Introduction: Regulatory macrophages (Mreg) have been shown to suppress polyclonal T cell proliferation in vitro and prolong allograft survival in a mouse model, and are being currently under evaluation in a clinical trial for living donor renal transplantation (The One Study). However whether they are capable of suppressing the xenogeneic response remain to be investigated. In this study the suppressive capacity of human Mreg in the xenogeneic response was evaluated in a parallel comparison with their suppressive potency in the allogeneic response in vitro.

Materials and Methods: CD14+ monocytes selected from human peripheral blood mononuclear cells (PBMC) were cultured with macrophage colony-stimulating factor (M-CSF) for 7 day with IFN-γ added at day 6 for Mreg induction. Mreg suppressive function was assessed by mixed-lymphocyte reaction (MLR) using irradiated pig and human PBMC as xenogeneic and allogeneic stimulator cells respectively, human PBMC as responder cells and autologous Mreg as suppressor cells. mRNA expression of IDO, IL-10, IFN-γ, iNOS and TGF-β by Mreg after MLR was measured by real-time PCR. Supernatants collected from MLR cultures were used for IDO activity assay by HPLC.

Results: Both proliferating allo and xeno reactive human responder cells were inhibited significantly by Mreg with stronger suppressive potency in the xenogeneic response even at a 1:32 ratio of Mreg: responder cells. While they expressed similar levels of IL-10 and TGF-b, and extremely low level of IFN-γ and iNOS gene expression, Mreg harvested from xeno but not allo MLR cultures demonstrated remarkably high levels of IDO mRNA detected by real-time PCR. Consistently, a stronger IDO activity measured by kynurenine/tryptophan ratio was revealed in the supernatants collected from xeno MLR cultures when compared to that from allo MLR cultures.

Discussion: Human Mreg showed a more potent suppression in the xeno-response compared to that they did in the allo-response. This stronger suppressive characteristic in the xeno-response may be attributedat least in part, to the higher levels of their IDO expression and activity, one of reported main mechanisms used by Mreg to regulate immune responses.

Conclusion: Human Mreg are more suppressive in vitro with IDO-involved mechanisms in the xeno- than in the allo-response, suggesting their potential in immunomodulation of the xenogeneic response.