Human Regulatory Macrophages

Abstract

Regulatory macrophages (M regs) are a novel type of suppressor macrophage which may be a particularly suitable cell for inducing tolerance of solid organ transplants. In this article, we provide a detailed description of the generation of human M regs from peripheral blood monocytes and methods for the assessment of their phenotype. The uniqueness of the human M reg is best appreciated when the M reg is compared to macrophages in other states of activation; therefore, protocols are provided for generating five comparator macrophage types which have been used as cell type-specificity controls in our work.

Cutting Edge: Immunological Consequences and Trafficking of Human Regulatory Macrophages Administered to Renal Transplant Recipients

“…For flow cytometry, harvested M regs were resuspended in ice-cold staining buffer (Dulbecco's modified PBS, 1% BSA, and 0.1% NaN 3 ) and blocked for 30 min with 10% FcR block (Miltenyi Biotec) before staining with fluorochrome-conjugated primary Abs for 1 h. 7-aminoactinomycin D exclusion was used for live/dead discrimination. To assess the T cell-suppressive capacity of M regs, CFSElabeled CD3 + T cells and M regs were set in direct coculture for 5 d. Subsequently, T cell proliferation and absolute numbers were assessed by flow cytometry, as described elsewhere (1). The mechanism of M reg-mediated T cell suppression was investigated in direct 1:1 M reg/T cell cocultures.…”

“…Efforts in our laboratory to develop a cell product for promoting transplant tolerance in the clinical setting have focused on a type of suppressor macrophage, the human regulatory macrophage (M reg) (1)(2)(3)(4)(5)(6)(7). M regs exhibit a number of properties that might make them particularly suitable for clinical purposes, in particular, the cells are fully differentiated and potently T cell suppressive (8).…”

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“…Regulatory macrophages are derived from purified human peripheral blood monocytes isolated by positive selection of CD14-expressing cells using magnetic bead selection. 25 When regulatory macrophages are cocultured with polyclonally activated allogeneic T cells in vitro, regulatory macrophages profoundly suppress proliferation of T cells. 26 Macrophages redivide to 3 phenotypes: (1) M1 macrophages are classically activated, damage graft endothelium, recruit additional leukocytes, and mediate tissue injury.…”

Laser Ablation–Inductively Coupled Plasma Mass Spectrometry: An Emerging Technology for Detecting Rare Cells in Tissue Sections

“…Human Mregs were generated as previously described (26). Briefly, CD14 + monocytes were isolated from Ficoll-prepared PBMCs by positive selection using anti-CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany).…”

“…Data were collected using a FACSCanto II cytometer (BD Biosciences), and the proportion of live Mregs, gated by physical characteristics, was analyzed using FlowJo v7.6.5 software. To assess the T cell-suppressive capacity of Mregs, allogeneic PHA-stimulated CFSElabeled CD3 + T cells and Mregs were set in direct 1:1 coculture for 5 d. Subsequently, T cell proliferation was quantified by flow cytometry, as previously described (26).…”