Generation of Induced Regulatory T Cells from Primary Human Naïve and Memory T Cells

1. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) from Buffy Coat

1. Acquire one unit of buffy coat from hospital or nearby blood center location. Our blood center provides us with about 40-60 mL of buffy coat per unit obtained from normal blood donors.
2. Pour blood into an autoclaved 500 mL glass bottle containing sterile PBS to dilute buffy coat. The final volume of PBS + buffy coat should be 250 mL.
3. Fill ten 50 mL conical tubes each with 20 mL Lymphoprep solution.
4. Gently overlay the Lymphoprep solution with 25 mL of the diluted buffy coat, being careful not to disturb the Lymphoprep solution.
5. Spin tubes for 30 minutes at room temperature at 500 x g. Make sure to turn off the centrifuge’s brake, so as to not disturb the lymphocyte fraction.
6. Collect the PBMCs at the interface between the Lymphoprep and the plasma-medium layers with a pipette. Aspirate the plasma-medium off until about 1 mL still covers the buffy coat layer containing the PBMCs. Use a 10 mL pipette to transfer PBMCs to a new 50 mL tube.
7. Wash cells twice with PBS and then resuspend cells in 50 mL of cold RPMI for counting on a hemocytometer. Typical recovery is 8 x 10⁸ – 1 x 10⁹ PBMCs.

This procedure can be scaled down for smaller volumes of blood. Dilution of whole blood samples is 1:1 in PBS.

2. Magnetic Negative Selection of Total CD4⁺ T Cells, CD4⁺ CD45RA⁺ naïve T Cells or CD4⁺ CD45RO⁺ Memory T Cells from PBMCs using EasySep Enrichment Kits (Stem Cell Technologies)

Steps to follow for 1 x 10⁸ to 4.25 x 10⁸ PBMCs.

1. Resuspend PBMCs to a final concentration of 5 x 10⁷ per mL in PBS containing 2% FBS and 1mM EDTA. Move cells to a fresh 14 mL polypropylene round bottom tube.
   1. Isolation of total human CD4⁺ T cells by negative selection: add human CD4⁺ T Cell Enrichment Cocktail of antibodies (50 μL per mL of PBMCs), mix and incubate for 10 minutes at room temperature.
   2. Isolation of human naïve T cells by negative selection: add 50 μL of anti-CD45RO antibody per mL of PBMC cell suspension, mix and incubate for 15 minutes at room temperature. Add enrichment cocktail provided with the kit (50 μL of per mL of PBMCs), mix and incubate at room temperature for 10 minutes.
   3. Isolation of human memory T cells by negative selection: add human Memory CD4⁺ T Cell Enrichment Cocktail of antibodies (50 μL per mL of PBMCs), mix and incubate for 10 minutes at room temperature.
2. Mix magnetic particles well to equally distribute them throughout the solution. Do not vortex nanoparticles from naïve kit.
3. Add the magnetic particles (100 μL per mL of PBMCs for total CD4⁺ and naïve T cell selection, 50 μL per mL of PBMCs for memory T cell selection). Mix by gently pipetting 2-3 times and incubate at room temperature for 10 minutes for naïve T cell selection or 5 minutes for memory or total CD4⁺ T cell selection.
4. Add PBS containing 2% FBS and 1 mM EDTA to bring the volume up to 10 mL per tube. Mix by gently pipetting 2-3 times before placing uncapped tube into silver EasySep magnet for 5, 10 or 2.5 minutes for total CD4⁺ T cells, naïve and memory subsets, respectively.
5. With tube still in EasySep magnet, pour liquid into new 50 mL tube to isolate cells of interest.
6. Repeat steps 2.5 and 2.6 for better recovery.

