Introduction: Human regulatory macrophages (Mregs) have been shown to suppress mitogen-driven T cell proliferation in vitro and are under evaluation as a cellular therapy in renal transplantation in THE ONE STUDY clinical trial. In this study, we investigated whether human Mregs were capable of suppressing the allogeneic response in vitro and protecting islet xeno-grafts in humanizing mice in vivo.
Methods: Human CD14+ cells were isolated from healthy donor peripheral blood monocytes (PBMCs) using CD14 magnetic beads. Mregs induction was performed by culturing CD14+ cells with M-CSF for 6 days and be stimulated with IFN-γ for a further 24 hr. The phenotype of Mregs was characterized by flow cytometry, and their suppressive activity was tested by mixed-lymphocyte reaction (MLR) using human PBMCs as responder cells, autologous Mregs as suppressor cells and irradiated allogeneic PBMCs as stimulator cells. In a separate transwell MLR setting, responder and allogeneic stimulator cells were cultured in the upper chamber and autologous Mregs were seeded in the lower chamber. Supernatants collected from MLR cultures were used for cytokine detection. Isolated the neonatal porcine islet cell clusters (NICCs) and cultured for 6 days. In vivo, every 5,000 IEQ NICCs were transplanted into NOD-SCID IL2R gamma -/- mice under the two sides of renal capsules. Adoptive transfer of human PBMCs and Mregs were suspended in PBS and injected into mice at doses of 10×10⁶ and 2×10⁶ by tail vein within 7 days after transplant. Graft rejection was defined as little or no intact graft with a heavy lymphocytic infiltrate on histological examination, survival was analyzed by H&E staining and endocrine secreting cells was analyzed by insulin staining.
Results: The resulting induced human Mregs demonstrated a typical Mregs phenotype, CD14^lowCD80^lowCD86⁺HLA-DR⁺CD83^-/lowCD16^-, and as a result, potent suppression of proliferating alloreactive PBMC even at Mregs: responder cells ratios >1:16. Separation of Mregs from responder and allogeneic stimulators in a transwell MLR system had the mild impact on their suppressive potency in the allogeneic response at all ratios of Mregs: responder cells tested. Upregulated levels of Mregs function related molecules TGF-b and IDO were detected in the supernatants collected from both non-transwell and transwell MLR cultures containing Mregs. In treated mice, the human PBMC were identified in the porcine-NICC-xenografts, as well as systemically in the spleens, blood, and kidneys. However, injected Mregs cannot be detected by flow cytometry, and there were no intact xenografts were found with mid-term.
Conclusions: In vitro induced human Mregs showed the capacity to suppress the allogeneic response in vitro and that was not cell-cell contact dependent. The porcine-NICC-xenografts cannot be protected by Human-Mregs in vivo as Mregs cannot proliferation. The suppressive function of the Mregs have more potency in vitro, but not in vivo.