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

Summary

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

Abstract

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.

Introduction

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 diseases^3,4,5. Meanwhile, inhibiting the function of Treg cells is regarded as an effective strategy to activate anti-tumor immunity^6,7. 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 disease^8,9,10. Moreover, studies on engineered Treg cells, including chimeric antigen receptor (CAR)-Treg cells and T-cell receptor (TCR)-Treg cells, are also gaining increasing attention^11,12.

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 “in vitro functional analysis of Treg cells” (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.

Protocol

All mouse experiments were authorized by the Animal Ethics Committee at West China Hospital, Sichuan University (Approval Nos. 20250814006, 20211212A). All mice used in this experiment were on a C57BL/6 genetic background.

1. Preparation of solutions

1. Prepare fluorescence-activated cell sorting (FACS) buffer: 1× PBS containing 0.5% bovine serum albumin (BSA).
2. Prepare magnetic-activated cell sorting (MACS) buffer: 1× PBS containing 0.5% BSA and 2 mM ethylenediamine tetraacetic acid (EDTA).
3. Prepare cell culture medium: RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, 55 µM β-mercaptoethanol, 100 U/mL penicillin, and 100 µg/mL streptomycin.

2. Preparation of C57BL/6 mouse lymphocytes

1. Take the spleen and peripheral lymph nodes (LNs) of a 6–8-week-old C57BL/6 mouse (either male or female) under sterile conditions and place them in a 50 mL centrifuge tube with 5 mL of FACS buffer.
   NOTE: The dissection of the spleen and LNs should be performed according to standard procedures.
2. Place a 70 µm cell strainer on a 50 mL centrifuge tube, then place the spleen and LNs on the strainer, and use a 5 mL syringe piston to grind the tissue gently for 1 min. During grinding, rinse the strainer 3x with FACS buffer. Make sure to grind all the tissue completely and rinse the cells into the centrifuge tube. After grinding, remove the strainer.
3. Centrifuge at 4 °C, 350 × g for 7 min.
4. Remove the supernatant, resuspend the cell pellet in 3 mL of red blood cell lysis buffer, and leave the suspension at room temperature for 3–5 min.
5. Immediately add FACS buffer to the centrifuge tube to a volume of 15–20 mL to terminate the lysis, and centrifuge at 4 °C, 350 × g for 7 min.
6. Remove the supernatant, add 10 mL of FACS buffer, and gently resuspend the cell pellet.
7. Take 10 µL of cell suspension and use trypan blue staining to count the total live lymphocytes.
   NOTE: These cells are CD45.2⁺ lymphocytes.

3. Preparation of CD45.1 mouse lymphocytes

1. Take the spleen and LNs of a 6–8-week-old CD45.1 mouse (either male or female) under sterile conditions and place them in a 50 mL centrifuge tube with 5 mL of FACS buffer.
2. Prepare lymphocytes from the CD45.1 mouse by following the same steps described above.
   NOTE: These cells are CD45.1⁺ lymphocytes.

4. Preparation of CD45.2⁺CD4⁺CD62L⁺ naïve T cells

NOTE: The entire process uses the CD4⁺CD62L⁺ T Cell Isolation Kit for MACS. The MACS buffer is pre-cooled at 4 °C, and all centrifugation conditions are 4 °C, 350 × g for 7 min.

