Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (T_reg) cells express forkhead box P3 (Foxp3) as a transcription factor for their development and function¹. Additionally, T_reg cells express various other molecules such as CD25², lymphocyte-activation gene 3 (LAG-3)³, glucocorticoid-induced tumor necrosis factor receptor⁴, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)⁵ on their surface or intracellular region. During chronic infection with various kinds of pathogens such as viruses^6,7, bacteria^8,9, and parasites^10-12, or in the course of cancer development^13,14, T_reg cells become differentiated into activated cells, displaying enhanced suppressive function targeting effector CD4⁺ and CD8⁺ T cells. A number of papers have suggested that expanded and activated T_reg cells contribute to the impaired CD8⁺ T cell response during friend retrovirus (FV) infection^15-17. FV-induced T_reg cells inhibit IFN-γ or granzyme B expression and cytotoxic reactivity of CD8⁺ T cells^15-17. Moreover, in a herpes simplex virus infection model, it was reported that depletion of CD4⁺CD25⁺ T_reg cells resulted in expansion of virus-specific CD8⁺ T cells and severe tissue damage by infiltration of immunopathogenic CD4⁺ T cells^18-20.

Mice infected chronically with the clone 13 strain of lymphocytic choriomeningitis virus (LCMV CL13)^21-24 have been widely used to characterize the phenotype and function of effector T cells (T_eff) and T_reg cells during chronic virus infection. During persistent LCMV infection, virus-specific T_eff cells progressively lose their effector function and become exhausted T (T_exh) cells. On the other hand, T_reg cells reinforce their ability to suppress virus-specific T cell response²⁵. The decrease in the functioning capacity of the T_eff cells can be explained by several factors such as upregulation of inhibitory receptors on T_eff cells, altered function of antigen-presenting cells, production of immunoregulatory cytokines, and increased frequency or enhanced function of T_reg cells²⁶. Among the factors involved in T cell suppression, programmed cell death protein-1 (PD-1)-expressing T_exh cells and T_reg cells have been widely considered as the hallmarks of antigen persistence and suppressive environment. Recently, it was reported that blockade of the PD-1 pathway and ablation of T_reg cells lead to enhanced T cell function and decreased viral load during LCMV chronic infection²⁷. Furthermore, T_reg cells are activated during chronic infection of mice with LCMV^23,25 and their suppressive function is strengthened²⁵. PD-1 is highly expressed on T_reg cells as well as T_exh cells, and the level of PD-1 expressed by T_reg cells correlates with the strength of their suppressive function to inhibit T cell proliferation²⁵.

Here, we describe a method to compare the characteristics of activated T_reg cells isolated from mice infected with LCMV CL13 and resting T_reg cells isolated from naïve mice. Furthermore, we explain a series of processes to separate activated T_reg cells and examine their ex vivo phenotype, as well as measure their suppressive activity in vitro.

In this study, mice were maintained in a specific pathogen-free facility of the Yonsei Laboratory Animal Research Center of Yonsei University. All animal experiments were conducted in accordance with the Korean Food and Drug Administration guidelines using protocols approved by the International Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center at Yonsei University.

1. Preparation of Solutions

1. Prepare 2% RPMI media by diluting fetal bovine serum (FBS) to 2% and penicillin-streptomycin to 1% in RPMI
2. Prepare complete RPMI media. To RPMI media add 10% of FBS, 1% of penicillin-streptomycin, 1% of L-glutamine, and 50 μM 2-mercaptoethanol.
3. Prepare fluorescence activated cell sorting (FACS) buffer. To do so, supplement phosphate-buffered saline (PBS) with 2% of FBS.
4. Prepare T cell isolation buffer. To do so, supplement PBS with 2% of FBS and 2 mM ethylenediaminetetraacetic acid.

