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Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection 1 . Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection 2 . Recent data suggest that suppressive macrophages facilitate successful long-term transplantation 3 and are required for the induction of transplantation tolerance 4 . This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function 5 . Due to the plasticity of macrophages, it is necessary to provide a methodology to isolate and characterize macrophages, depending on the tissue environment, and to define their functions according to different scenarios. Here, we describe a protocol for immune characterization of graft-infiltrating macrophages and the methods we used to functionally evaluate their capacity to inhibit CD8 + T proliferation and to promote CD4 + Foxp3 + Treg expansion in vitro.

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity