Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique

Summary

The present protocol combines ex vivo stimulation and flow cytometry to analyze polyfunctional T cell (T_PF) profiles in peripheral blood mononuclear cells (PBMCs) within Japanese encephalitis virus (JEV)-vaccinated children. The detection method and flow cytometry color scheme of JEV-specific TPFs were tested to provide a reference for similar studies.

Abstract

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" of a T cell needs to be assessed by function, and higher function is associated with more powerful immune protection. T cells that can simultaneously produce two or more cytokines or chemokines at the single-cell level are called polyfunctional T cells (T_PFs), which mediate immune responses through a variety of molecular mechanisms to express degranulation markers (CD107a) and secrete interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, or macrophage inflammatory protein (MIP)-1α. There is increasing evidence that T_PFs are closely related to the maintenance of long-term immune memory and protection and that their increased proportion is an important marker of protective immunity and is important in the effective control of viral infection and reactivation. This evaluation applies not only to specific immune responses but also to the assessment of cross-reactive immune responses. Here, taking the Japanese encephalitis virus (JEV) as an example, the detection method and flow cytometry color scheme of JEV-specific T_PFs produced by peripheral blood mononuclear cells of children vaccinated against Japanese encephalitis were tested to provide a reference for similar studies.

Introduction

Japanese encephalitis virus (JEV) is an important mosquito-borne virus belonging to the genus Flavivirus within the Flaviviridae family¹. Many Asia-Pacific countries have long faced enormous public health challenges due to the huge disease burden caused by Japanese encephalitis (JE), but this has improved dramatically with the increasing availability of various types of vaccinations². Adaptive protective immune responses evoked by natural infection or vaccination contribute to the prevention and antiviral regulation. Humoral immunity and cell-mediated immunity are classified as adaptive immunity, and the induction of the former has always been regarded as a key strategy in vaccine design, albeit with relatively limited understanding in the past³. However, the role of T cell-mediated immunity in limiting flavivirus dissemination and virus clearance has been increasingly focused on and extensively studied⁴. Furthermore, T cell immunity is not only indispensable in JEV-specific antiviral responses but also plays a prominent role in cross-protection from secondary infection with heterologous flaviviruses, which has been demonstrated in previous studies⁵. It is speculated that this effect may bypass potential antibody-mediated enhancement effects in infection⁵. Of note, such cross-reactive T cell immunity is important, especially in the absence of vaccines and antiviral drugs against flaviviruses. Although many studies have been performed to determine the contribution of T cells in JEV infection with respect to CD4⁺ and CD8⁺ T cells^6,7, the respective lineages secreting cytokines and their functional diversification remain undetermined, which means the elucidation of the exact functions of helper and killer T cells is hindered.

The scale of their antiviral defenses determines the quality of T cell responses. CD4⁺ or CD8⁺ T cells that can compatibly confer two or more functions, including cytokine secretion and degranulation, are characterized as polyfunctional T cells (T_PFs) upon specific stimulation at the single-cell level⁸. CD4⁺ T cells that produce single or multiple cytokines may have various effects and immune memories. For example, IL-2⁺ IFN-γ⁺ CD4⁺ T cells are more likely to form a long-term effective protective response than IL-2⁺ CD4⁺ T cells⁹, which can be used as an important parameter in evaluating the vaccination effect. The frequency of IL-2⁺ IFN-γ⁺ CD4⁺ T cells is increased in patients with long-term non-progression of acquired immune deficiency syndrome (AIDS), while CD4⁺ T cells in patients with AIDS progression are more inclined to produce IFN-γ alone due to the promoting effect of IL-2 on T cell proliferation¹⁰. Furthermore, a subset of IL-2⁺ IFN-γ⁺ TNF-α⁺ was shown to survive long-term in vivo and synergistically promote the killing function¹¹. Although CD8⁺ T cells are more likely to exhibit cytotoxic activity, some CD4⁺ T cells are also equipped with cytotoxic activity as an indirectly detected expression of surface CD107a molecules¹². In addition, certain T cell subsets express the chemokine MIP-1α, which is often secreted by monocytes to participate in T cell-mediated neutrophil recruitment¹³. Similarly, CD8⁺ T_PFs can also be used to characterize the versatility of the above markers. Studies have shown that the prime-boost strategy can effectively induce a prolonged period of T_PF protective effects¹³, which can enhance the protection elicited by vaccination. A central feature in examining the immune system is the ability of memory T cells to facilitate stronger, faster, and more effective responses to secondary viral challenges than naïve T cells. Effector memory T cells (T_EM) and central memory T cells (T_CM) are important T cell subsets that are often differentiated by the composite expression of CD27/CD45RO or CCR7/CD45RA¹⁴. T_CM (CD27⁺ CD45RO⁺ or CCR7⁺ CD45RA^-) tends to localize in secondary lymphoid tissues, while T_EM (CD27^- CD45RO⁺ or CCR7^- CD45RA^-) localizes in lymphoid and peripheral tissues^15,16. T_EM provides immediate but not sustained defense, whereas T_CM sustains the response by proliferating in the secondary lymphoid organs and generating new effectors¹⁷. Thus, given that memory cells can mediate specific and efficient recall responses to viruses, questions arise about the contribution of this subset of polyfunctions.