2. Isolation of Splenic Lymphocytes

1. Remove the spleens from naïve or LCMV CL13-infected mice as described previously¹⁹ and place them in 60 mm x 15 mm Petri dishes containing 10 ml of 2% RPMI media.
   NOTE: In this study experiments, 5 to 6 week-old C57B1L/6J female mice received 2 x 10⁶ plaque-forming units (p.f.u.) of LCMV CL13 through intravenous injection via the tail vein. Sacrifice the mice at day 16 post-infection (16 d p.i.)²⁵. Analyze ge-matched naive mice were on the same day.
2. Place a cell strainer into a 50 ml tube and rinse it with 2 ml of 2% RPMI media. Place the spleen on the 70 μm cell strainer and grind using the plunger of a syringe. Rinse the cell strainer with 2 ml of 2% RPMI media and remove it from the 50 ml tube.
3. Fill up the tube with 2% RPMI media and centrifuge at 300 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cell pellet in 1 ml of ACK lysing buffer. Incubate the samples at room temperature (RT) for 5 min.
   NOTE: The volume of ACK lysing buffer indicated above is for one spleen. Scale up the volume accordingly for pooled spleen samples.
4. Fill up the tube with 2% RPMI media and centrifuge at 300 x g for 10 min at 4 °C. Discard the supernatant and resuspend the cells at a density of 1 x 10⁷ cells/ml in complete RPMI media.
   NOTE: For live cell counting, stain cells with trypan blue and count only live cells that are not stained using hemocytometer.

3. Phenotyping of Splenic Conventional T (T_conv) Cells and T_reg Cells

NOTE: Before T_reg cell isolation, examine the phenotype of splenic lymphocytes isolated from naïve or infected mice by staining the cells with various antibodies and analyzing them by flow cytometry.

1. Transfer 50 μl of the cells (5 x 10⁵ cells) among total splenic lymphocytes into each well of a new u-bottom 96-well plate and add 150 μl of FACS buffer per well. Centrifuge at 300 x g for 2 min at 4 °C.
2. Discard the supernatant. Agitate the cell pellet and add 200 μl of FACS buffer per well. Centrifuge at 300 x g for 2 min at 4 °C (2 times).
3. Prepare the antibody cocktail for staining cell surface markers in 50 μl of FACS buffer per well with the following antibodies and reagents. Anti-CD4 violet dye, Anti-CD25 green dye, Anti-PD-1 violet dye, Anti-CD8 PerCP-Cy5.5 dye and cell viability detection reagent near-infrared (IR) fluorescent reactive dye.
   NOTE: To further analyze the phenotype of activated T_reg cells, anti-CD103^23,25 can be added for cell surface-marker staining.
4. Resuspend the cell pellet with 50 μl of antibody cocktail per well. Incubate for 20 min in the dark at 4 °C. Wash the cells twice with FACS buffer by centrifugation at 300 x g for 2 min at 4 °C.
5. After the final washing step, decant the supernatant and fix the cells for 20 min in the dark at 4 °C with 100 μl of fixation buffer prepared according to the manufacturer's protocol.
6. Wash the cells twice with the permeabilization wash buffer (prepared according to manufacturer's protocol) by centrifugation at 300 x g for 2 min at 4 °C.
7. Prepare the antibody solution for intracellular Foxp3 staining, and resuspend the cell pellet with 50 μl of Anti-Foxp3 antibody solution.
   NOTE: To further analyze the phenotypes of T_conv and T_reg cells, anti-CTLA can be added at this step.
8. Incubate for 20 min in the dark at 4 °C and repeat Step 3.6. After the final washing step, resuspend the cell pellet in 200 μl of FACS buffer and examine the phenotype of the cells by flow cytometer²⁵.
9. Gate the live cell population. To analyze the phenotypes of T_conv cells during LCMV CL13 infection, examine the frequency of Foxp3^-PD-1⁺ cells among CD4⁺ or CD8⁺ T cells.
   NOTE: Based on experimental results, the percentage of Foxp3^-PD-1⁺ is more than 50% in both CD4⁺ and CD8⁺ T cell populations at 16 d p.i. with LCMV CL13.
   1. To analyze the frequency and phenotypes of T_reg cells, examine the frequencies of Foxp3⁺ or Foxp3⁺PD-1⁺ cells among CD4⁺ T cells.
      NOTE: Based on experimental results, the percentage of Foxp3⁺ or Foxp3⁺PD-1⁺ cells is more than 20% in CD4⁺ T cell population, respectively.

4. Isolation of CD4⁺CD25⁺ T_reg Cells

NOTE: The volumes of all reagents indicated below are for a starting cell number of 1 x 10⁷ total splenocytes.