1. Collect the cell suspension from C57BL/6 mice, centrifuge and discard the supernatant, add 400 µL of MACS buffer and 100 µL of CD4⁺ T Cell Biotin-Antibody Cocktail to the centrifuge tube per 1 × 10⁸ initial total cells. Mix gently and incubate at 4 °C for 5 min in the dark.
2. Add 300 µL of MACS buffer and 200 µL of Anti-Biotin Microbeads to the centrifuge tube from step 4.1 per 1 × 10⁸ initial total cells. Mix gently and incubate at 4 °C for 10 min in the dark.
3. Add 10 mL of MACS buffer, centrifuge, and discard the supernatant. Resuspend the cell pellet in 2 mL of MACS buffer.
4. Place the LS column on the appropriate MACS sorting magnetic separator. Use 3 mL of MACS buffer to rinse the column. Wait until the buffer in the column completely drains before proceeding to the next step.
   NOTE: The maximum cell loading capacity of the LS column is 2 × 10⁹ cells, including both positive and negative cells, and the maximum number of positive cells it can capture is 1 × 10⁸. Usually, a single mouse only requires one LS column here.
5. Load the cell suspension from step 4.3 onto the LS column. When the cell suspension completely flows into the column, use 3 mL of MACS buffer to wash the column. Collect all the flowthrough liquid in a new 15 mL centrifuge tube.
   NOTE: These are the total CD4⁺CD25^– T cells.
6. Centrifuge the total CD4⁺CD25^– T cells obtained in step 4.5, discard the supernatant, and obtain the cell pellet.
7. Add 800 µL of MACS buffer and 200 µL of CD62L Microbeads to the cell pellet per 1 × 10⁸ initial total cells. Mix gently and incubate at 4 °C for 10 min in the dark.
8. Add 10 mL of MACS buffer to the centrifuge tube, centrifuge and discard the supernatant, and resuspend the cell pellet in 500 µL of MACS buffer.
9. Place the MS column on the appropriate MACS sorting magnetic separator and use 1 mL MACS buffer to rinse the column.
10. Load the cell suspension onto the MS column. When the suspension completely flows into the column, use 1 mL of MACS buffer to wash the column 2x.
11. Remove the MS column from the magnetic separator and place it on a new 15 mL centrifuge tube. Add 1 mL of MACS buffer to the column and immediately push the plunger firmly to elute the cells labeled with magnetic beads.
12. Count the cells, centrifuge and discard the supernatant, resuspend the cells in an appropriate volume of culture medium, and keep the tube on ice.
    NOTE: These cells are CD45.2⁺CD4⁺CD62L⁺ naïve T cells. Usually, approximately 8 × 10⁶ to 1 × 10⁷ CD4⁺CD25^–CD62L⁺ naive T cells can be purified from one mouse; inclusion of peripheral lymph nodes can increase the yield.
13. Determine the purity of the isolated cells: Take 5 × 10⁴ isolated CD4⁺CD62L⁺ T cells and resuspend them in 50 µL of antibody solution in a 1.5 mL centrifuge tube.
    NOTE: Prepare the antibody solution by adding the anti-mouse CD4 PerCP-Cy5.5 antibody, anti-mouse CD62L FITC antibody, and anti-mouse CD25 PE antibody into the FACS buffer at a 1:100 ratio.
14. Following the 20 min incubation at 4 °C in the dark, add 1 mL of FACS buffer to wash the cells. Centrifuge the cells, remove the supernatant, and resuspend the pellet in 200 µL of FACS buffer.
15. Detect the purity of the samples by flow cytometry.
    NOTE: For details of flow cytometer analysis, refer to the user manual of the instrument or the previously reported protocol¹⁴. The purity of CD4⁺CD25^–CD62L⁺ naive T cells should be greater than 95% in the isolated T cell sample (Figure 2).

5. Preparation of CD45.1⁺CD4⁺CD25⁺ Treg-enriched cells and antigen-presenting cell-enriched non-CD4⁺ cells

NOTE: The entire process uses the CD4⁺CD25⁺ Regulatory T Cell Isolation Kit for MACS, and the instructions in the kit have been optimized to simultaneously obtain antigen-presenting cell (APC)-enriched non-CD4⁺ cells and CD4⁺CD25⁺ Treg-enriched cells. The MACS buffer is precooled at 4 °C, and all centrifugation conditions are 4 °C, 350 × g for 7 min.

