Rapamycin-modified CD169low/-tolDC promotes skin graft survival in mice via IL-10Breg.

Skin allografts are prone to rejection because of their high immunogenicity. By achieving immune tolerance, the long-term survival of skin allografts can be extended without the need for immunosuppressants or with only short-term low-dose dependency. Tolerogenic dendritic cells (tolDCs) show a strong potential for graft tolerance. We explored the mechanism whereby rapamycin-modified CD169low/-tolDCs regulate interleukin-10 (IL-10) B regulatory (IL-10+ Breg) cell production and mediate the long-term survival of skin allografts in mice. CD169low/-tolDCs were obtained through flow cytometry sorting after treating the mesenchymal stem cell (MSC)-derived dendritic cells with a low dose of GM-CSF. A treatment regimen combining preoperative stimulation and postoperative adoptive infusion of CD169low/-tolDCs was used to treat an acute rejection (AR) mouse skin transplantation model-the adoptive infusion group. An equivalent dose of saline was administered to the control group. Survival and graft rejection rates were assessed. Mixed lymphocyte culture, flow cytometry, immunohistochemistry (IHC), and western blotting (WB) were used to elucidate the expression of different IL-10+ Breg subsets in mice treated with adoptive infusion therapy and the molecular mechanisms whereby CD169low/-tolDCs induce IL-10+ Breg production to mediate tolerance. Adoptive infusion of CD169low/-tol DCs markedly prolonged the rejection time after skin transplantation in mice and promoted graft survival. A significant increase was observed in local blood flow signals in the transplanted skin, along with mild local inflammation. Flow cytometric analysis revealed a positive correlation between high expression of IL-10+ Breg and the changes in Foxp3+ Tregs in vivo, primarily enriched in the CD19 + CD24 + CD27+ mBreg and CD19 + CD23 + CD27-CD24+ Breg subsets, with higher levels of IgM expression. Significant differences were observed compared with control mice. CD79b/NF-κB pathway was found to be involved in Breg production. CD24, CD23, CD79, BTK, NF-κB p50/p65, CD40, and IKKα levels in the adoptive infusion group were significantly increased compared with those in the control group. Adoptive infusion of CD169low/-MSCs/tolDCs may activate the NF-κB pathway through CD79/BTK-dependent and CD40/IKKα-independent pathways, inducing high expression of IL-10 + Breg and promoting graft survival of mouse skin transplantation.