Separation of Immune Cell Subpopulations in Peripheral Blood Samples from Children with Infectious Mononucleosis

Summary

We describe a method combining immunomagnetic beads and fluorescence-activated cell sorting to isolate and analyze defined immune cell subpopulations of peripheral blood mononuclear cells (monocytes, CD4⁺ T cells, CD8⁺ T cells, B cells, and natural killer cells). Using this method, magnetic and fluorescently labeled cells can be purified and analyzed.

Abstract

Infectious mononucleosis (IM) is an acute syndrome mostly associated with primary Epstein–Barr virus (EBV) infection. The main clinical symptoms include irregular fever, lymphadenopathy, and significantly increased lymphocytes in peripheral blood. The pathogenic mechanism of IM is still unclear; there is no effective treatment method for it, with mainly symptomatic therapies being available. The main question in EBV immunobiology is why only a small subset of infected individuals shows severe clinical symptoms and even develop EBV-associated malignancies, whilemost individuals are asymptomatic for life with the virus.

B cells are first involved in IM because EBV receptors are presented on their surface. Natural killer (NK) cells are cytotoxic innate lymphocytes that are important for killing EBV-infected cells. The proportion of CD4⁺ T cells decreases while that of CD8⁺ T cells expands dramatically during acute EBV infection, and the persistence of CD8⁺ T cells is important for lifelong control of IM. Those immune cells play important roles in IM, and their functions need to be identified separately. For this purpose, monocytes are separated first from peripheral blood mononuclear cells (PBMCs) of IM individuals using CD14 microbeads, a column, and a magnetic separator.

The remaining PBMCs are stained with peridinin-chlorophyll-protein (PerCP)/Cyanine 5.5 anti-CD3, allophycocyanin (APC)/Cyanine 7 anti-CD4, phycoerythrin (PE) anti-CD8, fluorescein isothiocyanate (FITC) anti-CD19, APC anti-CD56, and APC anti-CD16 antibodies to sort CD4⁺ T cells, CD8⁺ T cells, B cells, and NK cells using a flow cytometer. Furthermore, transcriptome sequencing of five subpopulations was performed to explore their functions and pathogenic mechanisms in IM.

Introduction

Epstein–Barr virus (EBV), a γ-herpesvirus also known as human herpes virus type 4, is ubiquitous in the human population and establishes lifelong latent infection in more than 90% of the adult population¹. Most EBV primary infection occurs during childhood and adolescence, with a fraction of patients manifesting with infectious mononucleosis (IM)², showing characteristic immunopathology, including an activated immune response with CD8⁺ T cells in blood and a transient proliferation of EBV-infected B cells in the oropharynx³. The course of IM may last for 2–6 weeks and the majority of the patients recover well⁴. However, some individuals develop persistent or recurrent IM-like symptoms with high morbidity and mortality, which is classified as chronic active EBV infection (CAEBV)⁵. In addition, EBV is an important oncogenic virus, which is closely related to a variety of malignancies, including epithelioid and lymphoid malignancies such asnasopharyngeal carcinoma, Burkitt's lymphoma, Hodgkin's lymphoma (HL), and T/NK cell lymphoma⁶. Although EBV has been studied for over 50 years, its pathogenesis and the mechanism by which it induces the proliferation of lymphocytes have not been fully elucidated.

Several studies have investigated the molecular signatures for the immunopathology of EBV infection by transcriptome sequencing. Zhong et al. analyzed whole-transcriptome profiling of peripheral blood mononuclear cells (PBMCs) from Chinese children with IM or CAEBV to find that CD8⁺ T cell expansion was predominantly found in the IM group⁷, suggesting that CD8⁺ T cells may play a major role in IM. Similarly, another study found lower proportions of EBV-specific cytotoxic T and CD19⁺ B cells and higher percentages of CD8⁺ T cells in patients with IM caused by primary EBV infection than in patients with IM caused both by EBV reactivation and other agents⁸. B cells are first involved in IM because EBV receptors are presented on their surface. Al Tabaa et al. found that B cells were polyclonally activated and differentiated intoplasmablasts (CD19⁺, CD27⁺ and CD20⁻, and CD138⁻ cells) and plasma cells (CD19⁺, CD27⁺ and CD20⁻, and CD138⁺) during IM⁹. Moreover, Zhong et al. found that monocyte markers CD14 and CD64 were upregulated in CAEBV, suggesting that monocytes may play an important role in the cellular immune response of CAEBV through antibody-dependent cellular cytotoxicity (ADCC) and hyperactive phagocytosis⁷. Alka et al. characterized the transcriptome of MACS sorted CD56^dim CD16⁺ NK cells from four patients of IM or HL and found that NK cells from both IM and HL had downregulated innate immunity and chemokine signaling genes, which could be responsible for the hyporesponsiveness of NK cells¹⁰. In addition, Greenough et al. analyzed gene expression of sorted CD8⁺ T cells from 10 PBMCs of individuals with IM. They reported that a large proportion of CD8⁺ T cells in IM were virus-specific, activated, dividing, and primed to exert effector activities¹¹. Both T cell-mediated, EBV-specific responses, and NK cell-mediated, nonspecific responses play essential roles during primary EBV infection. However, these studies only investigated the transcriptome results of the diverse mixture of immune cells or only a certain subpopulation of lymphocytes, which is not sufficient for the comprehensive comparison of the molecular characteristics and functions of different immune cell subpopulations in children with IM at the same disease state.

