The effect of decellularization of tracheal allografts on leukocyte infiltration and of recellularization on regulatory T cell recruitment

Tracheal transplantation without immunosuppressive therapy has been accomplished with a tissue-engineering approach using decellularized biological scaffolds in combination with recipient progenitor cells. The mechanisms of immune response directed towards these tracheal allografts have not been fully determined. In this study, we evaluated the immunogenicity of these grafts at the protein level, and functionally, in vitro and in vivo in a large animal model. Long-segment circumferential tracheal allografts were decellularized using two different protocols and recellularized using recipient mesenchymal stromal cells (MSC) and tracheal epithelial progenitor cells (TEC). Residual MHCI and MHCII immunostaining was found surrounding the submucosal glands despite cyclical decellularization. In an in vitro lymphocyte proliferation assay, CD4+ T cells continued to proliferate on decellularized pieces and this proliferation was inhibited by co-culture with autologous MSC. Allografts were heterotopically transplanted under a muscle flap in the neck of the recipients and decellularization was found to delay leukocyte infiltration but resulted in eventual cartilage degradation. Recellularization prevented this infiltration up to 3 weeks post-transplantation and allowed for preservation of the cartilage. The immune cells found within these grafts included a significant number of CD4+CD25+Foxp3+ regulatory T cells. Furthermore, gene expression of anti-inflammatory cytokines, such as IL-10 and TGF-β1, involved in proliferation, differentiation and function of regulatory T cells was found in these grafts. These results indicate that the immunological modification induced by recellularized tracheal scaffolds is an active process involving the recruitment of immunosuppressive cells, rather than simply the removal of donor-derived antigenic components.