Name: detection of polyfunctional t cells in children vaccinated with japanese encephalitis vaccine via the flow cytometry technique
Uploaded: 2022-09-23T15:00:00
Description: Watch this Scientific Journal Video about Detection of Polyfunctional T Cells in Children Vaccinated with Japanese Encephalitis Vaccine via the Flow Cytometry Technique at JoVE.com

R.W. was supported by National Natural Science Foundation of China (82002130), Beijing Natural Science Foundation of&#160;China (7222059). ZD.X. was supported by the CAMS Innovation Fund for Medical Sciences (2019-I2M-5-026).

Ethical approval for the present study was obtained by the Ethics Committee of Beijing Children&#39;s Hospital, Capital Medical University (Approval Number: 2020-k-85). Volunteers were recruited from Beijing Children&#39;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 unvaccin...

<ol>
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This protocol represents a feasible flow cytometry-based detection method for TPF 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 TPFs can be characterized by multicolor flow cytometry antibody staining. Compared with the conventional enzyme-linked immunospot assay method, flow cyto...

Japanese encephalitis virus (JEV) is an important mosquito-borne virus belonging to the genus Flavivirus within the Flaviviridae&#160;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 cells6,7, 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&#160;(TPFs)&#160;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-&#947;+ 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-&#947;+ 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-&#947; alone due to the promoting effect of IL-2 on T cell proliferation10. Furthermore, a subset of IL-2+ IFN-&#947;+ TNF-&#945;+ 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-1&#945;, which is often secreted by monocytes to participate in T cell-mediated neutrophil recruitment13. Similarly, CD8+ TPFs 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 TPF 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 na&#239;ve T cells. Effector memory T cells (TEM) and central memory T cells (TCM) are important T cell subsets that are often differentiated by the composite expression of CD27/CD45RO or CCR7/CD45RA14. TCM (CD27+ CD45RO+ or CCR7+ CD45RA-) tends to localize in secondary lymphoid tissues, while TEM (CD27- CD45RO+ or CCR7- CD45RA-) localizes in lymphoid and peripheral tissues15,16. TEM provides immediate but not sustained defense, whereas TCM 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 TPF 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>anti-human CD28</td>
			<td>Biolegend</td>
			<td>302934</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>anti-human CD49d</td>
			<td>Biolegend</td>
			<td>304339</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>APC anti-human MIP-1&#945;</td>
			<td>BD</td>
			<td>551533</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Automated cell counter</td>
			<td>BIO RAD</td>
			<td>TC20</td>
			<td>Cell count</td>
		</tr>
		<tr>
			<td>BD FACSymphony A5</td>
			<td>BD</td>
			<td>A5</td>
			<td>flow Cytometry</td>
		</tr>
		<tr>
			<td>BUV395 anti-human CD4</td>
			<td>BD</td>
			<td>563550</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BUV737 anti-human CCR7</td>
			<td>BD</td>
			<td>741786</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BUV737 anti-human CD27</td>
			<td>BD</td>
			<td>612829</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV421 anti-human CD8</td>
			<td>Biolegend</td>
			<td>344748</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV480 anti-human CD45RA</td>
			<td>BD</td>
			<td>566114</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV480 anti-human CD45RO</td>
			<td>BD</td>
			<td>566143</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV605 anti-human CD107a</td>
			<td>Biolegend</td>
			<td>328634</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV650 anti-human CD3</td>
			<td>BD</td>
			<td>563999</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>BV785 anti-human IL-2</td>
			<td>Biolegend</td>
			<td>500348</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Centrifuge Tube</td>
			<td>BD Falcon</td>
			<td>BD-35209715</td>
			<td>15 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td>
			<td>BD</td>
			<td>554714</td>
			<td>Cell fixation and permeabilization</td>
		</tr>
		<tr>
			<td>Density gradient medium</td>
			<td>Dakewe</td>
			<td>DKW-KLSH-0100</td>
			<td>Ficoll-Paque, human lymphocyte separation medium</td>
		</tr>
		<tr>
			<td>FITC anti-human IFN-&#947;</td>
			<td>Biolegend</td>
			<td>502506</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Gibco Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16000-044</td>
			<td>Fetal Bovine Serum</td>
		</tr>
		<tr>
			<td>Gibco RPMI-1640 medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>22400089</td>
			<td>cell culture medium</td>
		</tr>
		<tr>
			<td>High-speed&#160;centrifuge</td>
			<td>Sigma</td>
			<td>&#160;3K15</td>
			<td>Cell centrifugation for 15 mL centrifuge tube</td>
		</tr>
		<tr>
			<td>High-speed&#160;centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td>Cell centrifugation for 1.5 mL Eppendorf (EP) tube</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>1.5 mL microcentrifuge tube</td>
		</tr>
		<tr>
			<td>PE anti-human TNF-&#945;</td>
			<td>Biolegend</td>
			<td>502909</td>
			<td>Fluorescent antibody&#160;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>BI</td>
			<td>02-024-1ACS</td>
			<td>PBS</td>
		</tr>
		<tr>
			<td>Protein Transport Inhibitor (Containing Brefeldin A, GolgiPlug)</td>
			<td>BD</td>
			<td>555029</td>
			<td>blocks intracellular protein transport processes</td>
		</tr>
		<tr>
			<td>Protein Transport Inhibitor (Containing Monensin)</td>
			<td>BD</td>
			<td>554724</td>
			<td>blocks intracellular protein transport processes</td>
		</tr>
		<tr>
			<td>Round-bottom test tube</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td>5 mL test tube</td>
		</tr>
		<tr>
			<td>Trypan Blue Staining Cell Viability Assay Kit</td>
			<td>Beyotime</td>
			<td>C0011</td>
			<td>Trypan Blue Staining</td>
		</tr>
		<tr>
			<td>Zombie NIR Fixable Viability Dye</td>
			<td>Biolegend</td>
			<td>423106</td>
			<td>Dead cell stain</td>
		</tr>
	</tbody>
</table>