This paper describes a method that combines immunomagnetic beads and fluorescence-activated cell sorting (FACS) to isolate and analyze defined immune cell subpopulations of PBMCs (monocytes, CD4⁺ T cells, CD8⁺ T cells, B cells, and NK cells). Using this method, magnetic and fluorescently labeled cells can be purified using a magnetic separator and FACS or analyzed by flow cytometry. RNA can be extracted from the purified cells for transcriptome sequencing. This method will enable the characterization and gene expression of different immune cells in the same states of disease of individuals with IM, which will expand our understanding of the immunopathology of EBV infection.

Protocol

Blood samples were obtained from patients with IM (n = 3), healthy EBV carriers (n = 3), and EBV-uninfected children (n = 3). Volunteers were recruited from Beijing Children's Hospital, Capital Medical University, and all studies were ethically approved. Ethical approval was obtained by the Ethics Committee of Beijing Children's Hospital, Capital Medical University (Approval Number: [2021]-E-056-Y). Informed consent of patients was waived as the study only used the remaining samples for clinical testing. All data were fully deidentified and anonymized to protect patient privacy.

1. Isolation of PBMCs from peripheral blood

1. Collect fresh peripheral blood (2 mL) from patients with IM into K₃EDTA tubes by standard venipuncture.
   NOTE: The process needs to be rapid to maintain cell viability.
2. Dilute peripheral blood to twofold volume with phosphate-buffered saline (PBS) and layer it on top of human lymphocyte separation medium (density: 1.077 ± 0.001 g/mL) in a 15 mL centrifuge tube.
   NOTE: The volume ratio of blood, PBS, and separation medium was 1:1:1. Add the blood slowly to the separation medium, and centrifuge immediately to avoid blood settling into the separation medium.
3. Centrifuge at 800 × g for 20 min at room temperature. Transfer the middle layer (enriched PBMCs) to another 15 mL centrifuge tube.
   NOTE: After centrifugation, at the bottom of the tube are erythrocytes, the middle layer is the separation medium, the top layer is plasma, and between the plasma layer and the separation liquid layer were the PBMCs (including lymphocytes and monocytes).
4. Wash the PBMCs with 10 mL of PBS and centrifuge at 800 × g for 20 min at room temperature; discard the supernatant carefully.
5. Repeat the washing and centrifuging (step 1.4) 2 x. Resuspend the PBMCs with 1 mL of PBS in a 1.5 mL microcentrifuge tube and count the cells with a trypan blue-based, automated counter.

2. Isolation of CD14 ⁺ monocytes from PBMCs using CD14 microbeads

1. Prepare a buffer solution containing 0.5% fetal bovine serum (FBS) and 2 mM EDTA in PBS (pH 7.2). Keep the buffer cold (2−8 °C).
   NOTE: Degas the buffer before use as air bubbles could block the column. Keep the cells cold to prevent capping of the antibodies on the cell surface and non-specific cell labeling.
2. Centrifuge the PBMCs at 300 × g for 10 min at room temperature. Discard the supernatant carefully. Resuspend the cells with 80 µL of the buffer. Add 20 µL of CD14 microbeads to the cell suspension.
   NOTE: If there are ≤10⁷ PBMCs, use the volume indicated above. If there are >10⁷ PBMCs, proportionally increase all reagent volumes and the total volume.
3. Mix the CD14 microbeads and the cells well in the 1.5 mL microcentrifuge tube and incubate for 15 min in a 4 °C refrigerator. Wash the PBMCs with 1 mL of buffer and centrifuge at 300 × g for 10 min at room temperature. Discard the supernatant completely. Resuspend the cells with 500 µL of the buffer.
   NOTE: If there are ≤10⁸ PBMCs, use the volume indicated above. If there are >10⁸ PBMCs, proportionally increase the buffer volume.
4. Magnetic separation with columns:
   1. Put the column on the magnetic bead separator, and wash the column with 3 mL of buffer. Add the cell suspension (from step 2.3) to the column.
   2. Collect the unlabeled cells that pass through the column into a 15 mL centrifuge tube and wash the column with 3 mL of buffer. Wash the column with 3 x 3 mL of buffer. Collect the total effluent in the 15 mL centrifuge tube.
   3. Place the column in a new 15 mL centrifuge tube. Add 5 mL of buffer to the column. Push the plunger firmly into the column to immediately flush out the magnetically labeled cells into the 15 mL centrifuge tube.
   4. Centrifuge the magnetically labeled cells at 300 × g for 5 min and remove the supernatant. Resuspend the cells in 500 µL of PBS in a 1.5 mL microcentrifuge tube for use in subsequent transcriptome sequencing.

