Flow Cytometric Analysis for Identification of the Innate and Adaptive Immune Cells of Murine Lung

Summary

In this study, we present an effective and reproducible protocol to isolate the immune populations of the murine respiratory system. We also provide a method for the identification of all innate and adaptive immune cells that reside in the lungs of healthy mice, using a 9-color-based flow cytometry panel.

Abstract

The respiratory tract is in direct contact with the outside environment and requires a precisely regulated immune system to provide protection while suppressing unwanted reactions to environmental antigens. Lungs host several populations of innate and adaptive immune cells that provide immune surveillance but also mediate protective immune responses. These cells, which keep the healthy pulmonary immune system in balance, also participate in several pathological conditions such as asthma, infections, autoimmune diseases, and cancer. Selective expression of surface and intracellular proteins provides unique immunophenotypic properties to the immune cells of the lung. Consequently, flow cytometry has an instrumental role in the identification of such cell populations during steady-state and pathological conditions. This paper presents a protocol that describes a consistent and reproducible method to identify the immune cells that reside in the lungs of healthy mice under steady-state conditions. However, this protocol can also be used to identify changes in these cell populations in various disease models to help identify disease-specific changes in the lung immune landscape.

Introduction

The murine respiratory tract contains a unique immune system responsible for fighting pathogens and maintaining immune homeostasis. The pulmonary immune system consists of cellular populations with significant heterogeneity in their phenotype, function, origin, and location. Resident alveolar macrophages (AMs), originated mainly from fetal monocytes, reside in the alveolar lumen¹, while bone marrow-derived interstitial macrophages (IMs) reside in the lung parenchyma². IMs can be further subclassified by the expression of CD206. CD206⁺ IMs populate the peribronchial and perivascular area, while CD206^- IMs are located at the alveolar interstitium³. A few subclassifications of IMs have been proposed recently^3,4,5,6. Although IMs are less studied than AMs, recent evidence supports their crucial role in the regulation of the immune system of the lung⁷. In addition, CD206 is also expressed in alternatively activated AMs⁸.

Pulmonary dendritic cells (DCs) are another heterogeneous group of lung immune cells with respect to their functional properties, location, and origin. Four subcategories of DCs have been described in the lung: conventional CD103⁺ DCs (also known as cDC1), conventional CD11b⁺ DCs (also known as cDC2), monocyte-derived DCs (MoDCs), and plasmacytoid DCs^{9,10,11,12,13}. The first three subclasses can be defined as major histocompatibility complex (MHC) II⁺CD11c^+9,10,14,15. Plasmacytoid DCs express MHC II and are intermediately positive for CD11c but express high levels of B220 and PDCA-1^9,13,16. In naïve murine lungs, CD103 DCs and CD11b DCs are located in the airway interstitium, whereas plasmacytoid DCs are located in the alveolar interstitium¹⁷.

Two major populations of monocytes reside in the lung during steady state: classical monocytes and non-classical monocytes. Classical monocytes are Ly6C⁺ and are critical for the initial inflammatory response. In contrast, non-classical monocytes are Ly6C^- and have been widely viewed as anti-inflammatory cells^3,16,18. Recently, an additional population of CD64⁺CD16.2⁺ monocytes was described, which originate from Ly6C^- monocytes and give rise to CD206⁺ IMs³.

Eosinophils mainly appear in the lungs during helminth infection or allergic conditions. However, there is a small number of eosinophils in the pulmonary parenchyma during steady state, known as resident eosinophils. In contrast to the resident eosinophils, inflammatory eosinophils are found in the lung interstitium and bronchoalveolar lavage (BAL). In mouse models of house dust mite (HDM), inflammatory eosinophils are recruited into the lung after antigen-mediated stimulation. It has been proposed that resident eosinophils might have a regulatory role in allergy by inhibiting T helper 2 (Th2) sensitization to HDM¹⁹.

