Ex vivo-expanded natural CD4⁺CD25⁺ regulatory T cells synergize with host T-cell depletion to promote long-term survival of allografts

Foxp3⁺CD4⁺CD25⁺ natural regulatory T (nT_reg) cells have been shown in immunodeficient mice to suppress allograft rejection after adoptive cotransfer. We hypothesized that immunotherapy using ex vivo-expanded nT_reg could suppress allograft rejection in wild-type mice. Donor alloantigen (alloAg) specificity of naive splenic nT_reg was enriched in vitro by culturing with anti-CD3/CD28-coated Dynabeads plus bone marrow-derived dendritic cells (BM-DC) in the presence of interleukin (IL)-2 or IL-2 plus transforming growth factor (TGF)-β. On average, 96.2% fresh CD4⁺CD25⁺ nT _reg were intracellular Foxp3⁺. By d+20 in culture, 6.4% nT_reg were Foxp3⁺ following expansion with IL-2 alone, and 14.4% or 19.7% nT_reg were Foxp3⁺ when expanded with IL-2 plus 0.5 or 2.5 ng/mL TGF-β, respectively. In vitro, alloAg-enriched, TGF-β/IL-2-conditioned nT_reg exerted stronger donor alloAg-specific suppression than cells with IL-2 alone in mixed lymphocyte reaction (MLR) assays. In vivo, alloAg-enriched, TGF-β/IL-2-conditioned nT_reg expressed high-level Foxp3 following infusion, effectively overcame acute rejection and induced long-term survival of donor but not third-party heart allografts in peritransplant host T-cell-depleted mice. Long-term surviving allografts were noted to possess Foxp3⁺ graft-infiltrating cells of exogenous and endogenous origins. In conjunction with transient host T-cell depletion, therapeutic use of ex vivo-expanded nT_reg may be a practical means of preventing acute allograft rejection.