Prolongation Of Skin Allograft Survival By Intravenous Apoptotic Thymocytes Infusion

Objective: We investigated the immunomodulatory effect on skin allografts survival induced by intravenous donor derived apoptotic thymocytes infusion in a skin allograft mouse model, as well as its underlying cellular and molecular mechanisms involved in allograft tolerance induction, so that new strategies can be raised to prevent allograft rejection.Methods: Donor derived apoptotic thymocytes were obtained by dexamethasone(DEX) treatment. Cell apoptosis was confirmed by DNA Ladder assay and the Annexin V-FITC staining. The apoptosis of thymocytes from BALB/c mice were induced by dexamethasone(DEX) after pre-staining with PKH67. Thereafter the apoptotic cells were infused into C57BL/6 mice via caudal vein for clarifying the phagocytosis of appototic cells by recipient's dendritic cells. 12 hours latter, splenic cells isolated from C57BL/6 recipient mice were stained with PE labeled CD11c antibody, and imaged with a laser scanning confocal microscope(LSCM). Allogenic skin transplantation was performed 7 days after cells infusion, and the allograft survival was monitored, while leukocyte infiltration of skin grafts was examined by histology on day 7 post-transplantation. The proliferation capacity of T cells from recipient mice stimulated with donor spleen cells in vitro was evaluated by MLR. The mRNA levels for cytokines(e.g. IL-10, TGF-β) and FOXP3 expressions in the spleen of recipient mouse were detected using RT-PCR while the changes of DC subsets in recipient mouse were determined by flow cytometric analysis.Results:I. Induction of apoptotic thymocytes by dexamethasone(DEX) treatment and the phagocytosis of appototic thymocytes by recipient's spleen dendritic cellsThe apoptotic thymocytes were obtained by dexamethasone(DEX) treatment of thymocytes from donor BALB/c mice. Incubation of thymocytes with dexamethasone for 5 hours, the apoptosis was confirmed by DNA Ladder assay and the Annexin V-FITC staining. DNA Ladder can be seen clearly in the electrophoretogram. The percentage of apoptotic cells induced by DEX was 58.7%, whereas the control cells exhibited 32.4% of apoptotic cells.The pre-stained thymocytes from BALB/c mice with PKH67 were induced to apoptosis by DEX treatment, and then infused into C57BL/6 mice via caudal vein. After 12 hours, splenic cells isolated from C57BL/6 mice were stained with CD11c- PE. It was showed that the appototic cells were phagocytosed by recipient's dendritic cells, as evidenced by the existence of PKH67+CD11c+ DC.II. Donor apoptotic thymocytes infusion mediates prolongation of skin allograft survivalAllogenic skin transplantation was performed from normal BALB/c or C3H (third party) donors to C57BL/6 recipients mice treated with apoptotic thymocytes infusion while injection of C57BL/6 recipients with PBS or DEX as control. The survival of skin graft of recipient mice treated with apoptotic donor thymocytes (17.750±3.956) showed a marked prolongation in comparison with the controls( p﹤0.01), whereas there was no difference in the mean survival time between C3H mouse-derived skin graft of apoptotic cell-treated recipient mice (10.500±1.927) and BALB/c mouse donor-derived skin graft of PBS-treated recipient mice (10.250±1.389). Compared with third party and PBS groups, the survival of donor-derived skin graft of DEX-treated recipient mice (15.625±1.685 ) was longer. Histological analysis of allografts on day 7 post-transplantation revealed much lower inflammatory infiltration in donor-derived skin graft of apoptotic cell-treated recipient mice as compared to that in either C3H-derived skin graft of apoptotic cell-treated recipient mice or donor-derived skin graft of PBS-treated recipient mice. Donor-derived skin graft of DEX-treated recipient mice had moderate inflammatory infiltration. Next, we examined the proliferation effect of T cells from apoptotic cell-treated recipient mice in mixed lymphocyte reaction by CFSE labeling assays. Significant inhibitory effect against the proliferation of allogenic T cells was observed in apoptotic cell-treated group. Analysis of mRNA levels for cytokines and FOXP3 expressions in spleen of recipient mice showed that a significant increase of TGF-β,IL-10,Foxp3 expression was detected in apoptotic cell-treated group than PBS-treated group and C3H group. Furthermore, the percentage of CD11c+B220+DC was strikingly increased by treatment of recipients with donor apoptotic thymocytes infusion, and the CD11c+CD45RB+DC also up regulated in comparison with controls.Conclusions: These findings indicated that intravenous apoptotic thymocytes infusion prolonged murine skin allograft survival, and suggested that upregulation of regulatory T cell and some DC susets may play a crucial role in the induction of donor specific allograft tolerance.