Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

Summary

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.

Abstract

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.

Introduction

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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Protocol

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% penicillin-streptomycin (10,000 U/mL) by using 2 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 5 mM HEPES, and 0.05 mM 2-mercaptoethanol.

2. Allograft Isolation and single-cell Suspension

NOTE: The transplantation and anastomosis technique of the pulmonary artery and inferior cava vein was initially described by Corry and collaborators and can be visualized in Liu & Kang^10,11 (Figure 1A).

1. Anesthetize a transplanted mouse with 4-5% isoflurane in an induction chamber. Sacrifice it by cervical dislocation.
2. Make a midline abdominal incision with standard scissors and remove the abdominal contents to localize the aorta.
   NOTE: The transplanted heart will be on the right side of the recipient abdominal aorta (Figure 1B).
3. Pull out the graft gently using fine sharp-teeth forceps and place it immediately into ice-cold RPMI medium.
   NOTE: Using a surgery scissor to separate the graft from the aorta can damage the graft and cause some tissue to remain adhered to the recipient.
4. After all grafts have been collected, move them into a sterile culture hood for tissue processing. Place the graft in a petri dish and dice the tissue into small pieces (1 mm) using sterile, blunt, microsurgery scissors.
5. With sharp-teeth forceps, transfer the pieces to a 50 mL tube with 5 mL of collagenase A (0.1 mg/mL collagenase in sterile 1x PBS). Incubate it for 1 h in a 37 °C bath.
6. Add 5 mL of RPMI medium to neutralize the collagenase and transfer the sample through a 100 µm strainer with the help of the plunger of a 1 mL syringe.
7. Spin the sample down at 400 x g for 5 min at 4 °C.
8. Resuspend the pellet in 1 mL of ACK lysis buffer. Mix well and incubate for 5 min at 4 °C.
9. Add 1 mL of RPMI medium to neutralize the lysis buffer and spin the sample down at 400 x g for 5 m at 4 °C.
10. Resuspend the cell pellet in 200 µL of RPMI medium and transfer it to a 5 mL polypropylene round-bottom tube.
    NOTE: Polypropylene tubes support less adherence. Also, maintaining the cells at low temperature, such as 4 °C, reduces adhesion.

3. Isolation of Graft-infiltrating Macrophages Using Fluorescence-activated Cell Sorting

1. Block unwanted specific binding on myeloid cells by using Fc receptor blocking mAb (rat anti-mouse CD16/32). Add 1-2 µL per sample, 15 min prior to surface staining.
2. Stain with anti-mouse CD11b Percp/Cy5.5 (0.6 µg/µL final concentration in the RPMI medium), anti-mouse CD45 APC/eFluor780 (0.6 µg/µL), anti-mouse Ly6C APC (2 µg/µL), and anti-mouse Ly6G Pe/Cy7 (2 µg/µL). Cover the tubes with aluminum foil to protect the fluorescent antibodies from light and incubate the tubes in the refrigerator at 4 °C for 45 min.
   NOTE: For single-stain compensations, prepare a negative control tube (no stain) and tubes with cells labeled singly with each of the fluorochromes. To help set up the gate, isotype controls can be used especially for Ly6C (IgG2a) and Ly6G (IgG2b).
3. Wash the cells twice with RPMI medium and spin them at 400 x g for 5 min at 4 °C. Count the cells using trypan blue and a hemocytometer and dilute them in 1 mL of RPMI medium per 1 x 10⁶ cells. Before sorting, transfer the samples through a 5 mL, 70 µm cell strainer tube cap. Add DAPI (1 µg/mL) as a cell viability marker and proceed to sort.
4. Because macrophages are sensitive and fragile cells, set the sort conditions to 20 psi and use a 100 µm nozzle size to isolate macrophages using a 4-way purity mode.
5. Prepare collection tubes with 1 mL of RPMI medium in a 5 mL polypropylene tube.
6. Using the sorter software open new experiment and select blank experiment with a blank panel to define settings.
7. Set up a dot plot that displays the forward (FSC) versus (vs.) side scatter (SSC) and gate on all leukocytes, excluding debris and clumps with the lowest forward and side scatter. From this parent gate, create a new dot plot that displays SSC vs. DAPI and gate on DAPI cells.
   1. On this newly-gated population, create a dot plot that displays CD11b vs. CD45 and gate on double-positive (CD11b⁺ CD45⁺) macrophages and neutrophils.
   2. From here, create a final dot plot displaying Ly6C vs. Ly6G and gate the desirable populations: Ly6C^hi Ly6G^-, Ly6C^loLy6G^-, and Ly6C^intLy6G⁺ (Figure 2).
8. After sorting, check for purity and cell viability (>90%) and spin the collection tubes at 400 x g for 5 min at 4 °C. Count the cells using trypan blue and a hemocytometer and resuspend them at the desired concentration (e.g., 1 x 10⁶ macrophages/mL) in complete RPMI medium.
9. Plate 50 x 10³ macrophages per well in a 96-well round-bottom plate with a final volume of 100 µL of complete RPMI medium. Leave the cells undisturbed for at least 24 h at 37 °C and 5% CO₂.

