Correspondence to: R. A. Harris, Applied Immu-

Monocytes are highly abundant circulatory effector cells and play a vital role in driving or resolving inflammatory processes depending on their activation phenotype. We investigated and compared a panel of polarization protocols of blood-derived monocytes to achieve a stable, optimal and effective regimen for in vitro induction of immunosuppressive human macrophages, evaluating their surface receptor expression, cytokine profile, scavenging function and ability to suppress T-cell proliferation. Importantly, we assessed the effect of copolarization or secondary pro-inflammatory stimulation of a primary anti-inflammatory activation phenotype. A combination of IL-4/IL-10/TGF-b yielded a relatively stable and dominant immunosuppressive phenotype characterized by higher IL-10 production and down-regulated TNF-a, IL-6, CD86, CD274 and MHC II expression. Functionally, IL-4/IL-10/TGF-b-stimulated macrophages (M2) had a potent deactivating effect on a subsequent pro-inflammatory LPS/IFNc-activated macrophage (M1) stimulation and significantly suppressed T-cell proliferation. Monocytes derived from patients with chronic inflammatory diseases could be induced to be anti-inflammatory using this protocol. Pre-differentiation with GM-CSF or M-CSF was further demonstrated to enhance final M1/M2 activation status. Our findings indicate a robust polarization protocol for generation of specific immunosuppressive human monocyte-derived macrophages