In contrast to the rest of pulmonary myeloid cells, neutrophils express Ly6G but not CD68 and are characterized by a signature of the CD68^-Ly6G⁺ immunophenotype^16,20,21. Visualization studies have shown that during steady state, the lung reserves a pool of neutrophils in the intravascular compartment and hosts a considerable number of extravascular neutrophils²². Similar to eosinophils, neutrophils are not found in BAL at steady state; however, several forms of immune stimulation, such as LPS challenge, asthma, or pneumonia, drive neutrophils into the alveolar lumen, resulting in their presence in BAL^21,22,23.

A substantial number of CD45⁺ cells of the lung represent natural killer (NK), T cells, and B cells and are negative for most myeloid markers²⁴. In the lungs of naïve mice, these three cell types can be identified based on the expression of CD11b and MHC II¹⁸. Around 25% of pulmonary CD45⁺ cells are B cells, whereas the percentage of NK cells is higher in the lung than other lymphoid and non-lymphoid tissues^24,25,26. Among pulmonary T cells, a considerable fraction is CD4^-CD8^- and plays an important role in respiratory infections²⁶.

Because the lung hosts a very complex and unique immune system, several gating strategies for the identification of lung immune cells have been developed and reported^16,18,20,27. The gating strategy described herein provides a comprehensive and reproducible way to identify up to 12 different pulmonary myeloid and non-myeloid immune populations using 9 markers. Additional markers have been used to validate the results. Furthermore, a detailed method is provided for the preparation of a single-cell suspension that minimizes cell death and allows the identification of the most complete profile of the immune cell compartment of the lung. It should be noted that the identification of non-immune cells of the lung, such as epithelial cells (CD45^-CD326⁺CD31^-), endothelial cells (CD45^-CD326^-CD31⁺), and fibroblasts requires a different approach^28,29. Identification of such populations is not included in the protocol and method described here.

Protocol

All studies and experiments described in this protocol were conducted under guidelines according to the Institutional Animal Care and Use Committee (IACUC) of Beth Israel Deaconess Medical Center. Six to ten weeks old C57BL/6 mice of either sex were used to develop this protocol.

1. Surgical excision and tissue preparation

1. Euthanize the mouse by intraperitoneally injecting 1 mL of tribromoethanol (prepared according to standard protocol; Table of Materials).
   NOTE: CO₂ asphyxiation should be avoided in lung studies as it might cause lung injury and alter the features and properties of lung immune cells. Cervical dislocation should also be avoided as it might cause mechanical injury of the lung.
2. Transfer the mouse to a clean and dedicated area for surgical operation.
3. Stabilize the mouse dorsal side down by using needles or tape on the four extremities. Use 70% ethanol to sanitize the skin of the ventral area.
4. Perform an incision in the skin, from the neck to the abdomen. Carefully remove the skin from the thoracic area.
5. Carefully remove the sternum and ribs.
6. Flush the lungs by injecting 10 mL of cold PBS directly in the right ventricle, using an 18-21 G needle, until the lungs become completely white.
7. Carefully remove the thymus and heart without touching the lungs.
8. Gently detach the lungs from the surrounding tissues and transfer them to a tube with cold BSA buffer (Table 1).
   NOTE: Effort should be made to remove all adjacent fat from the lungs before further preparing the single-cell suspension, as this could bias the readouts.

