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.
1. Media Preparation
<ol>
	<li>Prepare complete RPMI 1640 medium with 10% FBS and 1% pe...

<ol>
	<li>Jose, M. D., Ikezumi, Y., van Rooijen, N., Atkins, R. C., Chadban, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+act+as+effectors+of+tissue+damage+in+acute+renal+allograft+rejection.">Macrophages act as effectors of tissue damage in acute renal allograft rejection.</a> Transplantation. 76 (7), 1015-1022 (2003).</li><li>Oberbarnscheidt, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-self+recognition+by+monocytes+initiates+allograft+rejection.">Non-self recognition by monocytes initiates allograft rejection.</a> J Clin Invest. 124 (8), 3579-3589 (2014).</li><li>Garcia, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytic+suppressive+cells+mediate+cardiovascular+transplantation+tolerance+in+mice.">Monocytic suppressive cells mediate cardiovascular transplantation tolerance in mice.</a> J Clin Invest. 120 (7), 2486-2496 (2010).</li><li>Conde, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DC-SIGN(+)+Macrophages+Control+the+Induction+of+Transplantation+Tolerance.">DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance.</a> Immunity. 42 (6), 1143-1158 (2015).</li><li>Ginhoux, F., Schultze, J. L., Murray, P. J., Ochando, J., Biswas, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+multidimensional+concept+of+macrophage+ontogeny,+activation+and+function.">New insights into the multidimensional concept of macrophage ontogeny, activation and function.</a> Nat Immunol. 17 (1), 34-40 (2016).</li><li>Quah, B. J., Wijesundara, D. K., Ranasinghe, C., Parish, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+fluorescent+target+arrays+for+assessment+of+T+cell+responses+in+vivo.">The use of fluorescent target arrays for assessment of T cell responses in vivo.</a> J Vis Exp. (88), e51627 (2014).</li><li>Tario, J. 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(70), e4287 (2012).</li><li>Poujol, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+evaluation+of+lymphocyte+transformation+test+based+on+5-ethynyl-2'deoxyuridine+incorporation+as+a+clinical+alternative+to+tritiated+thymidine+uptake+measurement.">Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2'deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement.</a> J Immunol Methods. 415, 71-79 (2014).</li><li>Last'ovicka, J., Budinsky, V., Spisek, R., Bartunkova, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cell Immunol. 256 (1-2), 79-85 (2009).</li><li>Corry, R. 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(6), e238 (2007).</li><li>Ochando, J., Conde, P., Bronte, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocyte-Derived+Suppressor+Cells+in+Transplantation.">Monocyte-Derived Suppressor Cells in Transplantation.</a> Curr Transplant Rep. 2 (2), 176-183 (2015).</li><li>Gallina, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+induce+a+subset+of+inflammatory+monocytes+with+immunosuppressive+activity+on+CD8++T+cells.">Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells.</a> J Clin Invest. 116 (10), 2777-2790 (2006).</li><li>Huang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gr-1+CD115++immature+myeloid+suppressor+cells+mediate+the+development+of+tumor-induced+T+regulatory+cells+and+T-cell+anergy+in+tumor-bearing+host.">Gr-1+CD115+ immature myeloid suppressor cells mediate the development of tumor-induced T regulatory cells and T-cell anergy in tumor-bearing host.</a> Cancer Res. 66 (2), 1123-1131 (2006).</li><li>Luan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytic+myeloid-derived+suppressor+cells+accumulate+in+renal+transplant+patients+and+mediate+CD4(+)+Foxp3(+)+Treg+expansion.">Monocytic myeloid-derived suppressor cells accumulate in renal transplant patients and mediate CD4(+) Foxp3(+) Treg expansion.</a> Am J Transplant. 13 (12), 3123-3131 (2013).</li><li>Tario, J. D., Muirhead, K. A., Pan, D., Munson, M. E., Wallace, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+immune+cell+proliferation+and+cytotoxic+potential+using+flow+cytometry.">Tracking immune cell proliferation and cytotoxic potential using flow cytometry.</a> Methods Mol Biol. 699, 119-164 (2011).</li></ol>

The authors have nothing to disclose.

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 ...

