin-resident DC from transplanted limbs were isolated at day 8 post-transplantation using our established methods and quantification of skin DC subsets were analyzed by flow cytometry.Skin DC isolated from Na´ve or 7-day topical FK506 treated untransplanted LEW rats were used as comparison groups. 2) To determinethe function of skin migratory DC on the T cell immune response, DC from each group were isolated in vitro and were characterized by subset and functional specialization by mixed lymphocyte reaction(MLR) and flow cytometric analysis. RESULTS: 1) After topical FK506 treatment, skin-resident dermal DC (DDC) declined with an elevation trend in Langerhans cells (LC) (Figure 1). 2) Migrated LC and mature skin DC were lower, while DDC were slightly higher in FK506-treated skin explant cultures compared to na´ve skin (Figure 2). 3) In MLR, skin-migrated DC inhibited effector T cell (Teff) proliferation and exhibited synergistic effects with regulatory T cells (Treg). 4) The addition of skin-migrated DC promoted Foxp3 expression in CD4+ T cells. In contrast, the addition of skin-resident DC decreased Foxp3 and promoted IL-17 expression. CONCLUSIONS: In vivo alterations of skin-resident DC subsets and ex vivo emigration and maturation of skin DC are affected by a short-term topical immunosuppression in VCA.There is differential contribution of skin-migrated and skin-resident DC to the effector T cell response in vitro. The determination of skin-migrated DC in regulating T-cell immune responses will provide a target for immunomodulation.
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