With the development of flow cytometry technology, it has become common to simultaneously detect markers of more than 10 clusters, phenotypes, and differentiation antigens, which is beneficial to more abundantly annotate the functional immunological features on individual T cells to reduce misinterpretation and difficulties in understanding of T cell phenotypes. This study used ex vivo stimulation and flow cytometry to analyze T_PF profiles in peripheral blood mononuclear cells (PBMCs) within JEV-vaccinated children. Applying this approach, the understanding of short- and long-term JEV-specific and even cross-reactive T cell immunity induced by vaccination will be expanded.

Protocol

Ethical approval for the present study was obtained by the Ethics Committee of Beijing Children's Hospital, Capital Medical University (Approval Number: 2020-k-85). Volunteers were recruited from Beijing Children's Hospital, Capital Medical University. Peripheral venous blood samples were obtained from apparently healthy children (2 years old) who had previously received a prime and boosted vaccination with live-attenuated JE SA14-14-2 vaccine for less than half a year (JE-vaccinated children, n = 5) and unvaccinated children (6 months old, n = 5). Informed consent of human subjects was waived as only the residual samples, after being clinically tested, were used in this study. To protect the privacy of the volunteers, all data were fully anonymized and de-identified.

1. Isolation of PBMCs from peripheral venous blood

1. Collect peripheral venous blood samples (2 mL) from JE-vaccinated and unvaccinated children in EDTA-K₂-anticoagulated tubes (see Table of Materials) by the standard venipuncture technique¹⁸.
2. Add 2 mL of phosphate-buffered saline (1x PBS) to dilute the peripheral blood.
   NOTE: The process is handled as soon as possible to maintain maximum survivability.
3. Add 4 mL of the density gradient medium (see Table of Materials) to a 15 mL centrifuge tube, and slowly transfer the diluted blood to the upper layer of the separation medium.
   NOTE: Do not mix the blood with the separation medium or destroy the contact surface formed by the two liquids; otherwise, the separation effect will not be achieved.
4. Centrifuge at 800 × g for 20 min at room temperature. Observe the significant layers after centrifugation.
5. Transfer the PBMCs (the middle layer) to another 15 mL centrifuge tube, and wash the PBMCs with 10 mL of RPMI-1640 medium containing 10% fetal bovine serum (FBS, see Table of Materials).
6. Centrifuge at 800 × g for 10 min at room temperature and discard the supernatant with a pipette carefully.
7. Resuspend the PBMCs with 1 mL of RPMI-1640 medium containing 10% FBS and count the cells with a trypan blue-based automated counter (see Table of Materials).

2. Stimulation of PBMCs by inactivated JEV particles to induce cytokine expression

1. Set two subgroups of the JEV stimulation and control groups in the JE-vaccinated and unvaccinated samples, respectively.
2. Adjust the number of PBMCs to 2 × 10⁶ cells/mL with RPMI-1640 medium containing 10% FBS, and seed the PBMCs in 24-well plates (2 × 10⁶ cells/1 mL medium per well); inoculate three wells for each group.
3. Stimulate the PBMCs of the JEV stimulation group with concentrated inactivated JEV particles⁵ (2 × 10⁵ PFU) for 16 h at 37 °C in the presence of monoclonal antibodies CD28 (1 µg/mL, 1:1,000) and CD49d (1 µg/mL), GolgiPlug (1 µg/mL, 1:1,000), and monensin (1 µg/mL, 1:1,000). For surface staining, use BV605-anti-CD107a (1 µg/mL, 1:1,000) (see Table of Materials).
   NOTE: The JEV was inactivated by UV irradiation as described previously¹⁹.
4. Stimulate the PBMCs of the control group without concentrated virus particles for 16 h at 37 °C in the presence of monoclonal antibodies CD28 (1 µg/mL, 1:1,000) and CD49d (1 µg/mL, 1:1,000), GolgiPlug (1 µg/mL, 1:1,000), and monensin (1 µg/mL, 1:1,000). Stain the surface with BV605-anti-CD107a (1 µg/mL, 1:1,000).