3. Separation of lymphocyte populations from PBMCs by fluorescently labeled antibody staining and FACS

1. Centrifuge the unlabeled cells (step 2.4.2) at 300 × g for 5 min and remove the supernatant. Resuspend the cells in 100 µL of PBS in a 1.5 mL microcentrifuge tube.
2. Add 2 µL of each labeled antibody (CD3, CD4, CD8, CD16, CD19, CD56) to the 100 µL of cell suspension (the volumes and conjugated fluorophore information of antibodies are shown in Table 1), incubate on ice for 30 min, and protect from light.
3. Wash the cells 2 x by adding 1 mL of PBS and centrifuging at 300 × g for 5 min at room temperature. Resuspend the cells in 500 µL of PBS in the 1.5 mL microcentrifuge tube. Vortex the cell suspension gently before acquiring data on a flow cell sorter cytometer.

4. Flow cytometry parameter setting

1. Take 100 µL of the cell suspension (see section 1) in 1.5 mL microcentrifuge tubes as required, and set up a negative control sample, a CD3 single-staining sample, a CD4 single-staining sample, a CD8 single-staining sample, a CD19 single-staining sample, and a CD56/CD16 staining sample.
2. Add 2 µL of the corresponding fluorescently labeled antibody per 100 µL of the cell suspension and vortex. Incubate on ice for 30 min in the dark. Centrifuge for 5 min at 300 × g and aspirate the supernatant. Resuspend the pellets with 500 µL of PBS and vortex.
3. Commission the sorting stream and delay the droplets using the fluorescent beads as follows:
   1. Open the cell sorting system, run the power-on program, install the 85 µm nozzle, and open the sorting stream. Set the sorting voltage to 4,500 V, and the Freq to 47.
   2. Adjust the parameters mainly by adjusting the main flow droplet breakpoint and the droplet delay according to the manufacturer's instructions¹². Set up the first droplet breakpoint position (Drop1) to 275, and the Gap to 8 in the Breakoff window. Turn on the Sweet Spot automatic sorting mode to allow the cytometry to automatically determine the droplet amplitude value to stabilize the stream.
   3. Adjust the droplet delay to 30.31 in the Side Stream window by loading the fluorescent beads, ensuring that the beads achieve a side flow deflection of >99% in either initial or fine tune mode.
4. Refer to the gating strategy shown in Figure 1 to perform gating as follows:
   1. Using a forward scatter-area (FSC-A)/side scatter-area (SSC-A) dot plot, draw the polygon gate (P1) to identify the intact lymphocyte population.
   2. Using an FSC-A/FSC-height (FSC-H) dot plot, draw the polygon gate (P2) to identify the single cells and exclude doublets (Figure 1A).
   3. Using a CD19 FITC-A/CD3 PerCP-Cy5.5-A dot plot, draw the rectangular gate (P3) to select CD3⁺ T cells and CD19⁺ B cells (Figure 1A,B).
   4. Using a CD4 APC-Cy7-A/CD8 PE-A dot plot, draw a rectangular gate to select CD4⁺ T cells and CD8⁺ T cells (cells with a high fluorescence for these markers, respectively).
   5. Using a CD56/CD16 APC-A/SSC-A dot plot, draw a rectangular gate to select CD56⁺/CD16⁺ NK cells (Figure 1C).
5. Adjust instrumental parameters using the negative control sample:
   1. Install the negative control tube onto the loading port and click Load in the Acquisition Dashboard. Select the Cytometer Settings in the software.
   2. In the Inspector window, click the Parameters tab, and adjust the voltages of FSC, SSC, and different fluorescent dyes; FSC: 231, SSC: 512, PE: 549, APC: 615, APC-Cyanine 7: 824, FITC: 555, PerCP-Cyanine 5.5: 663.
6. Adjust compensation using the single-stained samples¹³.
   1. Load the single-stained tubes onto the cytometry sequentially andselect Cytometer Settings in the software. Click the Compensation tab to adjust the compensation.
      ​NOTE: The compensation reference of flow cytometry is shown in Table 2.