2. Preparation of single-cell suspension

1. Transfer the lungs to an empty Petri dish and mince them with two fine scalpels. Transfer all the pieces of the minced lung to a new 50 mL conical tube. Use 5 mL of digestion buffer to wash the plate and add it to the 50 mL tube containing the minced lung (Table 1).
   NOTE: Digestion buffer should be prepared immediately before use. Use 5 mg/mL of collagenase²⁸. Combining 1 or 5 mg of collagenase with BSA buffer or protein-free PBS did not improve results (Supplemental Figure S1).
2. Secure the lid of the tube and digest the lung for 30 min on an orbital shaker at a speed of 150 rpm at 37 °C. Stop the reaction by adding 10 mL of cold BSA buffer.
3. After digestion, use an 18 G needle to mix and dissolve the lung pieces. Place a 70 µm filter strainer at the top of a new 50 mL conical tube.
   NOTE: Usage of a smaller micron filter might result in the loss of major myeloid populations.
4. Slowly transfer the digested lung mixture directly on the strainer. Use the rubber side of a 10 mL syringe plunger to smash the remaining lung pieces on the filter. Wash the processed material on the filter with BSA buffer.
5. Centrifuge the single-cell suspension at 350 × g for 8 min at 4 °C.
6. Carefully discard the supernatant and resuspend the cells in 1 mL of ACK lysis buffer. Mix well using a 1 mL pipet, and incubate for 90 s at room temperature.
7. Add 10 mL of cold BSA buffer to stop the reaction and centrifuge at 350 × g for 7 min at 4 °C.Carefully discard the supernatant and resuspend the pellet in Staining Buffer to count the cells using a hemocytometer.
8. Resuspend the cells at a concentration of 5 × 10⁶ cells/mL and use them for surface staining (see section 3).
   NOTE: For this purpose, plate the cells in a 96-well round-bottom plate followed by antibody staining and washes. If a plate centrifuge is not available, use flow tubes instead of plates. With this protocol, ~15-20 × 10⁶ cells per lung can be obtained from a 6-10-week-old C57BL/6 mouse of average size.

3. Surface antibody staining

1. Transfer 1 × 10⁶ cells in 200 µL per well in a 96-well plate. Centrifuge the plate at 350 × g for 7 min at 4 °C. In the meantime, prepare the Fc-block solution by diluting anti-16/32 antibody (1:100) in staining buffer (Table 1).
2. Resuspend the cells in 50 µL of the pre-prepared Fc-blocking solution (Table of Materials) and incubate for 15-20 min at 4 °C or on ice.
3. Add 150 µL of staining buffer and centrifuge the plate at 350 × g for 5 min at 4 °C. Meanwhile, prepare the surface antibody cocktail by diluting surface antibodies (1:100; Table 2) in staining buffer.
   NOTE: (i) Anti-16/32 antibody for Fc-blocking can be used with the surface antibodies in the same mixture. (ii) If fixable viability dye is used, add it to the surface antibody cocktail at a dilution of 1:1,000.
4. Resuspend the cells in 50 µL of the pre-prepared surface antibody cocktail and incubate for 30-40 min at 4 °C in the dark. Wash the cells with staining buffer twice.
   ​NOTE: If no intracellular staining is required, resuspend the cells in 200 µL of staining buffer and proceed directly to the acquisition of data on the flow cytometer. Alternatively, cells might be fixed and stored at 4 °C for acquisition later. We recommend using the cells for flow cytometry within 24 h.

4. Cell fixation and intracellular staining

1. Prepare the fixation/permeabilization buffer (Fix/Perm Buffer) by mixing three parts of fixation/permeabilization concentrate and 1 part of fixation/permeabilization diluent of the FoxP3/Transcription Factor Staining Buffer Set (Table 1).
2. Resuspend the cells in 50 µL of the pre-prepared Fix/Perm Buffer per well of the 96-well plate, where cells were plated as described in section 3, and incubate them for 20-25 min at 4 °C in the dark.
3. Dilute the 10x permeabilization buffer as 1: 10 in purified deionized water to prepare 1x permeabilization buffer.
4. Wash the cells once with 1x permeabilization buffer. Meanwhile, prepare the intracellular antibody cocktail by diluting intracellular antibodies (1:100) in 1 mL of permeabilization buffer.
5. Resuspend the cells using 50 µL of the pre-prepared surface antibody cocktail per cell of the 96-well plate and incubate for 40 min at 4 °C in the dark.
6. Wash the cells once with permeabilization buffer and once with staining buffer. After the final wash, resuspend the cells in 200 µL of staining buffer.
   NOTE: If no flow cytometer with plate reader is available, transfer the cells into flow cytometry tubes.
7. Acquire a minimum of 1.5 × 10⁶ cells per sample on the flow cytometer.
   NOTE: For single colors and unstained control samples, 0.5-1 × 10⁶ cells per sample will be sufficient. It is recommended to titer the individual antibodies used to achieve optimal staining and reduce costs. The present protocol has been optimized using Fix/Perm Buffer prepared using the FoxP3 staining buffer set. Because CD68 is a cytoplasmic and not a nuclear marker, other permeabilization solutions such as a low concentration of paraformaldehyde or cytofix/ cytoperm kits from various vendors might be sufficient.

