Alloantigen-Pulsed Host Dendritic Cells Induce T-Cell Regulation and Prolong Allograft Survival in a Rat Model of Hindlimb Allotransplantation

Abstract

Composite tissue allotransplantation is restricted due to the risks presented by long-term therapeutic immunosuppression. This study is conducted to investigate whether treatment with recipient immature dendritic cells (DCs) pulsed with donor alloantigens can prolong allograft survival and induce T-cell regulation in a rodent model. Orthotopic hindlimb transplants from Brown-Norway (RT1(n)) to Lewis (RT1(1)) rats were performed (day 0). DCs were propagated from the recipient bone marrow and pulsed with the donor alloantigen lysate. Group 1 (control group) did not receive any treatment. Groups 2 and 3 received cyclosporine A (CsA) at a concentration of 10 and 16 mg.kg(-1).day(-1), respectively, on days 0-20 following composite tissue allotransplantation. Group 4 received antilymphocyte serum (i.p. administered 4 d before and 1 d after transplantation) therapy. Group 5 received combined treatment with CsA (10 mg.kg(-1).day(-1), days 0-20) and donor alloantigen-pulsed recipient DCs (i.v. administered on days 7, 14, and 21). Group 6 received combined treatment with CsA (10 mg.kg(-1).day(-1) on days 0-20), antilymphocyte serum (administered i.p. 4 d before and 1 d after transplantation), and DCs (administered i.v. on days 7, 14, and 21). Graft rejection was defined as epidermolysis/desquamation of the donor skin. The mixed lymphocyte reaction was performed to determine the donor T-cell reactivity. Tissue samples were biopsied to analyze the histological changes, and flow cytometry was performed to quantify the donor T-cells. Allograft survival was significantly prolonged (>200 d) in Group 6 when compared with the other groups (P < 0.001). The mixed lymphocyte reaction performed for Group 6 revealed hyporesponsiveness of the T-cells to donor alloantigens. Flow cytometric analysis in Group 6 revealed a significant increase in the percentage of CD4(+)/CD25(+) and CD4(+)/foxP3(+) T-cells expression, and significant increase in the percentage of donor cells (RT1(n)) in the recipient peripheral blood. Immunohistochemical staining of allo-skin revealed a significant increase in the proportion of CD25(+) cells in the subcutaneous and dermis layers in Group 6, as compared to other groups. Treatment with donor alloantigen-pulsed recipient immature DCs in combination with transient immunosuppression prolongs allograft survival and induced tolerance by inducing T-cell hyporesponsiveness to donor alloantigens and increasing the CD4(+)/CD25(+) T-cell population.