detection of polyfunctional t cells in children vaccinated with japanese encephalitis vaccine via the flow cytometry technique

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 (TPFs), which mediate immune responses through a variety of molecular mechanisms to express degranulation markers (CD107a) and secrete interferon (IFN)-&#947;, tumor necrosis factor (TNF)-&#945;, interleukin (IL)-2, or macrophage inflammatory protein (MIP)-1&#945;. There is increasing evidence that TPFs 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 TPFs produced by peripheral blood mononuclear cells of children vaccinated against Japanese encephalitis were tested to provide a reference for similar studies.

Figure 1 shows&#160;the gating strategy used to divide the TCM or TEM of&#160;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...

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

The present protocol combines ex vivo stimulation and flow cytometry to analyze polyfunctional T cell (TPF)&#160;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.

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

T cell-mediated immunity plays an important role in controlling flavivirus infection, either after vaccination or after natural infection. The "quality" ...

The detection method and the flow cytometry color scheme of Japanese encephalitis virus-specific TPFS were tested to provide a reference for similar studies. Demonstrating the procedure will be performed by Linlin Zhang and Meng Zhang, two students from our laboratory. To begin, stimulate the PBMCs of the JEV stimulation group with concentrated inactivated JEV particles for 16 hours at 37 degrees Celsius in the presence of monoclonal antibodies, CD28 and CD49d, CD107a, GolgiPlug, and monensin.Then, stimulate the PBMCs of the control group without concentrated virus particles for 16 hours at 37 degrees Celsius in the presence of monoclonal antibodies, CD28 and CD49d, CD 107a, GolgiPlug and monensin. Collect the cell suspension from each group in a 1.5 milliliter microcentrifuge tube. Centrifuge at 500 g for five minutes at room temperature, and remove the supernatant with a pipette.Resuspend the cells in 1 milliliter of PBS. Add fixable viability dye to the cell suspension and incubate for 10 minutes at room temperature in the dark. Centrifuge at 500 g at room temperature for five minutes, and remove the supernatant.After resuspending the cells in 1 milliliter of PBS, again, centrifuge at 500 g at room temperature for five minutes and discard the supernatant as demonstrated previously. To perform cell surface marker staining, resuspend the cells in 100 microliters of PBS, and add two microliters of each surface marker antibody to the cell suspension in each tube. Incubate the tubes for 30 minutes at room temperature while protecting them from light.Centrifuge at 500 g for five minutes and remove the supernatant as demonstrated previously. Again, resuspend the cells in one milliliter of PBS. Centrifuge at 500 g for five minutes at room temperature and discard the supernatant carefully.To perform fixation and membrane breaking, resuspend the cells with 500 microliters of membrane-breaking fixative solution and fix the cells for 20 minutes in the dark at room temperature. Then centrifuge at 500 g for five minutes and remove the supernatant. After resuspending the cells in one milliliter of PBS, centrifuge for five minutes at 500 g at room temperature and remove the supernatant carefully, as demonstrated previously.Then, resuspend the cells in 100 microliters of PBS and add two microliters of each cytokine antibody to the cell suspension in each tube for intracellular cytokine staining. Incubate the tubes for 30 minutes at room