1. Collect the cell suspension from CD45.1 mice, centrifuge, and discard the supernatant, add 400 µL of precooled MACS buffer and 100 µL of CD4⁺CD25⁺ Regulatory T Cell Biotin-Antibody Cocktail to the centrifuge tube per 1 × 10⁸ initial total cells. Mix gently and incubate at 4 °C for 10 min in the dark.
2. Without washing, directly add 380 µL of precooled MACS buffer, 200 µL of Anti-Biotin Microbeads, and 20 µL of CD25-PE antibody per 1 × 10⁸ initial total cells to the centrifuge tube from step 5.1. Mix gently and incubate at 4 °C for 15 min in the dark.
3. Add 10 mL of MACS buffer to the centrifuge tube, centrifuge, and discard the supernatant, then resuspend the cell pellet in 500 µL of MACS buffer per 1 × 10⁸ initial total cells.
4. Place the LD column on the appropriate MACS sorting magnetic separator and rinse the column with 2 mL of MACS buffer. Wait until the buffer in the column completely drains before proceeding to the next step.
5. Slowly load the cell suspension from step 5.3 onto the LD column. When the cell suspension completely flows into the column, wash the column 2x with 2 mL MACS buffer. Collect all the flowthrough liquid in a new 15 mL centrifuge tube.
6. Remove the LD column from the magnetic separator and place it on a new 15 mL centrifuge tube. Add 3 mL of MACS buffer to the LD column, immediately push the plunger firmly to expel the Microbeads-labeled non-CD4⁺ cells. Count the cells, centrifuge, resuspend the cells in an appropriate volume of cell culture medium to achieve a cell concentration of 1 × 10⁷ cells/mL, and keep the tube on ice.
   NOTE: These non-CD4⁺ cells mainly consist of APCs such as macrophages, dendritic cells, and B cells. In this protocol, these cells are used as an APC-enriched non-CD4⁺ fraction.
7. Centrifuge the total CD4⁺ T cells collected from step 5.5, discard the supernatant, and obtain the cell pellet.
8. Add 900 µL of MACS buffer and 100 µL of Anti-PE Microbeads to the cell pellet per 1 × 10⁸ initial total cells. Mix gently and incubate at 4 °C for 15 min in the dark.
9. Place the MS column on the appropriate MACS sorting magnetic separator and rinse the column with 500 µL of precooled MACS buffer. Wait until the buffer in the column completely drains before proceeding to the next step.
10. Slowly load the cell suspension from step 5.8 onto the MS column. When the cell suspension completely flows into the column, wash the column 2x with 1 mL MACS buffer. Collect the flowthrough liquid in a new 15 mL centrifuge tube.
    NOTE: The cells in the flowthrough liquid at this time are CD4⁺CD25^– T cells.
11. Detach the MS column from the magnetic separator and position it over a fresh 15 mL centrifuge tube. Add 1 mL of MACS buffer to the column, then promptly depress the plunger to recover the magnetic bead-labeled CD4⁺CD25⁺ Treg-enriched cells.
12. After counting the cells, centrifuge the suspension and remove the supernatant. Resuspend the pellet in enough culture medium to obtain a final concentration of 2 × 10⁶ cells/mL, and keep the tube on ice.
    NOTE: These cells are CD45.1⁺CD4⁺CD25⁺ Treg-enriched cells. Usually, approximately 8 × 10⁵ to 1 × 10⁶ CD4⁺CD25⁺ cells can be purified from one mouse; inclusion of peripheral lymph nodes can increase the yield.
13. Determine the purity of the isolated CD4⁺CD25⁺ Treg-enriched cells: Take 5 × 10⁴ isolated CD4⁺CD25⁺ Treg-enriched cells and 5 × 10⁴ isolated APC-enriched non-CD4⁺ cells, respectively, and resuspend them in 50 µL of antibody solution.
    NOTE: Prepare the antibody solution by adding the anti-mouse CD4 PerCP-Cy5.5 antibody and anti-mouse CD25 PE antibody into the FACS buffer at a 1:100 ratio. Use the same anti-mouse CD25 PE antibody here to confirm that the CD4⁺CD25⁺ cells are adequately labeled and that there are no CD4⁺CD25⁺ T cells in the non-CD4⁺ fraction. For more stringent identification of Treg cells, intracellular staining with an anti-mouse Foxp3 PB antibody can also be carried out.
14. Following the 20 min incubation at 4 °C in the dark, add 2 mL of FACS buffer to wash the cells. Centrifuge the cells, remove the supernatant, and resuspend the pellet in 200 µL of FACS buffer.
15. Detect the purity of the samples by flow cytometry.
    NOTE: The frequency of CD4⁺CD25⁺ cells should be ≥ 95% to ensure that the purity of the Treg-enriched cell population meets the experimental requirements (Figure 2). Non-CD4⁺ cells used as the APC-enriched fraction should contain no more than 5% CD4⁺ T cells (Figure 2).

6. Labeling of naïve CD4⁺ T cells with cell proliferation dye

NOTE: All centrifugation conditions are 4 °C, 350 × g for 7 min. The PBS buffer is preequilibrated to room temperature before use. The labeling process is conducted under light protection to avoid dye quenching.

