In Vitro Functional Analysis of Regulatory T cells: Focus On Proliferation And Differentiation

Our lab studied T cell subset differentiation, metabolism, and interaction. Treg affect T cell crosstalk, maintains immune homeostasis, making Treg research crucial for treating autoimmune and inflammatory diseases. Traditional in vitro Treg suppression assays overlooked Treg-mediated effects and effector T cell differentiation.

This modified protocol evaluates both proliferation and polarization simultaneously. To begin, prepare naive T cells, regulatory T or Treg-enriched cells, and antigen-presenting cell-enriched non-CD4 positive cells. Centrifuge the naive CD4-positive T cell suspension at 350G for seven minutes at four degrees Celsius and discard the supernatant to obtain the cell pellet.

Dilute the cell proliferation dye eFluor 450 in PBS to obtain a concentration of 20 micromolar at room temperature. Add 0.5 milliliters of PBS per one times 10 to the power of seven cells to the tube. Keep the tube tilted at approximately 45 degrees and gently resuspend the cells by pipetting up and down 50 times.

Next, add an equal volume of the cell proliferation dye solution quickly into the cell suspension to obtain a final concentration of 10 micromolar. Hold the tube at a 45 degree angle and pipette the cells to thoroughly mix them with the cell proliferation dye. Immediately wrap the centrifuge tube with aluminum foil to avoid light exposure.

Incubate at room temperature for 20 minutes while gently vortexing them every five minutes to prevent cell aggregation. Next, add 10 milliliters of pre-cooled cell culture medium to the centrifuge tube and gently vortex the cells to terminate the staining. Then centrifuge the cells and discard the supernatant.

To wash the cells, add five milliliters of pre-cooled cell culture medium to the cell pellet. Repeat the centrifugation and resuspend the pellet in five milliliters of culture medium. Following cell counting, centrifuge the cells and resuspend in an appropriate volume of culture medium to achieve a cell concentration of two times 10 to the power of six cells per milliliter.

Keep the tube on ice. Calculate the number of samples required for the cell culture. Prepare an appropriate amount of cell culture medium containing 2.5 micrograms per milliliter anti-mouse CD3 antibody or the suitable induction cytokines.

Take prepared naive T cells and Treg-enriched cells and vortex them. Add 100 microliters of CD45.2 positive, CD4 positive naive T cells, and 100 microliters of CD45.1 positive APC-enriched non-CD4 positive cells to the 48-well plate. Transfer 100 microliters of CD45.1 positive, CD4 positive, CD25 positive Treg-enriched cells into the experimental wells and 100 microliters of cell culture medium into the control wells.

Then pipette 100 microliters of culture medium containing anti-mouse CD3 antibody and 100 microliters of culture medium containing the suitable induction cytokines into each well. Add PBS to the remaining empty wells to maintain the humidity of the plate and reduce edge effects. Incubate the 48-well plate in a 5%carbon dioxide incubator at 37 degrees Celsius for three days.

Gently pipette the cultured cells to resuspend them and transfer the cell suspension to a 1.5 milliliter centrifuge tube. Centrifuge the cells and remove the supernatant. Add 500 microliters of cell culture medium containing phorbol 12-myristate 13-acetate, ionomycin, and GolgiPlug protein transport inhibitor to each tube.

Gently vortex to resuspend the cells and incubate the cells in a 5%carbon dioxide incubator at 37 degrees Celsius for three hours. Following centrifugation, add 50 microliters of PBS containing one test of zombie yellow dye and 1:100 diluted anti-mouse CD16 or 32 antibodies to the pellet. Vortex the samples and incubate at room temperature in the dark for 15 minutes to label dead cells and block FC receptors.

Centrifuge the cells before discarding the supernatant. Add 50 microliters of surface antibody cocktail to the sample, vortex it, and incubate at four degrees Celsius in the dark for 20 minutes. Then add one milliliter of fluorescence-activated cell sorting or FACS buffer to each centrifuge tube.

After centrifugation and supernatant removal, add 300 microliters of fixation and permeabilization solution to each centrifuge tube and vortex the samples. Incubate the samples at four degrees Celsius in the dark for 20 minutes. Transfer on milliliter of permeabilization and wash buffer to each centrifuge tube followed by centrifugation and supernatant removal.

After repeating the wash, add 50 microliters of cytokine antibody cocktail to each sample and vortex the samples. Incubate the samples at four degrees Celsius in the dark for 40 minutes. Wash each sample with one milliliter of permeabilization and wash buffer.

Centrifuge the cells and remove the supernatant. Resuspend the cell pellet in 300 microliters of FACS buffer. Measure the proliferation and differentiation of responder T cells by flow cytometry.

Gate the lymphocyte population among the total cells to exclude cell debris and impurities. Select the single cell population among the lymphocytes based on scatter signals. Then identify the live cell population among the single cells, followed by the CD4 positive T cell population among the live cells.

Next, gate the CD45.1 positive regulatory T cell enriched population and the CD45.2 positive effector T cell population among the CD4 positive T cells. Then select the CD45.2 positive CD4 positive interferon gamma positive T helper 1 cells, CD45.2 positive CD4 positive interleukin-17A positive T helper 17 cells, and non-proliferated CD45.2 positive CD4 positive T cells among the CD45.2 positive T cells. Perform flow cytometry data acquisition for all samples.

Finally, analyze the flow cytometry data with FlowJo software using the same gating strategy as in flow cytometry data acquisition. The frequency of non-proliferated responder T cells was significantly increased under both T helper 1 and T helper 17 induction conditions after co-culture with regulatory T cell enriched cells. The differentiation of CD45.2 positive, CD4 positive interferon gamma, positive T helper 1 cells was significantly suppressed after co-culture with regulatory T cell enriched cells.

The frequency of CD45.2 positive, CD4 positive interleukin-17A positive T helper 17 cells increased when responder T cells were cultured with regulatory T cell enriched cells. In vitro functional analysis of Treg cells enable investigation of biodirectional effects between Treg and effector T cells on proliferation and polarization. The greatest challenge while performing this protocol is how to maintain the cell viability while ensuring the purity of Treg cells and naive T cells.

Following this process, ELISA and other biomedical assays can assess cytokine and metabolic effector production while FACS sorted cells enable downstream RNA-seq and proteomics analysis.