4. Isolation of T Cells Using Fluorescence-activated Cell Sorting

NOTE: C57BL/6-Foxp3tm1Flv/Jn is an X-linked targeted knock-in mouse strain that co-marks cells expressing the Foxp3 (forkhead box P3) gene with monomeric red fluorescent protein (mRFP).

1. Anesthetize and sacrifice C57BL/6 and C57BL/6-Foxp3tm1Flv/J (H-2b) mice as previously described in step 2.1. Isolate the spleen and lymph nodes (LN: inguinal, lumbar, axillar, brachial, and cervical) and rapidly place them in ice-cold RPMI medium.
2. In order to isolate the LN, place the mouse in a supine position, make a midline skin incision from bottom to top, carefully cutting the peritoneum and gently spreading the skin apart. Once all inguinal, lumbar, axillary, brachial, and cervical LN are collected (Figure 1C, colored in red), cut the peritoneum and isolate the spleen at the upper left side of the gut.
3. Disaggregate the spleen and LN using a 100 µm filter placed on top of a 50 mL tube. Using the plunger of a 1-mL syringe, gently press the tissue. Rinse the filter with RPMI medium as many times as necessary until it is clean.
   NOTE: LN and spleen from each mouse can be pooled together in the same tube.
4. Spin down at 400 x g for 5 min at 4 °C.
5. Resuspend the pellet in 2 mL of ACK lysis buffer. Mix well and incubate for 5 min at 4 °C.
6. Add 2 mL of RPMI medium to neutralize the lysis buffer. Spin down at 400 x g for 5 min at 4 °C.
7. Resuspend the cell pellet in 200 µL of RPMI medium and transfer it to a 5 mL polystyrene round-bottom tube.
8. Stain for anti-mouse CD4 APC (0.6 µg/µL) and/or anti-mouse CD8 PeCy7 (2 µg/µL). Cover the tubes with aluminum foil to protect the fluorescent antibodies from light, and incubate the tubes in the refrigerator at 4 °C for 45 min.
   NOTE: For single-stain compensations, prepare a negative control tube (no stain) and tubes with cells labeled singly with each of the fluorochromes.
9. Wash the cells twice with RPMI medium and spin them at 400 x g for 5 min at 4 °C. Count the cells using trypan blue and a hemocytometer.
   NOTE: CFSE dye is provided as carboxyfluorescein diacetate succinimidyl ester powder usually at 50 µg. Add 18 µL DMSO to a vial of CFSE for a final stock solution of 5 mM and store at -20 °C for several months.
10. For CFSE labeling, resuspend CD8 stained cells at a concentration of up to 10⁶ cells per mL in PBS at a final working concentration of 5 µM CFSE from the stock solution. Incubate them in a bath at 20 °C for 5 min as previously described⁶.
    NOTE: (CRITICAL STEP) For cells at a low concentration (≤10⁶ per mL), it is essential that the cells be labeled in the presence of added protein to buffer the toxic effects of CFSE. Therefore, PBS can contain 5% FBS. Note that if medium is used, free amino acids may lower the labeling efficiency by competing for CFSE.
11. Neutralize the CFSE with 2 mL of RPMI medium. Spin at 400 x g for 5 min at 4 °C. Add 1 mL of RPMI medium per 1 x 10⁶ CD8 T cells.
