Background: Regulatory macrophages (Mreg) have been experiencing a clinical trial in the One Study as a potential cell therapy for immunomodulation in transplantation. However the limited number of Mreg induced from individual adult peripheral blood mononuclear cells (PBMC) may restrict their application. Thus, exploring an alternative source for Mreg is necessary for the development of Mreg-based therapy. In this study we investigated the potential of cord blood derived Mreg for cellular immunotherapy.

Materials and Methods: CD14+ monocytes isolated from adult PBMC (APB) and cord blood (CB) were cultured with macrophage colony-stimulating factor (M-CSF) for 7 day with IFN-γ added at day 6 for Mreg induction. Mreg phenotype was characterized by flow cytometry, and their suppressive function was assessed by mixed-lymphocyte reaction (MLR) using anti-CD3/CD28 microbeads as stimulators, CFSE-labelled CD14- PBMC as responder cells and autologous and third-party APB- and CB-Mreg as suppressor cells. The non-specific effect of third-party (allo) CB-Mreg on naïve T cell proliferation and their immunogenicity were evaluated by coculturing T cells in the presence and absence of anti-CD3/CD28 microbeads with non- or irradiated CB-Mreg, respectively, followed by flow cytometry analysis.

Results: Mreg induced from both APB and CB showed no difference in their yield and phenotype. As a result, both APB- and CB-Mreg demonstrated similar potent suppression of proliferating polyclonal- and third-party (allo)-reactive CD14- PBMC, CD4+ and CD8+ T cells at ratios of 1:1 and 1:2 of Mreg: responder cells. Interestingly, CB-Mreg exhibited stronger suppression of proliferation of alloreactive CD14- PBMC and CD8+ T cells at a 1:4 ratio of Mreg: responder cells when compared to APB-Mreg. The proliferation of anti-CD3/CD28 microbeads stimulated but not non-stimulated allo T cells was impaired substantially by coculturing with CB-Mreg. However allo T cells did not proliferate when cultured with irradiated CB-Mreg.

Discussion: Compared to APB-Mreg, CB-Mreg had similar or stronger capacity to suppress the allogeneic response with low immunogenicity and no significant effect on naïve allo T cell proliferation in an in vitro alloimmune environment.

Conclusions: Our shows third-party CB-Mreg as a potential source for sufficient number of human Mreg used for clinical cellular therapy and may meet the demands of setting up a cell bank for transplantation immune tolerance.