Name: In Vitro Functional Analysis of Regulatory T cells: Focus On Proliferation And Differentiation (Video) | JoVE Journal
Uploaded: 2026-06-09T14:05:00.000+00:00
Duration: 10 min 21 s
Description: Learn a modified in vitro Treg suppression assay to evaluate both proliferation and differentiation of effector T cells using mouse T cells and flow cytometry. This protocol advances immune regulation research.

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5 minutes

This work was supported by the Sichuan Science and Technology Program (NO. 2026NSFSC0940), the 1&#183;3&#183;5 Project for Disciplines of Excellence, West China Hospital, Sichuan University (NO. ZYYC25010), and the China Postdoctoral Science Foundation (NO. 2025M781396). D.Z. sincerely wants to commemorate Dr. Sang-A Park, who passed away in 2018. The experimental workflow was created with BioRender.com.

The authors have no conflicts of interest to declare.

In this study, the results of the co-culture under Th1 induction conditions are consistent with previous reports that Treg cells can suppress Th1 cell proliferation and differentiation17,18. Treg-enriched cells also inhibited the proliferation of Tresp cells under Th17 induction conditions. However, under the Th17-polarizing conditions used here, the frequency of IL-17A+ Tresp cells increased in the presence of Treg-enriched cells, which differs from p...

CD4+CD25+Foxp3+ regulatory T (Treg) cells are important mediators of immune homeostasis1. Treg cells actively suppress excessive immune responses and maintain self-immune tolerance, thereby preventing immune reactions from damaging normal tissues and organs2. Manipulating Treg cells is considered a potential therapeutic strategy for treating inflammatory diseases such as autoimmune disorders, solid organ transplant rejection, graft-versus-host disease, and allergic diseases3,4,5. Meanwhile, inhibiting the function of Treg cells is regarded as an effective strategy to activate anti-tumor immunity6,7. Clinical studies have investigated adoptive regulatory T cell therapy or low-dose IL-2 treatment for autoimmune disorders, solid organ transplantation, and graft-versus-host disease8,9,10. Moreover, studies on engineered Treg cells, including chimeric antigen receptor (CAR)-Treg cells and T-cell receptor (TCR)-Treg cells, are also gaining increasing attention11,12.
To date, Treg cell therapy has not been widely established in routine clinical treatment. To support the clinical application of Treg cell therapy, systematic research and evaluation of its functions are important. A common strategy for evaluating the functions of regulatory T cells in vitro is the in vitro Treg suppression assay13, which can detect the suppressive function of Treg cells on effector T (Teff) cell proliferation. However, the standard in vitro Treg suppression assay does not evaluate the differentiation of Teff cells. If proliferation and differentiation of effector T cells can be evaluated simultaneously, more information about the function of regulatory T cells can be obtained.
Here, we present a modified in vitro Treg cell suppression assay, termed &#8220;in vitro functional analysis of Treg cells&#8221; (Figure 1). This protocol provides an in vitro co-culture strategy for investigating the effects of Treg cells on the proliferation and differentiation of Teff cells.