3. Ex vivo intracellular staining

1. Collect the cell suspension from each group in a 1.5 mL microcentrifuge tube, centrifuge at 500 × g for 5 min at room temperature, and remove the supernatant with a pipette.
2. Resuspend the cells in 1 mL of 1x PBS, add fixable viability dye (1 µL/mL, 1:1,000, see Table of Materials) to the cell suspension, and incubate for 10 min at room temperature in the dark. Centrifuge at 500 × g (at room temperature) for 5 min and remove the supernatant.
3. Resuspend the cells in 1 mL of 1x PBS. Centrifuge at 500 × g (at room temperature) for 5 min. Discard the supernatant carefully.
4. Perform cell surface marker staining.
   1. Resuspend the cells in 100 µL of 1x PBS, and add 2 µL of each surface markers antibody (BV650-anti-CD3, BUV395-anti-CD4, BV421-anti-CD8, BUV737-anti-CD27, and BV480-anti-CD45RO, dilution factor: 1:50, see Table of Materials) to the cell suspension in each tube.
      NOTE: The color fluorescent antibody staining protocol is shown in Table 1.
   2. Incubate the tubes (step 3.4.1) for 30 min at room temperature, and protect them from light. Centrifuge at 500 × g for 5 min and remove the supernatant.
      NOTE: The antibody of BUV737-anti-CD27 and BV480-anti-CD45RO can be replaced with BUV737-anti-CCR7 and BV480-anti-CD45RA as the annotation of the memory T cells.
5. Resuspend the cells in 1 mL of 1x PBS. Centrifuge at 500 × g for 5 min at room temperature. Discard the supernatant carefully.
6. Perform fixation and membrane breaking.
   1. Resuspend the cells with 500 µL of membrane-breaking fixative solution (see Table of Materials) and fix the cells for 20 min in the dark at room temperature. Centrifuge at 500 × g for 5 min and remove the supernatant.
7. Resuspend the cells in 1 mL of 1x PBS. Centrifuge for 5 min at 500 × g at room temperature and remove the supernatant carefully.
8. Perform intracellular cytokine staining.
   1. Resuspend the cells in 100 µL of 1x PBS, and add 2 µL of each cytokine antibody (FITC-anti-IFN-γ, PE-anti-TNF-α, BV785-anti-IL-2, and APC-anti-MIP-1α, dilution factor: 1:50, see Table of Materials) to the cell suspension in each tube.
      NOTE: The volumes and information on fluorophore conjugated antibodies are shown in Table 1.
   2. Incubate the tubes (step 3.8.1) for 30 min at room temperature in the dark. Centrifuge at 500 × g for 5 min and remove the supernatant.
9. Resuspend the cells in 1 mL of 1x PBS. Centrifuge at 500 × g for 5 min at room temperature, and remove the supernatant carefully. Add 500 µL of 1x PBS to resuspend the cells.

4. Flow cytometry set-up

1. Isolate the PBMC sample alone following the procedures described in step 1 as the control sample. Divide the cell suspension into 12 equal parts in 1.5 mL microcentrifuge tubes (100 µL/tube) as mentioned below.
   1. Set up unstained, APC-Cy7-live/dead fixable stained, BV650-anti-CD3 single-stained, BUV395-anti-CD4 single-stained, BV421-anti-CD8 single-stained, BUV737-anti-CD27 single-stained, BV480-anti-CD45RO stained, BV605-anti-CD107a, FITC-anti-IFN-γ stained, PE-anti-TNF-α single-stained, BV785-anti-IL-2 single-stained, and APC-anti-MIP-1α single-stained samples.
2. Add the cell surface markers and intracellular cytokine staining as described in step 3. For each single-staining sample, only add one of the fluorophores from the staining step. Add 500 µL of 1x PBS to resuspend the cells and vortex with low velocity.
3. Using an unstained sample, adjust the forward scatter (FSC), side scatter (SSC), and different fluorescent dye voltages.
   NOTE: For the present study, the following voltages were set. FSC: 440 V, SSC: 233 V, BV650: 575 V, APC-Cy7: 449 V, BUV395: 500 V, BV421: 402 V, BUV737: 585 V, BV480: 444 V, BV605: 457 V, FITC: 475 V, PE: 469 V, APC: 623 V, and BV785: 725 V.
4. Using the single-staining samples, adjust the flow cytometry compensation to eliminate the contamination signals between the different fluorophores.
   ​NOTE: The compensation parameters are shown in Table 2.

5. Gating strategy and data analysis

NOTE: In the present study, for this analysis, the central memory T cells (T_CM of CD8⁺ or CD4⁺ T cells as CD27⁺ CD45RO⁺ or CCR7⁺ CD45RA^- and the effector memory T cells (T_EM) of CD8⁺ or CD4⁺ T cells as CD27^- CD45RO⁺ or CCR7^- CD45RA^- were defined respectively¹⁶.