3. Cell Culture Conditions to Induce Regulatory T Cells

1. The day before steps 1 and 2, coat tissue culture plates by first diluting anti-CD3 antibody (OKT3 clone) to a concentration of 1 μg/mL in sterile PBS. For a 6 well plate add 2 mL of PBS + anti-CD3 per well, for a 12 well plate add 1 mL or for a 24 well plate add 500 μL per well. Keep the coated plate at 4 °C until use.
2. Prepare polarization medium by adding 10% heat-inactivated Fetal Bovine Serum (FBS),100 U/mL penicillin, 100 μg/mL streptomycin and 50 μM β-mercaptoethanol to RPMI-1640 pre-supplemented with 2.06 mM Glutamax-I and 25 mM HEPES buffer. Next, add 2 ng/mL TGF-β and 5 ng/mL IL-2. Here, one may add ligands or inhibitors of interest to the media. Resuspend cells from step 2 at a concentration of 2 x 10⁶ cells/mL of polarization medium.
3. Pre-warm plates at 37 °C and aspirate the PBS out of anti-CD3-coated wells before adding cells. Add 4 mL per well of cell suspension for a 6 well plate, 2 mL of cells for a 12 well plate or 1 mL of cells for a 24 well plate. Fill up empty wells with PBS to reduce evaporation of media. Incubate for 3 days at 37 °C / 5% CO₂.
4. On day 3 post plating, spin down plate in a centrifuge for 5 minutes at 500 x g. Without disturbing T cells on bottom of the plate, remove half of media and replace with fresh media. Alternatively, if the well becomes overcrowded with cells (concentration above 3 x 10⁶/mL), split the volume equally into another anti-CD3 coated plate and add fresh media back up to original volume. Incubate for two to three more days at 37 °C / 5% CO₂.

4. Fluorescence-Activated Cell Sorting (FACS) of Three Populations of T Cells

1. Prepare FACS washing buffer by adding 0.5% (w/v) bovine serum albumin (BSA) and 2 mM EDTA to sterile PBS. Place buffer at 4 °C to get cold.
2. Remove cells from cell culture well and rinse each well with 2 mL of PBS to ensure complete cell recovery. Place all cells in 50 mL polypropylene tubes and centrifuge at 500 x g for 10 minutes at 4 °C.
3. Count the cells on a hemocytometer to determine cell density.
4. Place 3-5 x 10⁵ cells per tube in four separate 5 mL round bottom polystyrene tubes for compensation controls. Place remaining cells in 50 mL polypropylene tubes, no more than 35 x 10⁶ cells per tube.
5. Spin down cells for 5 minutes at 4 °C and aspirate media. Resuspend cells to be sorted in 90 μL of cold washing buffer per 1 x 10⁶ cells. Resuspend in 100 μL of cold washing buffer the compensation control tubes.
6. To the cells to be sorted, combine 2 μL of anti-CD25-PE, 3 μL of anti-CD45RA-PE-Cy5 and 1 μL of anti-CD127-APC per 1 x 10⁶ cells. Add no antibody to the first compensation control tube, 2 μL of anti-CD25-PE to the second tube, 3 μL of anti-CD45RA^–PE-Cy5 to the third tube and1 μL of anti-CD127-APC to the fourth tube. Incubate all tubes on ice for 45 minutes in the dark.
7. Add 10 mL of cold washing buffer to the cells to be sorted and 1 mL to the compensation control tubes. Centrifuge all cells at 500 x g for 5 minutes at 4 °C.
8. Aspirate buffer and resuspend cells at a concentration of 1 x 10⁷ cells per mL of washing buffer. Add 1.5 μL of DNase II per mL of cells before filtering through a 40 μM nylon cell strainer. Move cells to multiple 5 mL round bottom polypropylene tubes with no more than 3.5 mL per tube. Resuspend compensation control cells in 300 μL of washing buffer.
9. Set compensation on MoFlo flow cytometer to minimize cross detection by the PE, APC and PE-Cy5 filters. Set gates to sort iTregs (CD25^HiCD127^-/LOWCD45RA^– cells), naïve (CD25-CD45RA⁺) and memory (CD25^–CD45RA^–) T cells into 5 mL round bottom polystyrene tubes containing 1 mL newborn calf serum.