1. Prepare the cells as described in section 2 using naive and LCMV chronically infected mice. Wash the cells by adding 10 ml of T cell isolation buffer. Centrifuge at 300 x g for 10 min at 4 °C. Discard the supernatant completely and resuspend the cell pellet in 40 μl of buffer.
2. For CD4⁺ T cell enrichment using the magnetic cell separation system, add 10 μl of biotin-antibody cocktail and mix well. Incubate for 10 min at 4 °C.
3. Add 30 μl of buffer, 20 μl of anti-biotin micro-beads for labeling of non-CD4⁺ T cells and 10 μl of CD25-PE antibody for fluorescent labeling of CD25⁺ cells. Mix well and incubate for 15 min in the dark.
4. Add 2 ml of buffer and pass the cells through a 40 μm cell strainer into a new 50 ml tube to remove cell debris. Wash the cells by centrifugation at 300 x g for 10 min at 4 °C. Discard the supernatant completely and resuspend the cell pellet in 500 μl buffer at a density of up to 1.25 x 10⁸ cells.
5. Apply the cells onto the column and collect the unlabeled cells that pass through the column. Wash the column by adding 2 ml of buffer and centrifuge at 300 x g for 10 min at 4 °C. Discard the supernatant completely and resuspend the isolated CD4⁺ T cells in 90 μl of buffer.
6. For magnetic labeling of CD25⁺ cells, add 10 μl of anti-PE microbeads, mix well, and incubate for 15 min in the dark at 4 °C.
7. Add 2 ml of buffer and wash the cells by centrifugation at 300 x g for 10 min at 4 °C. Discard the supernatant completely and resuspend the cell pellet in 500 μl of buffer at a density of up to 1 x 10⁸ cells.
8. To enrich the CD4⁺CD25⁺ T_reg cells, apply the cells onto the column for positive selection, and wash the column by adding 2 ml of buffer. Repeat the wash three times.
9. When the column is empty after the final washing step, add 1 ml of buffer onto the column, and flush out the magnetically labeled CD4⁺CD25⁺ cells using the plunger.
10. Repeat steps 4.8-4.9 to enhance the purity of the isolated CD4⁺CD25⁺ T_reg cells. Wash the cells with FACS buffer by centrifugation at 300 x g for 10 min at 4 °C. Discard the supernatant and resuspend the isolated CD4⁺CD25⁺ T_reg cells at a concentration of 2 x 10⁶ cells/ml in complete RPMI media.
11. To check the purity of the isolated-CD4⁺CD25⁺ T_reg cells, use 2 x 10⁵ cells from the total isolated-CD4⁺CD25⁺ T_reg cells. Stain the cells with 50 μl of FACS buffer containing anti-CD4 FITC and cell viability detecting reagent near-IR fluorescent reactive dye.
    NOTE: CD25 was already labeled with PE during isolation.
12. Incubate for 20 min in the dark at 4 °C. Wash the cells with FACS buffer by centrifugation at 300 x g for 2 min at 4 °C. After washing, resuspend the cell pellet in 100 μl of FACS buffer and check the purity by flow cytometry.
13. Gate the live-cell population. To analyze the purity of T_reg cells, confirm the percentage of CD4⁺CD25⁺ cells.
    NOTE: Based on experimental results, the percentage of CD4⁺CD25⁺ cells among purified cells is over about 80%.

5. Isolation of CD8⁺ T Cells and Labeling of CD8⁺ T Cells

NOTE: The volumes of all reagents indicated below are for a starting cell number of 1 x 10⁷ total splenocytes.

