Detection of Circulating and Tissue MDSC by Flow Cytometry Protocol

Introduction of Circulating and Tissue MDSC Test by FACS

Myeloid-derived suppressor cells (MDSC) have been a target for basic and clinical studies related to chronic inflammatory diseases such as autoimmunity, chronic obstructive pulmonary disease (COPD), transplantation, infections, and cancer. In cancer, MDSC play an important role in limiting immune responses and their accumulation is associated with tumor growth, deficient response to immunotherapy, and poor survival. Flow cytometry is a powerful tool for detailed analysis of MDSC. Although surface markers have been described and extensively utilized for the identification of MDSC in human samples as well as in mouse models of research, the strategy for isolating and analyzing those cells by flow cytometry differs between research groups.

The precise identification and phenotypification of MDSC are crucial for understanding their role in the pathophysiology and predict the outcome of the disease. MDSC are divided into three major subtypes based on their cell surface phenotype: granulocytic polymorphonuclear MDSC (PMN-MDSC), monocytic MDSC (M-MDSC), and early-stage MDSC (e-MDSC). Human PMN-MDSC are defined as CD11b^dim(or alternatively CD33^dim), CD14^neg, CD66b^pos(or CD15^pos), HLA-DR^neg whereas M-MDSC are CD11b^high, CD14^pos, HLA-DR^neg/Low, CD66b^neg. Early-stage MDSC (e-MDSC), a less characterized subtype is CD11b^dim, CD14^neg, CD66b^neg, HLA-DR^neg. Mouse MDSC are identified by expressing CD11b and Gr1, which is comprised of two components, Ly6C and Ly6G. The mouse PMN-MDSC subset is identified as CD11bpos, Gr1^pos, Ly6C^dim, Ly6G^pos, while M-MDSC are CD11b^pos, Gr1^pos, Ly6C^high, Ly6G^neg. The minimum criteria for defining MDSC include these phenotypic surface markers; however, to fully define these cells as MDSC, assessing the functional and, if possible, biochemical and molecular features should be incorporated. Despite the cost, the long period of coculture, and thereafter the evaluation of T cell response, functional analysis of the immunoregulatory activity of MDSC on T cell proliferation is frequently performed in research with rodents. Unfortunately, it is less common with human samples for practical reasons, for example, most likely insufficient amount of samples to purify MDSC and T cells.

MDSC are functionally suppressor cells by producing antiinflammatory cytokines, arginase-1 and reactive oxygen species (ROS) such as superoxide. Also, MDSC express inducible nitric oxide synthase (iNOS) and programmed death-ligand 1 (PD-L1), among other molecules.

Arginase-1 is an enzyme that converts L-arginine into L-ornithine and urea, leading to the depletion of L-arginine. The absence of this amino acid impairs the proliferation and function of T and NK cells abrogating the protective immune responses. ROS critically affects the functions of T cells, their proliferation, differentiation, and survival.

Here we present a protocol to identify and quantify MDSC from peripheral blood and dissociated tissue (e.g., tumor) from human and mouse by using a panel of antibodies that collects the minimal recommended surface markers to characterize the phenotypic features of MDSC. This panel includes fluorescently conjugated antibodies against CD3, CD19, CD20, CD56 (lineage cocktail), CD45, CD33, HLA-DR, CD66b, and CD14 in combination with anti-arginase-1 or with the fluorescent probe dihydroethidium (DHE) to indicate cellular superoxide content. With this immunophenotypic analysis, the presence of MDSC with potential suppressive capacity can be evaluated by flow cytometry. Also, this approach can be used as the basis for developing additional panels where can be included other relevant molecules such as PD-L1, LOX-1, iNOS, as well as additional ROS indicators such as mitochondrial O2^-(Mitosox) or intracellular H₂O₂ (DCFDA).

