Introduction: The high immunogenicity of skin has manifested as multiple acute rejection episodes in the majority of human vascularized composite allotransplantation (VCA) case. Skin dendritic cells (DC) are believed to play an important role in both the initiation and regulation of skin alloimmunity. 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 and examining their effect on T-cell immune responses are parts of our continued efforts to optimize targeted immunomodulation. 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.
Materials and Methods: 1) Hind-limb transplantation was performed from BN to Lewis rats and recipients were treated with topical FK506 (0.5mg/kg, 0.1% FK506 ointment, applied once daily). FK506 levels in blood and local skin were measured using liquid chromatography tandem-mass spectrometry. Skin-resident DC from transplanted limbs were isolated at day 8 post-transplantation and quantification of skin DC subsets was analyzed by flow cytometry. 2) Migrated skin DC from the limb of 7-day topical FK506 treated untransplanted and naïve Lewis rats were isolated in vitro after skin being cultured under a stimulation culture condition and were characterized by subsets and functional specialization using mixed lymphocyte reaction (MLR) and flow cytometric analysis.
Results and Discussion: 1) Limb allografts showed clinical signs of grade 2-3 rejection on day 8 post-transplantation. High skin levels (2110±177 ng/ml) and low blood levels (3.5±2.2 ng/ml) of FK506 were determined after 7-day topical treatment. Skin-resident dermal DC(DDC) declined with an elevation trend in Langerhans cells(LC) compared to no treatment group(Figure 1). Migrated LC and mature skin DC were lower, while DDC were slightly higher in FK506-treated skin compared to naïve skin(Figure 2). These data indicate the probable effect of topical immunosuppression on the modulation of skin DC subsets after VCA and skin DC migration and maturation could be inhibited by exposure to FK506. 2) In MLR, skin-migrated DC inhibited effector T cell (Teff) proliferation and exhibited synergistic effects with regulatory T cells (Treg). 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, suggesting there is differential contribution of skin-migrated and skin-resident DC to the Teff response in vitro.
Conclusion: 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. The determination of skin-migrated DC in regulating T-cell immune responses will provide a target for immunomodulation.