Results

Gating strategy
The first step of our gating strategy is the exclusion of the debris and doublets (Figure 1A). Careful exclusion of doublets is critical to avoid false-positive populations (Supplemental Figure S2). Then, immune cells are identified using CD45⁺, a marker for hematopoietic cells (Figure 1B). The live-dead stain can be added to exclude dead cells. However, this protocol results in the death of...

Discussion

Identification of pulmonary immune cells can be challenging because of the multiple immune cell types residing in the lung and their unique immunophenotypic characteristics compared to their counterparts residing in other tissues. In several pathologic conditions, cells with distinct phenotypic features appear in the lungs. For example, bleomycin-induced lung injury results in the recruitment of circulating monocyte-derived macrophages in the alveolar space, where they can remain for as long as one year and even persist ...

Materials

Name	Company	Catalog Number	Comments
10 mL syringe plunger	EXELINT	26265
18 G needles	BD Precision Glide Needle	305165
21 G needles	BD Precision Glide Needle	305195
50 mL conical tubes	Falcon	3520
70 μm cell strainer	ThermoFisher	22363548
96-well plates	Falcon/corning	3799
ACK Lysing Buffer	ThermoFisher	A10492-01
anti-mouse CD11b	Biolegend	101215	For details see Table 2
anti-mouse CD11c	Biolegend	117339 / 117337	For details see Table 2
anti-mouse CD45	Biolegend	103115	For details see Table 2
anti-mouse CD64	Biolegend	139319	For details see Table 2
anti-mouse CD68	Biolegend	137009	For details see Table 2
anti-mouse GR-1	Biolegend	108433	For details see Table 2
anti-mouse Siglec F	Biolegend	155503	For details see Table 2
AVERTIN	Sigma-Aldrich	240486
B220	Biolegend	103228	For details see Table 2
Bovine Serum Albumin (BSA)	Sigma-Aldrich	9048-46-8
CD103	Biolegend	121405 / 121419	For details see Table 2
CD24	Biolegend	138503	For details see Table 2
CD3	Biolegend	100205	For details see Table 2
Centrifuge
Collagenase Type 1	Worthington Biochemical Corp	LS004196
CX3CR1	Biolegend	149005	For details see Table 2
DNase I	Millipore Sigma	10104159001
Ethanol
F4/80	Biolegend	123133	For details see Table 2
FcBlock (CD16/32)	Biolegend	101301	For details see Table 2
Fetal Bovine Serum	R&D Systems
Fine Serrated Forceps	Roboz Surgical Instrument Co
Foxp3 / Transcription Factor Staining Buffer Set	ThermoFisher	00-5523-00
Futura Safety Scalpel	Merit Medical Systems	SMS210
Live/Dead Fixable Far Read Dead Cell Stain Kit	ThermoFisher	L34973	For details see Table 2
MERTK	Biolegend	151505	For details see Table 2
MHC-II	Biolegend	107621	For details see Table 2
NK1.1	Biolegend	108705	For details see Table 2
Orbital Shaker	VWR	Model 200
Petri dish	Falcon	351029
Refrigerated benchtop centrifuge	SORVAL ST 16R
Small curved scissor	Roboz Surgical Instrument Co