1. Prepare cells as described in section 2 using naïve mice. Wash the cells by adding 10 ml of T cell isolation buffer. Centrifuge at 300 x g for 10 min at 4 °C. Discard the supernatant completely, and resuspend the cell pellet in 40 μl of buffer.
2. For CD8⁺ T cell isolation using the magnetic cell separation system, add 10 μl of biotin-antibody cocktail for non-CD8⁺ T cell labeling and mix well. Incubate for 5 min at 4 °C.
3. Add 30 μl of buffer and 20 μl of anti-biotin microbeads. Mix well and incubate for 10 min at 4 °C.
4. Apply the cells onto the column and collect the unlabeled cells that pass through the column. Wash the column by adding 2 ml of buffer three times.
5. Centrifuge at 300 x g for 10 min at 4 °C. Discard the supernatant completely and resuspend the isolated CD8⁺ T cells in 2 ml of buffer.
6. To check the purity of the isolated cells, prepare 50 μl of antibody solution in FACS buffer. Resuspend the 2 x 10⁵ cells among isolated CD8⁺ T cells in 50 μl of antibody solution.
   NOTE: To prepare antibody solution, add anti-CD8 FITC, and cell viability detection reagent (near-IR fluorescent reactive dye) into FACS buffer.
7. Incubate for 20 min in the dark at 4 °C. Wash the cells with FACS buffer by centrifugation at 300 x g for 2 min at 4 °C. After washing, resuspend the cell pellet in 100 μl of FACS buffer, and check the purity of the cells by flow cytometry.
8. Gate the live-cell population. To check the purity of CD8⁺ T cells, confirm the percentage of CD8⁺ T cells.
   NOTE: Based on experimental results, the percentage of CD8⁺ cells among purified cells is over about 90%.
9. Add PBS to the isolated CD8⁺ T cells. Centrifuge at 300 x g for 10 min at 4 °C. Discard the supernatant. Resuspend the isolated CD8⁺ T cells at a concentration of 1 x 10⁷ cells/ml in PBS.
10. To label the CD8⁺ T cells for the in vitro suppression assay, dilute cell proliferation tracking violet dye in PBS to obtain a concentration of 5 μM at RT.
    NOTE: The approximate excitation and emission peaks of the cell proliferation tracking violet dye used in the study are 405 and 450 nm, respectively.
11. Mix well equal volumes of cell proliferation tracking violet dye (5 μM) and cell suspension (1 x 10⁷ cells/ml of CD8⁺ T cells) in a 15 ml tube, and incubate at 20 min at 37 °C. Vortex the tube every 10 min.
12. Fill up the tube with cold complete RPMI media, and leave the tube for 10 min at RT. Centrifuge at 300 x g for 10 min at RT. Discard the supernatant completely, and resuspend the cells at a concentration of 2 x 10⁶ cells/ml with pre-warmed complete RPMI media. Incubate the cells for 15 min at RT.

6. Setting Up the In Vitro Suppression Assay Using CD4⁺CD25⁺ T_reg and CD8⁺ T Cells

1. To prepare anti-CD3/CD28-coated beads, transfer the appropriate volume of magnetic beads to a 15 ml of tube (2.5 μl/1 x 10⁵ cells). Add an equal volume of PBS and mix. Wash by centrifugation at 300 x g for 2 min at 4 °C and discard the supernatant. Dilute the magnetic beads in complete media (50 μl/well).
2. Aliquot 50 μl of CD4⁺CD25⁺ T_reg cells per well of u-bottom 96-well plate (1 x 10⁵ cells/well). Add 50 μl of CD8⁺ T cells as responder T (T_resp) cells per well (1 x 10⁵ cells/well). Add 50 μl of diluted anti-CD3/CD28-coated beads into per well.
   NOTE: In this step, label and set up control wells as follows: "unstimulated CD8⁺ T cell only" with no anti-CD3/CD28-coated beads; "CD8⁺ T cell only" with anti-CD3/CD28-coated beads; "CD8⁺ T cell only" with anti-CD3/CD28-coated beads; "T_reg cell only" with anti-CD3/CD28-coated beads. T_reg cells can be diluted by complete media and co-cultured with T_resp cells in a different ratio of T_resp cells:T_reg cells (1:0.25-1:1).
3. Add 50 μl or appropriate volume of media into all wells to total volume of 200 μl. Cover the plate with foil and incubate in a CO₂ incubator at 37 °C for 72 hr.

7. Analysis of CD8⁺ T Cell Proliferation & Cytokine Production from CD8⁺ T Cells

1. For the cytokine production analysis, after 3 days of culture, separate the supernatant of each well into another plate and perform enzyme-linked immunosorbent assay (ELISA).
   NOTE: The supernatant may be aliquoted and stored at -70 °C. In this experiment, anti-mouse IFN-γ antibody-coated plate was used to detect IFN-γ production according to the manufacturer s protocol. To determine IFN-γ production of proliferating CD8⁺ T cells on a single cell level, intracellular cytokine staining can be performed.
2. After separating the supernatant from each well, wash the plate containing the cells with FACS buffer and centrifuge at 300 x g for 2 min at 4 °C (3 times).
3. After washing, discard the supernatant. Resuspend the cell pellet with 50 μl of antibody cocktail for staining of proliferated CD8⁺ T cells. Incubate for 20 min in the dark at 4 °C.
   NOTE: To prepare antibody cocktail, add anti-CD4 FITC, anti-CD8 PerCP-Cy5.5, and cell viability detection reagent (near-IR fluorescent reactive dye) into FACS buffer.
   NOTE: Antibodies against various markers such as CD44 or CD69 can be combined with other antibodies to confirm activation of CD8⁺ T cells. Remember that CD8⁺ T cells have already been labeled with cell proliferation tracking violet dye at Step 5.10.
4. Wash twice by centrifugation at 300 x g for 2 min at 4 °C. After the final washing step, discard the supernatant, and fix the cells for 20 min in the dark at 4 °C with 100 μl of fixation buffer.
5. Wash twice by centrifugation at 300 x g for 2 min at 4 °C. Resuspend the cells with 200 μl of FACS buffer and measure the proliferation of cell proliferation tracking violet dye-labeled CD8⁺ T cells by flow cytometry.
   1. Gate the CD8⁺ T cell population among the live cells. Measure the percentages of divided and undivided cells according to the dilution of the cell proliferation tracking violet dye and CD8⁺ T cell dilution according to the following equation. % Inhibition = [(% of proliferated CD8⁺ T cells in the absence of T_reg cells - % of proliferated CD8⁺ T cells in the presence of T_reg cells)/(% of proliferated CD8⁺ T cells in the absence of T_reg cells)] x 100. Further analyze the data by using the flow cytometry software²⁵.