temperature in the dark. Again, centrifuge at 500 g for five minutes, and remove the supernatant as demonstrated previously.Once again, resuspend the cells in one milliliter of PBS. Centrifuge at 500 g for five minutes at room temperature and remove the supernatant carefully as demonstrated previously. Then, add 500 microliters of PBS to resuspend the cells.After isolating the PBMC sample alone, as demonstrated previously, divide the cell suspension into 12 equal parts in 1.5 milliliter microcentrifuge tubes described in the manuscript. Similarly, after adding the cell surface markers and intracellular cytokine antibody, as demonstrated previously, add 500 microliters of PBS to resuspend the cells and vortex with low velocity. Using an unstained sample, adjust the forward scatter, side scatter and different fluorescent dye voltages, while adjust the flow cytometry compensation to eliminate the contamination signals between the different fluorophores, using the single staining samples.Draw a polygon gate through the forward scatter area, side scatter area dot plot to select the intact lymphocyte population, while excluding the debris, and a rectangular gate through the forward scatter area, forward scatter width dot plot to select the single cells. Next, draw a rectangular gate through the live, dead side scatter area dot plot to select the live cells, and a rectangular gate through the CD3 side scatter area dot plot to identify the CD three positive T-cells. Then, draw a quad gate through the CD4, CD8 dot plot to identify the CD4 positive or CD8 positive T-cells and through the CD45RO, CD27 dot plot to subdivide the CD4 positive or CD8 positive T-cells into TCM and TEM.After drawing the gates of CD107a, interferon gamma, tumor necrosis factor alpha, interleukin 2 and microphage inflammatory protein one alpha from the TCM or TEM of the CD8 positive or CD4 positive T-cells to determine the frequency of different response patterns respectively. Load the samples onto the cytometry sequentially. The gating strategy was employed to identify the TCM or TEM of CD8 positive or CD4 positive T-cells and their subsets using the forward scatter area width, then side scatter area dot plot.The poly functional characterization of CD4 positive and CD8 positive T-cell responses to JEV indicated that increased levels of CD107a, interferon gamma, tumor necrosis factor alpha and interleukin 2 were detected in the CD8 positive TCM cells of the vaccinated children after JEV stimulation compared to those in unvaccinated children. However, the level of macrophage inflammatory protein one alpha did not differ between the two groups. Higher levels of CD107a, and interferon gamma were detected in the CD8 positive TEM cells of the vaccinated group under JEV stimulation compared with the unvaccinated group.Whereas tumor necrosis factor alpha, interleukin 2 and macrophage inflammatory protein one alpha, were not significantly elevated in those positive cells. The JEV antigen successfully induced higher levels of CD107a, interferon gamma, tumor necrosis factor alpha, and macrophage inflammatory protein one alpha in the CD4 positive TCM cells of the vaccinated group compared with the unvaccinated group. However, the proportion of interleukin 2 positive cells did not differ between the two groups under JEV stimulation.The proportion of CD107a positive, interferon gamma positive and tumor necrosis factor alpha positive subsets, sub CD4 positive TEM cells in the vaccinated group was higher in the presence of JEV, than in the unvaccinated group. The T-cell immunogenicity of the stimuli and the compatible color matching strategies are critical steps in this protocol.

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