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