Anti-CD3 mAbs induce CD4+ FoxP3+ Regulatory T(Treg) cells and alleviate acute rejection of the pulmonary allografts in mice

Objective By using mouse orthotopic lung transplant model, we investigated the immune mechanisms of anti-CD3 induced lung allograft protection. Our study intends to further dissect the features of lung transplant immunology and to provide a novel therapeutic insight for the clinical application of anti-CD3 mAbs after lung transplantation. Methods Murine orthotopic allogeneic lung transplants were performed in C57BL/6 wild type(WT) mice using major histocompatibility complex(MHC) fully mismatched BALB/c donors. Syngeneic transplants were also performed in WT C57BL/6 mice using C57BL/6 donors. For immunosuppressive therapy, allograft recipients received 50g dose of anti-CD3 by intraperitoneal injection on days 2, 3, 4, 5, 6 and 9 post-operation(n=4). At day 10, histopathologic characteristics and rejection status of the pulmonary grafts were assessed. The severity of acute rejection was graded by the pathological score, and T cell and neutrophil infiltration in the pulmonary grafts was evaluated by immunohistochemical(IHC) staining for CD3 and myeloperoxidase(MPO) respectively. Real-time RT-PCR was performed for FoxP3, IL-17A and IFN-γ expression in the pulmonary grafts. The percentage of FoxP3+ Treg in total CD4+ T lymphocytes from the recipient spleens was analyzed by FACS. Results 10 days after transplantation, histopathologic examination demonstrated that there is no apparent acute rejection observed in the pulmonary isografts, whereas allografts from untreated recipients have marked inflammatory cell infiltration and pulmonary parenchyma lesion. IHC staining for CD3 and MPO showed that the allograft-infiltrating cells of perivascular layers are mainly T lymphocytes, and the cells around the small airways are mostly neutrophils. Anti-CD3 treatment significantly alleviated the acute rejection of pulmonary allografts, when compared with the untreated group. Real-time RT-PCR showed that the expression levels of IL-17A and IFN-γ in allografts were markedly elevated compared to those in isografts, and anti-CD3 increased the expression of FoxP3, and reduced the expression of IL-17A and IFN-γ in the pulmonary allografts. FACS analysis of splenocytes showed that the percentage of Treg in total CD4+ T lymphocytes increased significantly in the anti-CD3 treated allograft recipients, as compared with the isograft and untreated allograft recipients. Conclusion Anti-CD3 mAbs may alleviate acute rejection of the pulmonary allografts by promoting FoxP3 expression and Treg development. Key words: Orthotopic lung transplantation; Acute rejection; Anti-CD3 monoclonal antibodies(Anti-CD3 mAbs); Regulatory T(Treg) cells