Cord Blood Derived Regulatory Macrophages (Mreg) – An Alternative Source for Mreg-based Cell Therapy in Transplantation

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Abstract

Introduction

Regulatory macrophages (Mreg) have been experiencing a clinical trial in the One Study to test their potential in immunomodulation in allotransplantation. 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 required for the development of Mreg as a practicable cell therapy. In this study the potential of cord blood (CB) derived Mreg used as a cellular immunotherapy in allotransplantation was investigated.

Materials and Methods

CD14+ monocytes isolated from adult peripheral blood (APB) or CB were cultured with M-CSF for 7 day with IFN-γ added at day 6 for Mreg induction. Mreg phenotype was characterized by flow cytometry, and in vitro suppressive function was assessed by mixed lymphocyte reaction (MLR) in both transwell and non-transwell systems. The possible molecular mechanism(s) involved were studied after MLR culture.

Results

Both APB- and CB-derived Mreg revealed a similar phenotype and showed no difference in their capacity to suppress proliferating allo-reactive responder CD14- PBMC at a 1:1 ratio of Mreg: responder cells in both transwell and non-transwell MLR assays, suggesting the involvement of their indirect suppression mechanism(s). However, CB-Mreg were more suppressive than APB-Mreg when ratios of Mreg: responder cells increased from 1:2 through to 1:16 in MLR. Moreover, CB-Mreg harvested from the MLR cultures with both allogeneic stimulators and responder cells exhibited much higher expression of Mreg functional molecule indoleamine-2,3-dioxygenase (IDO) than that by APB-Mreg determined by real-time PCR and western blot, and demonstrated upregulated levels of IDO transcription factor p-stat1 (Signal transducer and activator of transcription 1) examined by western blot when compared to their counterparts cultured with stimulators or responder cells alone, suggesting the induction of stat1 phosphorylation by allogeneic stimulated responder cells which may result in increased IDO expression in CB-Mreg and consequently, lead to their more potent suppressive function in MLR. The IDO-involved mechanism was then confirmed by including IDO inhibitor epacadostat in the MLR assays, showing reduced capacity of Mreg to suppress the allogeneic response in a dose dependent manner when compared to that detected in the MLR without adding epacadostat.

Discussion

Compared to their APB-Mreg, CB-Mreg were more potent in suppression of the allogeneic response in vitro due, at least in part, to their upregulated IDO expression. However, their in vivo function needs be further assessed in an appropriate humanized mouse model.

Conclusion

Our study demonstrates CB-derived Mreg as a potential source for large-scale preparation of human Mreg to meet the demands for clinical cell therapy in immunomodulation in transplantation.

Conclusion

This project was supported by NSFC 81201171 and Hunan Provincial Natural Science Foundation of China 2017JJ3423

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