We generated mice with persistent virus infection by injecting them with 2 x 10⁶ p.f.u. of LCMV CL13 intravenously. To investigate the phenotypic changes in T_reg cells and T_conv cells during chronic virus infection, splenic lymphocytes obtained from naïve and infected mice were stained with various antibodies and analyzed by flow cytometry. At 16 d p.i., PD-1 was upregulated in both Foxp3^-CD4⁺ T_conv (Figure 1A, upper panel) and Foxp3^-CD8⁺ T_conv (Figure 1B, upper panel) cells. The frequency of Foxp3⁺CD4⁺ T_reg cells was two times higher in the LCMV CL13-infected mice than in the naïve mice (Figure 1A). In particular, most of the Foxp3⁺CD4⁺ T_reg cells displayed the activated phenotype, expressing high levels of PD-1 at this time point (Figure 1A).

For the in vitro suppression assay, a considerable number of CD8⁺ T_conv cells and CD4⁺CD25⁺ T_reg cells were required. To obtain 1 x 10⁷ cell proliferation tracking violet dye-labeled CD8⁺ T cells, splenocytes from two spleens of naïve mice were pooled. To obtain 2 x 10⁶ or 3 x 10⁶ of CD4⁺CD25⁺ T_reg cells, at least three spleens from naive and LCMV CL13-infected mice, respectively, were pooled. CD8⁺ T cells as T_resp cells were separated successfully without significant contamination with other immune cells (Figure 2A). T_reg cells could also be isolated, with a dominant population of CD25⁺CD4⁺ T cells with more than 80% purity (Figure 2B).

To compare the suppressive function of naïve and chronic T_reg cells, the isolated T_reg and T_resp cells were incubated together under stimulation with anti-CD3/CD28-coated beads at 37 °C for 3 days. % inhibition of CD8⁺ T_resp cell proliferation was increased in a T_reg cell dese-dependent manner (Figure 3A). When CD8⁺ T_resp cells were co-cultured with chronic T_reg cells in a ratio of 1:1, proliferation of CD8⁺ T_resp cells were significantly inhibited (Figure 3B). IFN-γ production by CD8⁺ T_resp cells were also significantly inhibited when CD8⁺ T_resp cells were co-cultured with activated T_reg cells from chronically infected mice rather than with resting T_reg cells from naïve mice in a T_reg cell dose-dependent manner (Figure 3C).

Figure 1: Phenotypes of CD4⁺ and CD8⁺ T cells in the spleen of naïve mice or LCMV CL13-infected mice at 16 d p.i. (A) Expression of PD-1 or CD25 on Foxp3^-CD4⁺ T_conv and Foxp3⁺CD4⁺ T_reg cells. (B) Expression of PD-1 or CD25 on Foxp3^-CD8⁺ T_conv cells. Splenic lymphocytes were stained with antibodies against CD4, CD8, CD25, PD-1, and Foxp3. Data are representative of three independent experiments. Panel A has been modified from²⁵ as a reference. Please click here to view a larger version of this figure.

Figure 2: Purities of T_resp cells isolated from naïve mice and T_reg cells isolated from naïve or LCMV CL13-infected mice. (A) Percentage of CD8⁺ T cells after isolation. (B) Percentage of CD25-expressing CD4⁺ T_reg cells after isolation. Each quadrant in (B) was determined by the expression levels of CD4 and CD25. Data are representative of three independent experiments. Please click here to view a larger version of this figure.