Methods of Circulating and Tissue MDSC Test by FACS

Isolation of Peripheral Blood Mononuclear Cells (PBMC)

1. Draw peripheral blood on a vacutainer tube containing K2EDTA (see Note 6).
2. Dilute blood 3 times with 1XPBS.
3. Carefully layer 35 mL of diluted human blood over 15 mL of Ficoll-Paque in a 50 mL conical tube, or 4 mL of diluted mouse blood over 2 mL of Ficoll-Paque in a 15 mL conical tube. The layering must be performed very slow in drop motion. Blood and Ficoll-Paque should stay as two different layers.
4. Centrifuge at 400Xg for 20 min at room temperature (RT) in a swinging-bucket rotor without brake (ascending rate: 1–3; descending rate: 1–3).
5. Aspirate the monolayer (PBMC) formed in the interphase between Ficoll and Plasma/PBS and transfer it to a new 50 mL tube.
6. Wash cells by adding 20 mL of 1XPBS to the collected PBMC monolayer. Mix and centrifuge at 300Xg for 10 min at RT. Carefully remove completely the supernatant.
7. Add 2 mL ACK lysing buffer. Gently resuspend the cells by pipetting, and incubate for 3–5 min at RT.
8. Add 10 mL of 1XPBS, mix, centrifuge (300–400Xg for 10 min at RT), and decant the supernatant.

Figure 1. Schematic illustration of isolating of PBMC from whole peripheral blood by a density gradient centrifugation using Ficoll-Paque and tissue/tumor infiltrating-leukocytes by discontinuous Percoll gradient.

9. Visually evaluate the absence of red cells in the pellet of PBMC. If red cells are still present, repeat steps 7 and 8.
10. Decant the supernatant and resuspend the pellet in 5–10 mL of washing buffer (see Note 7).
11. Quantify viable cells on a hemocytometer or automated cell counter by using trypan blue-stained cell suspension.

Mechanical and Enzymatic Dissociation for Extraction of Immune Cells from Solid Tissues

1. Place the tissue into a 100 mm petri dish, record the tissue weight (to calculate absolute number per gram of weight) and add half of the dissociation medium for tissue digestion (0.5 mL per gram of tissue).
2. Mince the tissue with scissors or scalpel into small fragments.
3. Transfer to 50 mL Falcon tube and rinse the petri dish with half of the dissociation medium (0.5 mL per gram of tissue) and transfer to the tube. The total volume of media should be 1 mL per gram of tissue.
4. Incubate for 30 min at 37°C (ideally under continuous rotation or mixing every 5 min; see Note 8).
5. After incubation, gently pipette up and down with 1 mL pipette. Transfer the suspension to 70–100 μm nylon mesh strainer and complete the dissociation by using a 3 mL syringe plunger and gently completely disrupt the tissue.
6. Pass the cellular suspension through the nylon mesh strainer into a 50 mL Falcon tube.
7. Wash cells twice with 20 mL 1XPBS and pellet the cells by centrifugation at 300–400Xg for 10 min.
8. To enrich leukocytes from unwanted cells such as epithelial, stroma cells, and cell debris in the cellular suspension after tissue dissociation, two Percoll cell separation gradient is recommended. Suspend the cells in 15 mL 40% Percoll, mix well and then very carefully add 15 mL 80% Percol at the bottom of the 50 mL falcon tube. The 80% Percoll will displace the 40% Percoll and an interface between the two layers is visible.
9. Centrifuge at 325Xg at RT for 23 min without brake (ascending rate: 5; descending rate: 5).
10. Collect the cells at the interface between 40% and 80% Percoll.
11. Wash cells three times by adding 5 volumes 1XPBS to 1 volume of Percoll cell suspension and pellet the cells by centrifugation at 300Xg for 10 min.
12. Suspend cells in 2–5 mL washing buffer and keep on ice.
13. Quantify viable cells on a hemocytometer or automated cell counter by using trypan blue-treated cell suspension.

Staining Cells with Viability Dye (See Note 9)

1. Transfer 1X10⁶ PBMC to a flow cytometry tube passing through the flow cytometry cap with 35 μm strainer mesh. Centrifuge and resuspend the cells in 1 mL PBS containing viability stain. For optimal performance, the concentration of the dye must be optimized by titrating the dye. In general: 1 μL is added for every 1 mL of cell suspension (dilution 1:1000).
2. Vortex immediately and incubate the mixture for 5–7 min at 37°C, 10–15 min at room temperature, or 30–60 min at 2–8°C protected from light.
3. Wash cells twice with 2 mL of staining buffer by centrifuging at 300–400Xg for 5 min at 4°C.
4. Decant the supernatant and gently loosen the cell pellet by vortexing briefly.

