Name: functional characterization of regulatory macrophages that inhibit graft-reactive immunity
Uploaded: 2017-06-07T12:30:00
Description: Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

We acknowledge the technical contributions of the Flow Cytometry, Microsurgery, and Bio- repository/Pathology Centers of Research Excellence at Mount Sinai. This work was supported by the COST Action BM1305: Action to Focus and Accelerate Cell Tolerogenic Therapies (A FACTT), the Mount Sinai Recanati/Miller Transplantation Institute developmental funds, Ministerio de Ciencia e Innovacion SAF2013-48834-R and SAF2016-80031-R J.O.

In this study, mice are housed in accordance with the United States Department of Agriculture guidelines and the recommendations of the Public Health Service Guide for the Care and Use of Laboratory Animals. All experimental techniques involving animal use were performed in accordance with Institutional Animal Care and Utilization Committee (IACUC)-approved protocols of the Mount Sinai School of Medicine.
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This protocol describes the methods we used to immunocharacterize graft-infiltrating myeloid cell subsets in an experimental murine model of heart transplantation, which is also applicable to other tissues in different murine experimental models. Low-pressure cell sorting at 20 psi was the preferred method to isolate a good yield of pure cell subsets. Maintaining the purity of each myeloid subset is critical to establish conclusive results of the suppressive capacity between the different myeloid populations. However, ot...

This protocol describes in vitro techniques to study the function of tissue-infiltrating macrophages isolated from cardiac allografts, according to their ability to modulate T-cell responses. Widely described in the literature, fluorescent cell-tracking dyes in combination with flow cytometry, are powerful tools to study the suppressive function of specific cell types in vitro and in vivo. Our protocol follows the carboxyfluorescein succinimidyl ester (CFSE) method for monitoring lymphocyte proliferation in vitro.
When a CFSE-labeled cell divides, its progeny acquires half the number of carboxyfluorescein-tagged molecules6. The corresponding decrease in cell fluorescence by flow cytometry can be used to assess cell division, monitoring the capacity of suppressive macrophages to modulate T-cell immune responses. Since CFSE is a fluorescein-based dye, it is compatible with a broad range of other fluorochromes, making it applicable to multi-color flow cytometry. The appropriate choice of fluorochromes for phenotyping is also important to avoid excessive spectral overlap and the inability to recognize antibody-positive cells, particularly with visible protein dyes such as CFSE7.
There are many advantages of using fluorescent dyes over alternative techniques that measure cell proliferation, such as the thymidine incorporation assay, which uses radiolabeled thymidine (TdR)8. This assay utilizes tritiated thymidine (3H-TdR) that is incorporated into new strands of chromosomal DNA during mitotic cell division. A safety concern associated with this assay is the use of radioisotopes, since a scintillation beta-counter is used to measure the radioactivity in DNA recovered from the cells in order to determine the extent of cell division. Methodologically, the tritiated thymidine assay is not flexible enough to fit important clinical laboratory constraints such as low number of cells and delayed analysis after staining. On the contrary, CFSE staining has been shown to prevent cell proliferation and to interfere with critical activation markers, such as CD69, HLA-DR and CD259. Therefore, understanding advantages and limitations of each methodology, particularly in multicolor studies where multiple dyes are used to track different cell types, is critical for obtaining accurate and reproducible results.

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functional characterization of regulatory macrophages that inhibit graft-reactive immunity

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the allograft early after transplantation, mount a graft reactive response against the transplanted organ, and initiate organ rejection2. Recent data suggest that suppressive macrophages facilitate successful long-term transplantation3 and are required for the induction of transplantation tolerance4. This suggests a multidimensional concept of macrophage ontogeny, activation, and function, which demands a new roadmap for the isolation and analysis of macrophage function5. 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.

The representative results show the gating strategy described in the above protocol. Results also display the analysis of the T-cell proliferation activity after the co-culture with graft-infiltrating macrophages. The in vitro suppressive capacity of macrophage subsets was analyzed in Figure 4. The results indicate that the Ly6CloLy6G- macrophages obtained from tolerized recipients are suppressive. The results also indicate that Ly6Cint...

Watch this Scientific Journal Video about Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity at JoVE.com

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Macrophages are plastic cells of the hematopoietic system that have a crucial role in protective immunity and homeostasis. In this report, we describe optimized in vitro techniques to phenotypically and functionally characterize graft-infiltrating regulatory macrophages that accumulate in the transplanted organ under tolerogenic conditions.

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

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