Figure 3: Effect of activated T_reg cells on CD8⁺ T cell response. (A) % inhibition of CD8⁺ T cells co-cultured with T_reg cells in a T_reg cell dose-dependent manner. (B) Proliferation of CD8⁺ T cells co-cultured with T_reg cells in 1:1 ratio. (C) IFN-γ production of CD8⁺ T cells co-cultured with T_reg cells in a T_reg cell dose-dependent manner. Proliferation of CD8⁺ T cells was measured by dilution of cell proliferation tracking violet dye in proliferated CD8⁺ T cells and IFN-γ secretion was evaluated by ELISA. Cell proliferation tracking violet dye-labeled CD8⁺ T cells were stimulated with anti-CD3/CD28-coated beads for 3 days in the absence or presence of T_reg cells isolated from either naïve or chronically infected mice. In the histogram, the numbers without and with parenthesis indicate the proliferation percentage of CD8⁺ T cells co-cultured with and without T_reg cells, respectively. Filled gray peaks in the histogram indicate proliferation of CD8⁺ T cells co-cultured without T_reg cells. Each group was designed in triplicates. The bars represent mean + SEM. This figure has been modified from²⁵ as a reference. Please click here to view a larger version of this figure.

Although only a small number of T_reg cells exist in mice and humans, it is important to understand their function as they play a crucial role in regulating the immune response and maintaining immune tolerance. The number and suppressive functions of T_reg cells increases during a chronic virus infection^15-20 as well as cancer progression^13,14. This is probably due to continued antigen stimulation. To evaluate the T_reg cells function under antigen persistence and disease development, their suppressive activity needs to be measured.

Here, we describe a protocol to analyze the ex vivo phenotype of T_reg cells and measure their suppressive activity using an in vitro co-culture system of CD8⁺ T cells and T_reg cells. Critical steps in the current protocol are analysis and isolation of T_reg cells. Live and freshly isolated T_reg cells show clear suppressive activity in vitro. We also examined the phenotypes of T_conv and T_reg cells during a chronic virus infection. T_conv cells, i.e., Foxp3^-CD4⁺ and CD8⁺ T cells showed a decrease in cellular activity after the chronic virus infection (Figure 1). The PD-1 expression was upregulated in both the T_conv cell populations. Increase in T_reg cell number and up-regulation of PD-1 expression are the hallmarks of immune response during a chronic virus infection. Additionally, anti-bodies for other inhibitory receptors such as TIM-3 and CTLA-4 can be used in combination with PD-1 to examine the T cell phenotype by FACS after choric virus infection.

In order to investigate the suppressive activity, T_resp cells and T_reg cells were co-cultured at a ratio of 1 to 1. Co-culture with T_reg cells from chronically infected mice significantly reduced CD8⁺ T cell proliferation and IFN-γ secretion when compared to T_reg cells from naïve mice. This implies that the proliferation and cytokine production of CD8⁺ T cells co-cultured with T_reg cells are inversely related to the suppressive function of T_reg cells. Although this protocol recommends the use of a magnetic cell separation system for T_reg cell isolation, contamination with non-T_reg cells cannot be ruled out. The T_reg cells obtained from Foxp3-GFP reporter mice^28-30 are better candidates to accurately investigate the function of T_reg cells than magnetic cell separation system, as CD25 is often overexpressed on activated CD4⁺ T_conv as well as T_reg cells.

The suppressive function of T_reg cells has been evaluated by various in vitro suppression assays^16,28,31-34. The advantage of the in vitro suppression assay is that it evaluates the direct effect of T_reg cells on the inhibition of the CD8⁺ T cell response, because only T_resp cells are co-cultured with T_reg cells. In addition to CD8⁺ T cells, the CD25^-CD4⁺ T cells can be used instead of T_resp cells²⁵. The effects of T_reg or T_conv cells on immune cells such as dendritic cells or natural killer cells can be evaluated by varying the experimental conditions.

Molecules such as the CD103³⁵, LAG-3³⁶, glycoprotein A repetitions predominant³⁷, CD43³⁸, CD11a³⁸, or killer cell lectin-like receptor subfamily G member 1³⁸ have been used as markers of activated T_reg cells in specific diseases. In this protocol, we used PD-1 as an indicator to identify activated T_reg cells during chronic virus infection. This protocol can be used for multifaceted analyses (phenotypic and functional) of T_reg cells under specific conditions.