Regulatory T Cells Promote Tolerance during the Early Post-Operative Period in Murine Osteomyocutaneous Vascularized Composite Allotransplantation
Madonna R. Anggelia1,2, Hui-Yun Cheng1, Wen-Yu Chuang3, Chih-Hung Lin4, Gerald Brandacher5, Fu-Chan Wei1, Nicholas Do1, WP Andrew Lee5, Cheng-Hung Lin1.
1Center for Vascularized Composite Allotransplantation, Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Chang Gung Medical College and Chang Gung University, Taoyuan, Taiwan, 2Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University,, Taoyuan, Taiwan, 3Department of Pathology, Chang Gung Memorial Hospital, Chang Gung Medical College and Chang Gung University, Taoyuan, Taiwan, 4Department of Plastic and Reconstructive Surgery, Chiayi Chang Gung Memorial Hospital, Taoyuan, Taiwan, 5Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
PURPOSE:Regulatory/suppressive immune cells, such as CD4+CD25+FoxP3+regulatory T cells (Tregs),have been demonstrated to mediate allograft tolerance in various transplant models. However, their role in vascularized composite allotransplantation (VCA) has not been specifically defined. This study determines the relevant molecular mechanisms, origin (donor vs recipient), location, and activity time frame of Tregs in mediating the induction of allograft tolerance.METHODS:Osteomyocutaneous (OMC) allografts from Balb/c were transplanted into 34 C57BL/6 mice. Immunosuppressive protocol consisted of 1 mg anti-CD154 (POD 0), 0.5 mg CTLA4Ig (POD 2), and 3mg/kg/day rapamycin for 7 days then reduced to 3mg/kg every other day for 3 weeks. Recipients were organized into 5 groups based on time point of Tregs depletion using anti-CD25 antibody: Group 1 (control), no Tregs depletion (n=10); Group 2, depletion on POD 0 (n=7); Group 3, POD 30 (n=7); Group 4, POD 90 (n=7); and Group 5, with anti-CD25 and Tregs isolated from tolerant mice at POD 30 (n=3). Intracellular markers and cytokines associated with Tregs activation were measured. Ratios of Tregs to rejection mediating T cell subpopulations (Th1, Th2, & Th17) were assessed by flow cyometry. To observe Tregs origins, Balb/c -Tg(FoxP3-GFP) mice were used as donor and the presence of FoxP3+GFP+ cells were then determined by flow cytometry and immunohistochemistry. To confirm function of Tregs in the allograft, skin from na´ve Balb/c or tolerated OMC allografts were grafted onto Rag2-/- mice in the presence of adoptive-transferred effector T(Teff) cells. Tolerance/rejection was assessed clinically and median survival time (MST) recorded.RESULTS:Intracellular markers (GATA-3+, T-bet+ and Helios+) and cytokines (IL-10+, TGF-β+, and IL-35+) associated with Tregs activation were elevated in tolerant animals vs animals experiencing rejection. Tolerant animals showed increased ratios of Tregs/Th1 cells and Tregs/Th17 cells but not of Tregs/Th2 cells. Tregs in the circulation, secondary lymphoid organs, and OMC allograft skin were of recipient origin in all animals though a higher amount was found in tolerated allografts. Tregs from the tolerant grafts circulated in Rag2-/- recipient mice and delayed adoptive transferred Teff-mediated rejection (MST=37 vs 52 days). Allograft survival was significantly shortened in the groups with Treg-depletion on POD0 (3 of 7, MST=90) and POD 30 (5 of 7, MST=104, p<0.05) compared to the un-depleted control. However, allograft survival was unaffected with Tregs depletion on POD 90. Tregs depletion-mediated rejection was rescued with adoptive transfer of Tregs from allograft tolerant animals (Group5)(MST > 200) (Fig 1).CONCLUSION:Recipient Tregs are crucial for VCA tolerance in the early post-operative period and could be utilized as a cellular therapy to improve VCA outcomes
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