1. Draw a polygon gate through the FSC-area (FSC-A)/SSC-area (SSC-A) dot plot to select the intact lymphocyte population while excluding the debris (Figure 1A).
2. Draw a rectangular gate through the FSC-A/FSC-width (FSC-W) dot plot to select the single cells (Figure 1B).
3. Draw a rectangular gate through the live/dead/SSC-A dot plot to select the live cells (Figure 1C).
4. Draw a rectangular gate through the CD3/SSC-A dot plot to identify the CD3⁺ T cells (Figure 1D).
5. Draw a quad gate through the CD4/CD8 dot plot to identify the CD4⁺ or CD8⁺ T cells (Figure 1E).
6. Draw a quad gate through the CD45RO/CD27 dot plot to subdivide the CD4⁺ or CD8⁺ T cells into T_CM (CD27⁺ CD45RO⁺) and T_EM (CD27^- CD45RO⁺) (Figure 1F-I).
7. Draw the gates of CD107a, IFN-γ, TNF-α, IL-2, and MIP-1α from the T_CM or T_EM of the CD8⁺ or CD4⁺ T cells to determine the frequency of different response patterns, respectively.
8. Load the samples onto the cytometry sequentially. Use a stopping gate²⁰ to acquire 1 × 10⁶ lymphocytes.
   NOTE: The individual user can collect more than 1 × 10⁶ cells. This number was a compromise between the time taken and the collection of enough cells to provide meaningful results.

Results

Figure 1 shows the gating strategy used to divide the T_CM or T_EM of CD8⁺ or CD4⁺ T cells from a representative JEV stimulation group of JE-vaccinated children. The FSC-A/SSC-A dot plot is used to identify lymphocytes, and the FSC-A/FSC-W dot plot is used to identify single cells. Viable cells are selected on the live/dead/SSC-A dot plot. The CD3/SSC-A dot plot is used to identify the CD3⁺ T cells. The CD4/CD8 dot plot is used t...

Discussion

This protocol represents a feasible flow cytometry-based detection method for T_PF profiles in the PBMCs of children vaccinated with the JEV vaccine SA14-14-2. This study used the venous blood PBMCs of both vaccinated and unvaccinated children as research materials. With the stimulation of PBMCs with the JEV antigen, those amplified antigen-specific T_PFs can be characterized by multicolor flow cytometry antibody staining. Compared with the conventional enzyme-linked immunospot assay method, flow cyto...

Materials

Name	Company	Catalog Number	Comments
anti-human CD28	Biolegend	302934	Antibody
anti-human CD49d	Biolegend	304339	Antibody
APC anti-human MIP-1α	BD	551533	Fluorescent antibody
Automated cell counter	BIO RAD	TC20	Cell count
BD FACSymphony A5	BD	A5	flow Cytometry
BUV395 anti-human CD4	BD	563550	Fluorescent antibody
BUV737 anti-human CCR7	BD	741786	Fluorescent antibody
BUV737 anti-human CD27	BD	612829	Fluorescent antibody
BV421 anti-human CD8	Biolegend	344748	Fluorescent antibody
BV480 anti-human CD45RA	BD	566114	Fluorescent antibody
BV480 anti-human CD45RO	BD	566143	Fluorescent antibody
BV605 anti-human CD107a	Biolegend	328634	Fluorescent antibody
BV650 anti-human CD3	BD	563999	Fluorescent antibody
BV785 anti-human IL-2	Biolegend	500348	Fluorescent antibody
Centrifuge Tube	BD Falcon	BD-35209715	15 mL centrifuge tube
Cytofix/Cytoperm Fixation/Permeabilization Solution Kit	BD	554714	Cell fixation and permeabilization
Density gradient medium	Dakewe	DKW-KLSH-0100	Ficoll-Paque, human lymphocyte separation medium
FITC anti-human IFN-γ	Biolegend	502506	Fluorescent antibody
Gibco Fetal Bovine Serum	Thermo Fisher Scientific	16000-044	Fetal Bovine Serum
Gibco RPMI-1640 medium	Thermo Fisher Scientific	22400089	cell culture medium
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
Microcentrifuge tubes	Axygen	MCT-150-C	1.5 mL microcentrifuge tube
PE anti-human TNF-α	Biolegend	502909	Fluorescent antibody
Phosphate Buffered Saline (PBS)	BI	02-024-1ACS	PBS
Protein Transport Inhibitor (Containing Brefeldin A, GolgiPlug)	BD	555029	blocks intracellular protein transport processes
Protein Transport Inhibitor (Containing Monensin)	BD	554724	blocks intracellular protein transport processes
Round-bottom test tube	BD Falcon	352235	5 mL test tube
Trypan Blue Staining Cell Viability Assay Kit	Beyotime	C0011	Trypan Blue Staining
Zombie NIR Fixable Viability Dye	Biolegend	423106	Dead cell stain