The overall goal of this in vitro procedure is to phenotypically and functionally characterize macrophage populations isolated from allografted hearts in a mass transplantation model. These methods can help answer key questions in the immunity we find about how to improve success in allograph survival and tolerance. The main advantage of this technique is that it allows the study of how a specific tissue infiltrated cardiac allograph isolated microphages subset modulate host tissues response.After generating a single-cell suspension from the allograft, block any non-specific binding on the myeloid cells with one to two microliters of FC receptor blocking monoclonal antibody for 15 minutes. At the end of the incubation, stain the cells with the appropriate fluorescence conjugated myeloid cell surface antibodies for 45 minutes at four degrees Celsius protected from light. Then wash the cells two times in RPMI medium by centrifugation.And re-suspend the pellet in trypan blue for counting. Next, dilute the cells to a one time 10 to the sixth cells per milliliter concentration in fresh medium. And filter the cells through a 70 micron strainer into five-milliliter round-bottom tube.Just before sorting, stain the cells with DAPI as a viability marker and set the flow cytometer to the appropriate macrophage sorting parameters. Place five-milliliter conical tubes containing one milliliter of RPMI medium for collecting the sorted cells and load the sample tube onto the cytometer. In the flow cytometer software, select a blank experiment with a blank panel, and set up a forward versus side scatter dot plot.Gate on the leukocytes, excluding the cell debris and clumps with the lowest forward and side scatter. From this parent gate, create a new dot plot that displays side scatter versus DAPI and gate on the DAPI negative cells. On this newly gated population, create a dot plot that displays CD11b versus CD45, and gate on the CD11b CD45 double positive macrophages and neutrophils.Then create a final dot plot display Ly6C versus Ly6G to allow gating of the desirable population and sort the cells. At the end of the sort, confirm the purity and viability of the samples. Collect the sorted cells by centrifugation, and re-suspend the pellet for counting in fresh RPMI medium.Dilute the macrophage populations to the appropriate concentration for seeding five times 10 to the fourth macrophages per 100 microliters of complete medium per well, in a 96 well round-bottom plate. Then place the cells in a cell culture incubator for at least 24 hours at 37 degrees Celsius and 5%CO2. To isolate the T, put the knock-in mouse in the supine position.First make a mid-line skin incision, carefully cutting the peritoneum and gently spreading apart the skin. Collect the peripheral lymph nodes and the spleen, and place the tissues in a 100-micrometer filter on top of a 50-milliliter conical tube. Using the plunger of a 1-milliliter syringe, gently press the lymphoid tissues through the filter, and rinse the filter with fresh RPMI medium until the mesh is clean.Collect the single-cell suspension by centrifugation, and re-supsend the pellet in two milliliters of ACK lysis buffer. After five minutes at four degrees Celsius, neutralize the lysis buffer with two milliliters of fresh RPMI medium, and centrifuge the cells. Re-suspend the pellet in 200 microliters of fresh medium, and transfer the cells to a five-milliliter round-bottom tube for straining with the appropriate T-cell surface-staining antibodies.After 45 mintues at four degrees Celsius protected from light, wash the cells two times in fresh RPMI medium and count the cells by trypan blue exclusion. Next, dilute the CD8-stained cells to a one times 10 to the sixth cells per milliliter concentration in CFSE for five minutes in a 20-degree Celsius water bath. At the end of the incubation, neutralize the CFSE with two milliliters of RPMI medium, and collect the cells by centrifugation.Re-suspend the cells at one times 10 to the sixth CD8-stained T-cells per milliliter of RPMI medium concentration, and filter all of the cell samples through a 70-micrometer cell strainer into five-milliliter round-bottom tubes. Then label the cells with DAPI and prepare the flow cytometer for sorting as just demonstrated. For CD4 positive T-cell isolation, set up a forward versus side scatter dot plot for gating on the lymphocytes, excluding the debris as well as the large granular myeloid cells.From this parent gate, create a dot plot that displays side scatter versus DAPI, and gate on the DAPI negative cells. On this newly-gated population, create one dot plot that displays side scatter versus CD4, and one dot plot that displays CD8 versus CFSE. Then sort the gated T-cell populations.At the end of the sort, confirm the purity and viability of the samples, and collect the cells by centrifugation for counting. Dilute the cells to a two times 10 to the sixth T-cells per milliliter of RPMI concentration, and 100 microliters of T-cells in complete RPMI medium to each well of macrophages for a 96-hour co-culture at 37 degrees Celsius and 5%CO2. Finally, add five microliters PBS wash T-cell activator CD3 CD28 magnetic beads to each well to activate and expand the T-cells.Ly6Clo Ly6G-macraphages obtained from tolerized recipients exert a suppressive effect on CD8 positive T-cells, with a modest suppressive activity also observed from Ly6C intermediate Ly6CG positive cells. Only Ly6Clo Ly6G-cells promote the in vitro expansion of CD4 positive Foxp3 positive TReg, however. Together, these data support the conclusion that graft infiltrating CD11B positive Ly6Clo Ly6G negative macrophages possess many of the properties reported to be associated with monocyte-derived suppressor cells, including their ability to inhibit CD8 T-cell proliferation and to promote CD4 positive Foxp3 positive TReg expansion.While attempting this procedure, it's important to maintain the purity in order to establish conclusive data about the suppressive capacity of each macrophage subset. Following this procedure, other methods like using commercial enrichment gates can be used for isolating different leukocyte populations of interest. Macrophage sorting is the longest part of this procedure, but when it has been mastered, this part of the technique can be complete in eight hours if performed properly.After its development, this technique paved the way for researchers in the field of immunology to explore the suppressive capacity of infiltrating macrophages in solid organ transplanting sim model. After watching this video, you should have a good understanding of how to characterize different macrophages population in selected from mouse heart allograph.

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JoVE Journal

Immunology and Infection

8.1K Görüntüleme.  Icahn School of Medicine at Mount Sinai. The overall goal of this in vitro procedure is to phenotypically and functionally characterize macrophage populations isolated from allografted hearts in a mass transplantation model. These methods can help answer key questions in the immunity we find about how to improve success in allograph survival and tolerance. The main advantage of this technique is that it allows the study of how a specific tissue infiltrated cardiac allograph isolated microphages subset modulate host tissues response....