<table><tbody><tr><td>1.5 mL centrifuge tube</td><td>Thermo Fisher Scientific</td><td>3457IW</td><td>or equivalent</td></tr><tr><td>12.5 &amp;mu;L Filtered Pipette Tips</td><td>Thermo Fisher Scientific</td><td>94420043</td><td>or equivalent</td></tr><tr><td>1250 &amp;mu;L Filtered Pipette Tips</td><td>Thermo Fisher Scientific</td><td>94420813</td><td>or equivalent</td></tr><tr><td>15 mL Conical Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>339650</td><td>or equivalent</td></tr><tr><td>200 &amp;mu;L Filtered Pipette Tips</td><td>Thermo Fisher Scientific</td><td>94420318</td><td>or equivalent</td></tr><tr><td>48-well plates</td><td>Corning Incorporated</td><td>3548</td><td>or equivalent</td></tr><tr><td>5 mL syringe piston</td><td>Shifeng Medical</td><td>SB2-061(A)</td><td>or equivalent</td></tr><tr><td>50 mL Conical Centrifuge Tubes</td><td>Thermo Fisher Scientific</td><td>339652</td><td>or equivalent</td></tr><tr><td>50 &amp;mu;L Filtered Pipette Tips</td><td>Thermo Fisher Scientific</td><td>94420258</td><td>or equivalent</td></tr><tr><td>70 &amp;mu;m cell strainer&amp;nbsp;</td><td>Biosharp Life Sciences</td><td>BS-70-CS</td><td>or equivalent</td></tr><tr><td>Anti-mouse CD28 antibody</td><td>BioXcell</td><td>BE0015-1</td><td>or equivalent</td></tr><tr><td>Anti-mouse CD3 antibody</td><td>BioXcell</td><td>BE0001-1</td><td>or equivalent</td></tr><tr><td>Anti-mouse CD4 PerCP-Cy5.5 (RM4-5)</td><td>Thermo Fisher Scientific</td><td>45-0042-82</td><td>or equivalent</td></tr><tr><td>Anti-mouse Foxp3 PB (FJK-16s)</td><td>Thermo Fisher Scientific</td><td>48-5773-82</td><td>or equivalent</td></tr><tr><td>Anti-mouse IFN-gamma PE (XMG1.2)</td><td>BioLegend</td><td>505808</td><td>or equivalent</td></tr><tr><td>Anti-mouse IL-17A PE-Cy7 (eBio17B7)</td><td>Thermo Fisher Scientific</td><td>25-7177-82</td><td>or equivalent</td></tr><tr><td>Beckman CytoFLEX S Flow Cytometer</td><td>Beckman Coulter</td><td>N/A</td><td>for flow cytometric data acquisition of immune cell samples</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>B2064</td><td>or equivalent</td></tr><tr><td>C57BL/6 mice</td><td>Shanghai Model Organisms Center, Inc</td><td>SM-001</td><td>for isolation of primary lymphocytes and T cell subsets</td></tr><tr><td>CD25 Monoclonal Antibody (PC61.5), PE</td><td>Thermo Fisher Scientific</td><td>12-0251-83</td><td>or equivalent</td></tr><tr><td>CD4+CD25+ Regulatory T Cell Isolation Kit, mouse</td><td>Miltenyi Biotec</td><td>130-091-041</td><td>for cell sorting</td></tr><tr><td>CD4+CD62L+ T Cell Isolation Kit, mouse</td><td>Miltenyi Biotec</td><td>130-106-643</td><td>for cell sorting</td></tr><tr><td>CD45.1 mice</td><td>Shanghai Model Organisms Center, Inc</td><td>NM-KI-210226</td><td>for isolation of primary lymphocytes and T cell subsets</td></tr><tr><td>CD45.1 Monoclonal Antibody (A20), FITC</td><td>Thermo Fisher Scientific</td><td>11-0453-85</td><td>or equivalent</td></tr><tr><td>CD45.2 Monoclonal Antibody (104), APC-eFluor 780</td><td>Thermo Fisher Scientific</td><td>47-0454-82</td><td>or equivalent</td></tr><tr><td>CD62L (L-Selectin) Monoclonal Antibody (MEL-14), FITC&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11-0621-85</td><td>or equivalent</td></tr><tr><td>Cytofix/Cytoperm Fixation/Permeabilization Solution Kit</td><td>BD Biosciences</td><td>554714</td><td>or equivalent</td></tr><tr><td>eBioscience Cell Proliferation Dye eFluor 450</td><td>Thermo Fisher Scientific</td><td>65-0842-90</td><td>or equivalent</td></tr><tr><td>ethylenediamine tetraacetic acid (EDTA)</td><td>Solarbio Life Sciences</td><td>E1170</td><td>or equivalent</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>A5669701</td><td>or equivalent</td></tr><tr><td>FlowJo 10.6.2</td><td>BD Biosciences</td><td>N/A</td><td>software for flow cytometry data analysis</td></tr><tr><td>Golgi-Plug Protein Transport Inhibitor</td><td>BD Biosciences</td><td>555029</td><td>or equivalent</td></tr><tr><td>HEPES (1 M)</td><td>Thermo Fisher Scientific</td><td>15630080</td><td>or equivalent</td></tr><tr><td>Human TGF-beta1</td><td>PEPROTECH</td><td>100-21C</td><td>or equivalent</td></tr><tr><td>Ionomycin calcium salt</td><td>Millipore Sigma</td><td>I3909</td><td>or equivalent</td></tr><tr><td>LD Columns</td><td>Miltenyi Biotec</td><td>130-042-901</td><td>for cell sorting</td></tr><tr><td>L-Glutamine (200 mM)&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>25030081</td><td>or equivalent</td></tr><tr><td>LS Columns</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>for cell sorting</td></tr><tr><td>MACS MultiStand</td><td>Miltenyi Biotec</td><td>130-042-303</td><td>for cell sorting</td></tr><tr><td>MS Columns</td><td>Miltenyi Biotec</td><td>130-042-201</td><td>for cell sorting</td></tr><tr><td>OctoMACS Separators</td><td>Miltenyi Biotec</td><td>130-042-109</td><td>for cell sorting</td></tr><tr><td>PBS, pH 7.4</td><td>Thermo Fisher Scientific</td><td>10010049</td><td>or equivalent</td></tr><tr><td>PE Rat IgG1, &amp;kappa; Isotype Ctrl Antibody</td><td>BioLegend</td><td>400408</td><td>or equivalent</td></tr><tr><td>PE/Cyanine7 Rat IgG2a kappa Isotype Control Antibody</td><td>Thermo Fisher Scientific</td><td>25-4321-82</td><td>or equivalent</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>or equivalent</td></tr><tr><td>PMA</td><td>Millipore Sigma</td><td>P8139</td><td>or equivalent</td></tr><tr><td>Purified anti-mouse CD16/32 Antibody</td><td>BioLegend</td><td>101302</td><td>or equivalent</td></tr><tr><td>QuadroMACS Separator</td><td>Miltenyi Biotec</td><td>130-090-976</td><td>for cell sorting</td></tr><tr><td>Recombinant mouse IL-12 p70</td><td>PEPROTECH</td><td>210-12</td><td>or equivalent</td></tr><tr><td>Recombinant Mouse IL-6</td><td>R&amp;amp;D Systems</td><td>406-ML</td><td>or equivalent</td></tr><tr><td>Red Blood Cell Lysis Buffer (ACK Lysing Buffer)</td><td>Thermo Fisher Scientific</td><td>A1049201</td><td>or equivalent</td></tr><tr><td>RPMI 1640 medium&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>11875093</td><td>or equivalent</td></tr><tr><td>Sodium Pyruvate (100 mM)</td><td>Thermo Fisher Scientific</td><td>11360070</td><td>or equivalent</td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermo Fisher Scientific</td><td>15250061</td><td>or equivalent</td></tr><tr><td>UltraComp eBeads Compensation Beads&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>01-2222-42</td><td>or equivalent</td></tr><tr><td>Zombie Yellow Fixable Viability Kit</td><td>BioLegend</td><td>423104</td><td>or equivalent</td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M3148</td><td>or equivalent</td></tr></tbody></table>

