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.

Background: 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.
Aim of the Study: 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.
Methods: 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 _IFNγ0 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.
Results: Mreg and Mreg _IFNγ0 showed no differences in cell size and volume. However, phenotypically Mreg _IFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. Mreg _IFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of Mreg _IFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, Mreg _IFNγ0 differed significantly from Mreg. Mreg _IFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased.
Conclusion: Exposing CD14 ⁺ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (Mreg _IFNγ0 ). Mreg _IFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.
(© 2024. The Author(s).)