1. Take the naïve CD4⁺ T cell suspension, centrifuge, discard the supernatant, and obtain the cell pellet.
2. Dilute the Cell Proliferation Dye eFluor 450 in PBS to obtain a concentration of 20 µM at room temperature.
3. Add 0.5 mL of PBS per 1 × 10⁷ cells. Keep the tube tilted at approximately 45° and gently resuspend the cells by pipetting up and down 50x.
4. Add an equal volume of cell proliferation dye solution quickly into the cell suspension, so that the final concentration of the cell proliferation dye is 10 µM. Keep the tube tilted at approximately 45° and mix the cells by pipetting up and down 50x to ensure full contact between the cells and the cell proliferation dye.
5. Immediately wrap the centrifuge tube with aluminum foil to avoid light exposure. Incubate at room temperature for 20 min. Gently vortex the cells every 5 min to prevent cell aggregation.
6. Add 10 mL of precooled cell culture medium to the centrifuge tube, gently vortex to terminate the staining, then centrifuge, and discard the supernatant.
7. Add 5 mL of precooled cell culture medium to the cell pellet, centrifuge, and discard the supernatant to wash the cells.
8. Add 5 mL of precooled cell culture medium to the cell pellet again and resuspend the cells.
9. Count the cells, centrifuge and discard the supernatant, resuspend the cells in an appropriate volume of culture medium to achieve a cell concentration of 2 × 10⁶ cells/mL, and keep the tube on ice.

7. Setting up the in vitro functional analysis of Treg cells

1. Calculate the number of samples required for the cell culture, prepare an appropriate amount of cell culture medium containing 2.5 µg/mL anti-mouse CD3 antibody or the indicated induction cytokines, and gently vortex the prepared CD45.2⁺CD4⁺CD62L⁺ naïve T cells, CD45.1⁺CD4⁺CD25⁺ Treg-enriched cells, and APC-enriched non-CD4⁺ cells.
   NOTE: Use CD45.2⁺CD4⁺CD62L⁺ naïve T cells as responder T (Tresp) cells. This example experiment studies the effect of Treg cells on the differentiation and proliferation of Th1 and Th17 cells. Therefore, there are a total of four samples for the cell culture: Tresp cells cultured under Th1 or Th17 induction conditions, in the absence or presence of Treg-enriched cells.
2. Add 100 µL of CD45.2⁺CD4⁺ naïve T cells (2 × 10⁵ cells per well) to the 48-well plate.
3. Add 100 µL of CD45.1⁺ APC-enriched non-CD4⁺ cells (1 × 10⁶ cells per well) to the 48-well plate.
4. Add 100 µL of CD45.1⁺CD4⁺CD25⁺ Treg-enriched cells (2 × 10⁵ cells per well) to the experimental wells, and add 100 µL of cell culture medium to the control wells. The Treg-enriched cell:Tresp ratio in the experimental samples is 1:1.
   NOTE: The ratio of Treg cells to Tresp cells can be set as needed, or a panel of multiple ratios can be designed. The number of Tresp cells is fixed in the co-culture, while the number of Treg cells can be changed as needed. Typically, the ratios of Treg to Tresp in the experimental samples include 1:1, 1:2, 1:4, 1:8, 1:16, and 0:1. Furthermore, each experimental condition can be prepared in triplicate to minimize random experimental error. Here, for the sake of clarity, based on previous research experience^15,16, a 1:1 ratio is used as an example.
5. Add 100 µL of cell culture medium containing 2.5 µg/mL anti-mouse CD3 antibody to each well.
   NOTE: The final concentration of anti-mouse CD3 antibody is 0.5 µg/mL. In addition to the experimental samples, set a blank control sample without anti-mouse CD3 antibody. In this sample, add 100 µL of T cell culture medium without anti-mouse CD3 antibody to determine the fluorescence intensity of the non-proliferated T cells, or the intensity of peak 0. This allows accurate calibration of the proliferation distribution.
6. Add 100 µL of cell culture medium containing the indicated cytokines to each well to make the final volume of each well 500 µL.
   NOTE: In this experiment, recombinant mouse IL-12 p70 at a final concentration of 10 ng/mL is used to induce Th1 cells, and recombinant human TGF-β1 at a final concentration of 2 ng/mL and recombinant mouse IL-6 at 50 ng/mL are used to induce Th17 cells.
7. Add PBS to the remaining empty wells (500 µL/well) to maintain the humidity of the plate and reduce edge effects.
8. Incubate the 48-well plate in a 5% CO₂ incubator at 37 °C for 3 days.
   NOTE: In this co-culture system, soluble anti-mouse CD3 antibody provides the primary T cell receptor (TCR) signal for T cell activation, and APC-enriched non-CD4⁺ cells provide costimulatory signals to fully activate T cells. TCR and costimulatory signals can also be triggered by plate-coated anti-mouse CD3 antibody and soluble anti-mouse CD28 antibody. Researchers can choose the stimulation method as needed.