in vitro functional analysis of regulatory t cells: focus on proliferation and differentiation

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

Regulatory T (Treg) cells play key roles in maintaining immune homeostasis. Abnormalities in Treg cell function are associated with various diseases, including autoimmune diseases, organ transplant rejection, infection, and cancer. Manipulating Treg cells is considered a potential therapeutic strategy for treating inflammatory diseases, including autoimmune disorders, solid organ transplant rejection, graft-versus-host disease, and allergic diseases. Classic in vitro Treg cell suppression assays mainly assess the inhibitory effect of Treg cells on effector T (Teff) cell proliferation. However, in addition to proliferation, T cell differentiation is important for T cells to perform their immune functions. Therefore, this protocol provides a modified in vitro Treg cell suppression assay for investigating the effects of Treg cells on the proliferation and differentiation of Teff cells. In this protocol, the preparation of mouse Treg cells and naive T cells is first introduced, followed by the co-culture strategy for studying the regulatory effects of Treg cells on the proliferation and differentiation of Teff cells, including Th1 and Th17 cells, and finally, the staining strategy for flow cytometry after co-culture is presented. This in vitro functional analysis of Treg cells can be used to investigate the effects of Treg cells on T cell proliferation and polarization.

In the representative experiment, naïve T cells were used as Tresp cells to investigate the effects of Treg-enriched cells on the proliferation and differentiation of Th1 and Th17 cells. In this experiment, Tresp cells were activated by anti-mouse CD3 and APC-enriched non-CD4+ cells, and then differentiated into Th1 cells under IL-12 p70 induction or into Th17 cells under TGF-β1 and IL-6 induction. Under these two induction conditions, changes in the proliferation and differentiation of Tresp cells were studie...

This protocol provides an in vitro co-culture strategy for investigating the effects of regulatory T cells on effector T cell proliferation and differentiation.

Regulatory T (Treg) cells play key roles in maintaining immune homeostasis. Abnormalities in Treg cell function are associated with various diseases, ...

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.

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Learn a modified in vitro Treg suppression assay to evaluate both proliferation and differentiation of effector T cells using mouse T cells and flow cytometry. This protocol advances immune regulation research.

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

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