Introduction: Great efforts from teams around the world have made composite tissue allotransplantation (CTA, or vascularized composite allotransplantation, VCA in short) a clinical reality. However, chronic application of immunosuppresants is still critical for long-term survival of the allografts. To circumvent this, major research efforts have been focused on establishing the allograft-specific tolerance. A specific subset of T cells termed regulatory T cells (Treg), with expression of CD4+CD25+Foxp3+, was found to be involved in suppressing the immune response. Tregs have been increasingly documented to suppress alloantigen-driven rejection especially in organ transplantation studies. However, less is known about functions of Tregs in composite tissue allotransplantation. Methods: The hindlimb osteomyocutaneous flap allotransplantation was performed on a rat model with BN donors and LEW recipients. The recipients were conditioned with two doses of antilymphocyte serum (ALS) in addition to daily injection of cyclosporine A for 7 days. CD4+CD25+ splenocytes were collected from the recipients with MACS magnetic microbeads technique in two steps. Firstly, splenic CD4+ cells were sorted out using negative selection by antibodies against non-CD4 cells. Secondly, CD25+ cells were sorted from CD4+ cells using positive selection. The in vitro suppressive potential of sorted CD4+CD25+ cells were assayed with mixed lymphocyte reaction. The in vivo effects were evaluated by adoptive transfer to allotransplantation receipients. Flap appearance was observed daily to monitor the rejection. Blood cell population was characterized with flow cytometry. Results: Two-step sorting greatly increased the purity of CD4+CD25+ cells from 7% to 91%. Out of these CD4+CD25+ cells, 61% expressed Treg marker Foxp3, in contrast to 6% in whole splenocyte population. When added into the coculture of Lewis and BN splenocytes, Tregs suppressed alloantigen-stimulated proliferation of LEW splenocytes in a dose-dependent manner. The in vivo effects of Tregs were evaluated by adoptive transfer to the allotransplantation recipients. Without conditioning by ALS and cyclosporine A, fresh-isolated CD4+CD25+ -infused rats showed signs of rejection sooner than controls. However, after alloantigen-stimulation, via coculture with BN splenocytes for 72 hours prior to infusion, the Tregs greatly delayed the onset of rejection in combination with ALS and cyclosporine A. One third of the allotransplantation recipients with alloantigen-stimulated Treg infusion had the allografts survive longer than 150 days. Conclusion: The protocol to isolate Tregs from rat splenocytes was developed with good purity. Isolated Tregs showed immnunosuppressive function in vitro. In vivo, alloantigen-stimulated Tregs in combination of ALS and cyclosporine A prolonged the survival of allotransplanted grafts.
Read full abstract