5. Cell sorting and collecting data  via flow cytometry

1. Vortex the cell suspension (separated PBMCs according to sections 1–3) briefly to resuspend the cells before loading the tube into the cytometer. Keep the remaining tubes on ice.
2. Add 200 µL of FBS to four collection flow tubes to avoid sticking of the sorted cells to the tube wall and place them in the cytometer collection chamber.
3. Collect CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells, CD3⁻CD19⁺ B cells, and CD3⁻ CD56⁺/CD16⁺ NK cells separately from the sample of a patient with IM in the four flow tubes (Figure 2A).
4. Centrifuge the separated cells at 300 × g for 5 min and remove the supernatant. Add 200 µL of RNA isolation reagent to the cells for transcriptome sequencing.
5. Separate the immune cell subpopulations of samples from healthy EBV carriers and EBV-uninfected children according to the above steps (Figure 2B, C).

Results

Reference of the gating strategy
The gating strategy used to sort the four lymphocyte subpopulations is shown in Figure 1. Briefly, lymphocytes are selected (P1) on a dot plot showing the granulosity (SSC-A) versus size (FSC-A). Then, single cells are selected (P2) on a dot plot showing the size (FSC-A) versus forward scatter (FSC-H), while doublet cells are excluded. CD3⁺ T cells (P3) and CD19⁺ B cells (Figure 1B) ar...

Discussion

This protocol represents an efficient way to sort peripheral blood immune cell subpopulations. In this study, venous blood samples from patients with IM, healthy EBV carriers, and EBV-uninfected children were selected as the research objective. This work using the peripheral blood of patients of IM mainly focuses on analyzing and determining the proportions of different cell subsets through multi-color flow cytometry. Transcriptome sequencing is mainly used for the detection and analysis of a certain subpopulation of lym...

Materials

Name	Company	Catalog Number	Comments
APC anti-human CD16	Biolegend	302012	Fluorescent antibody
APC anti-human CD56 (NCAM)	Biolegend	362504	Fluorescent antibody
APC/Cyanine7 anti-human CD4	Biolegend	344616	Fluorescent antibody
Automated cell counter	BIO RAD	TC20	Cell count
BD FACSAria fluorescence-activated flow cell sorter-cytometer (BD FACSAria II)	Becton, Dickinson and Company	644832	Cell sort
CD14 MicroBeads, human	Miltenyi Biotec	130-050-201	microbeads
Cell ctng slides	BIO RAD	1450016	Cell count
Centrifuge tubes	Falcon	35209715	15 mL centrifuge tube
EDTA (≥99%, BioPremium)	Beyotime	ST1303	EDTA
Ethylene diamine tetra acetic acid (EDTA) anticoagulant tubes	Becton, Dickinson and Company	367862	EDTA anticoagulant tubes
FITC anti-human CD19	Biolegend	302206	Fluorescent antibody
Gibco Fetal Bovine Serum	Thermo Fisher Scientific	16000-044	Fetal Bovine Serum
High-speed centrifuge	Sigma	3K15	Cell centrifugation for 15 mL centrifuge tube
High-speed centrifuge	Eppendorf	5424R	Cell centrifugation for 1.5 mL Eppendorf (EP) tube
Human lymphocyte separation medium	Dakewe	DKW-KLSH-0100	Ficoll-Paque
LS Separation columns	Miltenyi Biotec	130-042-401	Separation columns
Microcentrifuge tubes	Axygen	MCT-150-C	1.5 mL microcentrifuge tube
MidiMACS Separator	Miltenyi Biotec	130-042-302	Magnetic bead separator
PE anti-human CD8	Biolegend	344706	Fluorescent antibody
PerCP/Cyanine5.5 anti-human CD3	Biolegend	344808	Fluorescent antibody
Phosphate Buffered Saline (PBS)	BI	02-024-1ACS	PBS
Polystyrene round bottom tubes	Falcon	352235	5 mL tube for FACS flow cytometer
TRIzol reagent	Ambion	15596024	Lyse cells for RNA extraction
Trypan Blue Staining Cell Viability Assay Kit	Beyotime	C0011	Trypan Blue Staining