12. Before sorting, transfer the sample through a 70 µm cell strainer on a 5 mL tube cap. Add 50 µL of 1x DAPI (1 µg/mL) as a cell viability marker and proceed to sorting.
    NOTE: 1x means 1:1 ratio of the reagent with respect other constituent.
13. Set the sort conditions at 20 psi and 100 µm nozzle size to isolate double-positive CD8⁺CFSE⁺ T cells (Figure 3A).
14. Prepare collection tubes with 1 mL of RPMI medium in a 5 mL polypropylene tube and proceed to sort.
15. Using the sorter software open new experiment and select blank experiment with a blank panel to define settings.
16. For CD4⁺ T cell isolation, set up a dot plot that displays the FSC vs. SSC and gate on lymphocytes, excluding debris as well as large granular cells, such as dendritic cells.
    1. From this parent gate, create a new dot plot that displays SSC vs. DAPI and gate on DAPI cells.
    2. On this newly-gated population, create a dot plot that displays SSC vs. CD4 to isolate all CD4⁺ cells (Figure 3B). Alternatively, an anti-CD3 mAb can be used to ensure isolation of pure T cell populations.
       NOTE: A small fraction of all CD4⁺ T cells (5-10%) should be Foxp3⁺mRFP⁺.
17. After sorting, check for purity and cell viability (>90%) and spin the collection tubes at 400 x g for 5 min at 4 °C. Count the cells using trypan blue and a hemocytometer. Resuspend using 1 mL of complete RPMI medium per 2x10⁶ T cells.
18. Set up suppression assay by culturing T cells with previously-sorted macrophages. Plate 10 x 10⁴ CD4⁺ T and 10 x 10⁴ CD8⁺CFSE⁺ T cells in the same well in a final volume of 100 µL of complete RPMI medium. Incubate at 37 °C and 5% CO₂ for 96 h.
    NOTE: Macrophages and T cells were used in a 1:4 ratio.
19. Stimulate T cell division by washing T cell-activator CD3/CD28 magnetic beads in 2 mL of PBS before using them in order to clear the azide-containing buffer. Add 5 µL of mouse T cell-activator CD3/CD28 beads per well.
    NOTE: Magnetics beads are similar in size to antigen-presenting cells and are coupled to anti-CD3 and anti-CD28 mAb, offering a simple method for the activation and expansion of T cells.

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Results

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 Ly6C^loLy6G^- macrophages obtained from tolerized recipients are suppressive. The results also indicate that Ly6C^int...

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Discussion

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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Materials

Name	Company	Catalog Number	Comments
RPMI 1640 Media	Life Technologies	11875119
10% FBS	Life Technologies	26140-079
1,000x 2-mercaptoethanol	Life Technologies	21985023
100x Pen/Strep	Life Technologies	15140122
100x L-glutamine	Life Technologies	25030081
100x Non Esential amino acids	Corning	25025039
1 M HEPES buffer	Corning	25060CL
100 mM Sodium Pyruvate	ThermoScientific	SH30239.01
Penicilin/Streptavidyn	ThermoScientific	15140122
Collagenese A	Roche	70381322
DPBS (w/o calcium&magnesium)	Corning	21031CV
70 µm 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