5. Suppression Assay

1. Make suppression assay media by adding 100 U/mL of penicillin, 100 μg/mL of streptomycin, 5 ng/mL of IL-2 and 2 ng/mL of TGF-β to AIM-V.
2. The day of the assay, purify heterologous CD4⁺ T cells from buffy coat as indicated in steps 1 and 2. These will be the target cells for the suppression assay and does not contain CD25⁺ Treg cells, as can be seen in Figure 2, Day 0.
3. Label cells with CellTrace kit as per manufacturer’s instructions, except using only 1 μL of 5 mM stock solution per mL of cells instead of 2 μL. Keeping out from direct light, add 18 μL of the DMSO supplied by the CellTrace kit to one vial of CFSE to make a 5 mM stock solution. Resuspend the required number of target cells (to a maximum of 1 x 10⁷) in prewarmed PBS + 0.1% (w/v) BSA to a final concentration of 1 x 10⁶ cells/mL. Add 1 μL of 5 mM CFSE per mL of cells and incubate in a 37 °C water bath for 5 minutes. Add 5 volumes of complete, ice cold RPMI with 10% FBS to quench staining and incubate on ice for 5 minutes. Wash cells twice more with cold complete RPMI and resuspend 1 x 10⁵ cells per 100 μL of suppression assay media.
4. Treg suppression inspector beads are at a stock concentration of 2 x 10⁷ beads/mL. Pellet a number of beads equal the total number of cells per experiment by quick centrifugation in an eppendorf tube. Wash beads once with RPMI and re-pellet. After aspiration of RPMI, resuspend beads so that the appropriate amount of beads per well are in 8 μL of suppression assay media.
5. To a 96 well round bottom tissue culture plate, add CFSE-stained cells (1 x 10⁵ cells/mL), inspector beads and polarized and sorted cells (1 x 10⁵ cells/mL) in fresh suppression assay media to a desired target (CFSE stained):effector (sorted) ratio in a final volume of 200 μL. All conditions are set in triplicates.
6. Prepare the first of two control conditions by adding 100 μL of CFSE stained cells, 8 μL of inspector beads and 1 x 10⁵ of fresh, unstained cells in 92 μL suppressor assay medium per well. Prepare the second control with the same cellular components as above but without Treg inspector beads.
7. Cover plate in aluminum foil and incubate at 37 °C / 5% CO₂ for five days.
8. In the dark, collect cells from each well by pipetting and place in a 5 mL round bottom polystyrene tube. Centrifuge cells at 500 x g for 5 minutes at 4 °C, aspirate media, and resuspend in 300 μL cold FACS washing buffer from step 4. Analyze the first 3 x 10⁴ CFSE⁺ events from the live lymphocyte gate representing target cells in a histogram with Cell Quest software.

6. Representative Results

Example of flow cytometric pseudocolor dot plots over a five-day time-course monitoring iTreg differentiation based on the relative co-expression of CD25 with FoxP3, CTLA-4 and CD45RA can be seen in Figure 2. The histogram in Figure 3 shows a successful suppression assay in which sorted iTregs (CD25^HiCD45RA^– CD127^-/LOW cells) are the only subset from a five-day culture that has acquired regulatory/suppressor ability. Figure 4 shows the induction of Tregs from a naïve T cell pool (top panels) and a memory T cell pool (bottom panels), after five-day culture in standard iTreg medium. CFSE staining of initial cells demonstrate that either naïve (top right panel) or memory T cells (bottom right panel) differentiate to iTregs (highest FoxP3-expressing cells) only after several rounds of cell division.

Figure 1. Schematic of experimental procedure. PBMCs are separated out of human peripheral blood via gradient centrifugation before magnetic negative selection of CD4⁺CD25^– T cells. After five to six days in culture, cells undergo FACS and are co-incubated with heterologous CFSE labeled target cells to measure suppressor activity.

Figure 2. Purified human primary CD4⁺CD25^– T cells are cultured in iTreg medium. An aliquot of cells is collected just after isolation (day 0) and at days 1, 3 and 5 of cell culture to monitor the progress of CD45RA, FoxP3, CTLA-4 and CD25 markers. The iTreg profile corresponds to CD45RA^–, FoxP3^Hi, CTLA-4^Hi and CD25^Hi (highlighted in the in-graph window).

Figure 3. Purified CD4⁺ CFSE labeled cells (1 x 10⁵/well) are cultured with Treg suppression inspector beads in the presence of sorted naïve, memory or iTreg cells (3 x 10⁴/ well). After five days, cells are harvested and the CFSE profile of the stained cells is analyzed by flow cytometry. The presence of iTreg cells completely abolishes the proliferation of CD4⁺ T cells. Numbers are indicative of percentage of CFSE-labeled cells that have undergone division.

Figure 4. Purified human primary naïve (CD4⁺CD25^–CD45RA⁺) and memory (CD4⁺CD25^–CD45RO⁺) T cells are stained with CFSE and cultured in iTreg medium. After five days, cells are phenotyped and the cell division rates estimated. As indicated in Figure 2, the iTreg subset differentiated from both subsets corresponds to CD45RA^–CD25^Hi FoxP3^Hi and CTLA-4^Hi (latter two not shown). Comparative CFSE staining profiles identify iTregs (here as the highest expressing FoxP3 T cells) as the most proliferative cells during the five-day culture.