Surface Markers Staining with Fluorochrome- Conjugated Antibodies

1. After staining cells with the viability dye, resuspend the cells in 50 μL of staining buffer containing Fc Receptor Binding Inhibitor (diluted at 1:50 ratio) and incubate for 5 min. See Note 10.
2. Add 50 μL of Brilliant Stain Buffer Plus containing the antibody cocktail (in a concentration of 0.1 to 10 μg/mL per antibody, defined during the standardization step by titrating the antibodies; see in Note 11). Include the necessary additional tubes for nonspecific control staining by matching fluorescent Ig isotype control or FMO (see Notes 12 and 13).
3. Incubate for 30 min at 4°C protected from light.
4. Wash the cells with 1–2 mL of washing buffer by centrifugation at 300–400Xg for 5 min at 4°C.
5. Resuspend cell in 150 μL of ice-cold washing buffer and keep on ice to avoid free-floating DNA fragments and to reduce cell clumping.
6. Keep the cells at 4 °C in the dark until the time for the collection of data in the flow cytometer (a process termed "acquisition").
7. If the acquisition is not performed immediately and overnight storage is required, preserve cells by resuspending them in 500 μL of 1% paraformaldehyde or stabilizing fixative (see Note 14) and incubate for 30–60 min at 4°C, wash and store the fixed cells in 250 μL staining buffer at 4°C until acquisition analysis.

Staining for Intracellular Arginase-1

The detection of intracellular proteins such as arginase-1 (see Note 15) by flow cytometry requires cell fixation and permeabilization (see Note 16).

1. After the staining of cell surface markers is completed (steps 1– 6), wash one more time with 2 mL of staining buffer and centrifuge cells at 350Xg for 5 min.
2. Decant the supernatant and loosen the cell pellet by vortexing briefly.
3. Add 1 mL of freshly prepared 1XFix/Perm buffer working solution to the tube and mix by vortexing for approximately 3 s. Incubate samples at 4°C for 40 min protected from light.
4. Add 1 mL of 1XPerm/Wash buffer directly to the cells and pellet the cells by centrifugation at 400Xg and 4°C for 7–10 min. Decant the supernatant.
5. Wash the cells again by adding 2 mL of 1XPerm/Wash buffer to the cells, centrifuge, and decant the buffer.
6. Add 100 μL of 1_ Perm/Wash Buffer containing the fluorescent antibody anti-Arginase-1 or the staining control (e.g., matching fluorescent Ig isotype or FMO, see Note 12) to cell samples. Mix by vortexing for 10 s and incubate at 4°C for 45–60 min protected from light (see Note 17).
7. After incubation, vortex samples for 3 s and wash twice by adding 2 mL of 1XPerm/Wash Buffer. Centrifuge cells and decant the buffer.
8. Resuspend the cell pellet in 200 μL of PBS or staining buffer. Acquire data using a flow cytometer.

ROS Detection by Staining with Dihydroethidium (DHE) Indicator

1. Incubate 1X10⁶ cells with prewarmed 2.5 μM DHE in RPMI at 37°C for 10 min.
2. Wash twice with 2 mL of 1XPBS.
3. Proceed for staining the surface markers.

Acquisition of Data in the Flow Cytometer and Analysis

1. Identify the myeloid cell population by its forward-scatter (indicates cell size) and side-scatter (granularity or complexity) characteristics.
2. Determine the level of autofluorescence of the cell suspension and set the voltages by using unstained cells as a negative population.
3. Distinguish cells from debris and cell doublets (double discrimination) to prevent false-positive events by gating according to side-scatter and forward-scatter.
4. Identify alive cells by gating on the negative population according to the viability dye.
5. Apply the gating strategy set by using FMO controls (see Note 12).
6. Generate a compensation matrix by using samples stained with a single fluorochrome. Single staining will allow to detect the level of spectral overlap between different fluorophores to be used and reveals which overlap needs to be compensated.
7. Distinguish myeloid population by gating off the lineage cocktail marker (optional).
8. Display in a density or dot plot the combination of the following markers. For human PBMC: HLA-DR vs CD33, and CD66b vs CD14 and gate the populations (see Note 13). For mouse cells: CD45 vs side-scatter, CD11b vs Gr1, and Ly6C vs Ly6G.
9. Acquire the appropriate number of events. Since the number of MDSC in PBMC vary subject-to-subject, starting from a very low percentage as 0.1 the acquisition of a large number of events is required to have a statistically significant positive population.

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