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Abstract

Introduction

Protocol

Representative Results

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Materials

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Immunology and Infection

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Published: June 7th, 2017

DOI:

10.3791/54242

1Department of Medicine, Icahn School of Medicine at Mount Sinai, 2Immunología de Trasplantes, Centro Nacional de Microbiología, Instituto de Salud Carlos III

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 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.

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....

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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.

1. Media Preparation

  1. Prepare complete RPMI 1640 medium with 10% FBS and 1% pe.......

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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.......

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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.......

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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.

....

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Name Company Catalog Number Comments
RPMI 1640 Media Life Technologies 11875119
10 % FBS Life Technologies 26140-079
1000X 2-mercaptoethanol Life Technologies 21985023
100X Pen/Strep Life Technologies 15140122
100X L-glutamine Life Technologies 25030081
100X Non Esential amino acids Corning 25025039
1M HEPES buffer  Corning 25060CL
100mM Sodium Pyruvate ThermoScientific SH30239.01
Penicilin/Streptavidyn ThermoScientific 15140122
Collagenese A Roche 70381322
DPBS (w/o calcium&magnesium) Corning 21031CV
70 micron Cell strainer Fisher 22363548
ACK lysis buffer Life Technologies A10492-01
DAPI Sigma 32670-5mg-F
CFSE-FITC Invitrogen C34554
Dynabeads Mouse T cell activator CD3/CD28 Life Technologies 11452D
96 well  U-bottom plate Corning 353077
Antibodies:
anti-Ly6C-APC eBioscience 175932-80
anti-Ly6G-Pe/Cy7 Biolegend 127617
anti-CD11b-Percp/Cy5.5 eBioscience 45011282
anti-CD45-APC/Cy7 eBioscience 47045182
anti-CD4-APC eBioscience 17004181
anti-CD8-P/ Cy7 eBioscience 25008181
LSRII Flow Cytometer BD Bioscience
FACSDiva Software BD Bioscience
C57BL/6-Foxp3tm1Flv/J  The Jackson Laboratory 008374
C57BL/6 The Jackson Laboratory 000664
Balb/c The Jackson Laboratory 000651

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