Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (Mreg IFNγ0 ) for potential cell therapeutic applications

Macrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14 monocytes by using a defined cell culture medium and a stimulus of IFNγ. To scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype. Differentiation of human CD14 monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to Mreg using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling. Mreg and Mreg showed no differences in cell size and volume. However, phenotypically Mreg exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. Mreg revealed reduced expression of IDO and PD-L1 (P