8. In vitro functional analysis of Treg cells

NOTE: All centrifugation conditions are 4 °C, 350 × g for 7 min.

1. Gently pipette the cultured cells to resuspend them and transfer the suspension to a 1.5 mL centrifuge tube. Centrifuge the cells and remove the supernatant.
2. Add 500 µL of cell culture medium containing 5 ng/mL PMA, 1 µg/mL ionomycin, and 1:1,000 diluted GolgiPlug Protein Transport Inhibitor to each tube, gently vortex to resuspend the cells, and incubate them in a 5% CO₂ incubator at 37 °C for 3 h.
3. Centrifuge, discard the supernatant, add 50 µL of PBS containing one test of Zombie Yellow dye and 1:100 diluted anti-mouse CD16/32 antibody to each sample, vortex, and incubate at room temperature in the dark for 15 min to label dead cells and block Fc receptors.
4. Centrifuge, discard the supernatant, add 50 µL of surface antibody cocktail, vortex, and incubate at 4 °C in the dark for 20 min.
   NOTE: Add anti-CD4 PerCP-Cy5.5 antibody, anti-CD45.1 FITC antibody, and anti-CD45.2 APC-eFluor 780 antibody to the FACS buffer at a 1:200 ratio to prepare the surface antibody cocktail.
5. Add 1 mL of FACS buffer to each centrifuge tube, centrifuge, and discard the supernatant.
6. Add 300 µL of Fixation and Permeabilization Solution to each centrifuge tube, vortex, and incubate at 4 °C in the dark for 20 min.
7. Add 1 mL of 1× Permeabilization/Wash (Perm/Wash) Buffer to each centrifuge tube, centrifuge, and discard the supernatant; then repeat this wash step once to ensure complete removal of residual fixative.
8. Add 50 µL of cytokine antibody cocktail, vortex, and incubate at 4 °C in the dark for 40 min.
   NOTE: Add anti-IFN-γ PE antibody and anti-IL-17A PE-Cy7 antibody to the Perm/Wash Buffer at a 1:100 ratio to prepare the cytokine antibody cocktail.
9. Wash each sample with 1 mL of Perm/Wash buffer, centrifuge the cells, and remove the supernatant. Resuspend the pellet in 300 µL of FACS buffer.
10. Measure the proliferation and differentiation of Tresp cells by flow cytometry. Use the following gating strategy (Figure 3): first, gate the lymphocyte population among the total cells and exclude cell debris and impurities; second, gate the single-cell population among the lymphocytes based on scatter signals; third, gate the live cell population among the single cells; fourth, gate the CD4⁺ T cell population among the live cells; fifth, gate the CD45.1⁺ Treg-enriched cell population and CD45.2⁺ Teff cell population among the CD4⁺ T cells; sixth, gate CD45.2⁺CD4⁺IFN-γ⁺ Th1 cells, CD45.2⁺CD4⁺IL-17A⁺ Th17 cells, and non-proliferated CD45.2⁺CD4⁺ T cells among the CD45.2⁺ T cells.
11. Perform flow cytometry data acquisition and analyze the flow cytometry data.
    1. Use cultured T cells labeled with cell proliferation dye eFluor 450 as the cell proliferation dye single-stained control. Prepare the Zombie Yellow dye single-stained control with cultured T cells that were not labeled with cell proliferation dye. Prepare the other single-stained controls with compensation beads for every fluorophore used.
    2. Use T cells labeled with cell proliferation dye eFluor 450 and cultured without anti-mouse CD3 antibody to define the gate of non-proliferated cells.
    3. Use cultured T cells labeled with cell proliferation dye and stained with Zombie Yellow dye, all cell surface antibodies, and isotype controls of anti-mouse IFN-γ PE antibody and anti-mouse IL-17A PE-Cy7 antibody to define gates of IFN-γ⁺CD4⁺ and IL-17A⁺CD4⁺ T cells.

Results

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...

Discussion

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 differentiation^17,18. 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...

Materials

List of materials used in this article

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