Retroviral Transduction of Helper T Cells as a Genetic Approach to Study Mechanisms Controlling their Differentiation and Function

All mouse work performed in these protocols was undertaken according to the Animals Scientific Procedures Act, UK under the animal Project License 70/6965.

1. Retroviral Production

Prior to proceeding obtain all required approvals for producing genetically modified organisms and the use of retroviruses in mammalian cells.

1. Growth of HEK 293T Cells
   1. Grow HEK 293T cells on 10 cm tissue culture dishes in HEK 293T medium (Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), 100 units/ml Penicillin 0.1 mg/ml Streptomycin, and 2 mM L-Glutamine). For general cell passage, split cells 1:10 when they reach confluence by removing medium and pipetting the cells off the plate with fresh HEK 293T medium.
      NOTE: Cells should reach confluence in 2-3 days. 293T HEK cells attach loosely to the plate so trypsinization is not required.
   2. 24 hr prior to transfection, split a confluent plate of cells 1:10 to a 10 cm plate (one plate per transfection) so that the next day the cells are ~50% confluent.
2. Transfection by Calcium Phosphate Precipitation
   1. Approximately 1 hr prior to transfection, remove medium from cells and carefully add 9 ml of fresh HEK 293T medium to prevent cells detaching from the plate.
   2. Prepare 2x Hepes Buffered Saline (2x HBS) and a fresh 2.5 M CaCL₂ solution as described in Table 1. Thaw DNA stocks. For the most efficient transfections use plasmid prep quality DNA.
   3. In a 6 ml round bottom or 15 ml conical tube add 5 µg of retroviral DNA (pMIG)⁴, 5 µg of Helper virus DNA (pCL-Eco)⁷, and H₂O to 420 µl. Add 80 µl of 2.5 M CaCl₂ bringing the final volume to 500 µl.
      Note: This along with the next step (addition of 2x HBS) can be done on a regular bench as long as general sterile technique is used.
   4. Slowly add 500 µl of 2x HBS drop by drop to the above DNA mixture while gently vortexing so that the solution is continuously mixed and the CaPO₄ precipitates in even-sized crystals.
   5. Transfer to a tissue culture hood and slowly add the CaPO₄ DNA mixture (1 ml) dropwise to the 9 ml of medium covering the cells while constantly and gently swirling the plate. Once again be careful not to detach cells from the plate. Place the cells back into the incubator.
      NOTE: At this point the CaPO₄ precipitate will be readily visible under a microscope as small crystals about the size of bacteria. These are easily seen after 30-60 min when the crystals have had a chance to settle to the bottom of the plate.
3. Collection of Virus in Culture Supernatants.
   1. The following day (anywhere from 12-24 hr after transfection) remove the medium and feed the cells with 10 ml of fresh HEK 293T medium once again being careful not to dislodge the cells from the plate.
      NOTE: This dilutes out lymphocyte inhibitory substances that the HEK 293T cells secrete into the medium during the transfection.
   2. In the evening of the second day (~24 hr post transfection) feed cells with 3.5 ml of fresh HEK 293T medium. Be careful that the plate is placed exactly level in the incubator so that one side is not left dry.
   3. Collect medium ~12 hr later (store at 4 °C) and feed with 3.5 ml of fresh HEK 293T medium. Repeat collection 2 more times at roughly 12 hr intervals so that about 10 ml of medium is collected. This is the virus stock.
   4. Spin out any residual HEK 293T cells and cellular debris by centrifuging at 600 x g for 5 min. Alternatively, filter using a 0.45 µm, low protein binding, syringe filter. Store virus at 4 °C, and for best titers, use within a day or two, as the titer will slowly diminish at 4 °C. Do not freeze, as this significantly decreases the titer.

2. Isolation of Primary Naïve CD4⁺ T Cells

1. Isolation of Leukocyte Cells
   1. Euthanize mice by cervical dislocation, CO₂ asphyxiation, or other humane protocol appropriate to the animal handling rules of the institution.
   2. Dissect freshly euthanized mice and remove spleen and desired lymph nodes ¹⁵. Place the organs in wells of a 6 well tissue culture plate containing R10 medium (RPMI medium with 10% heat inactivated FBS, 100 units/ml Penicillin, 0.1 mg/ml Streptomycin, 2 mM L-Glutamine, and 0.1% β-mercaptoethanol). Use a cell culture hood for dissection of mice and all subsequent manipulations to minimize chance of contamination in cultures.
      NOTE: Keep the R10 medium cold and perform all the downstream purification steps at 4 °C.
   3. Place a 70 µm cell culture strainer into a 50 ml conical tube containing approximately 5 ml of R10 medium. Use one strainer for the spleen and lymph nodes of up to three mice.
   4. Add spleen and lymph nodes to the cell strainer and macerate using the end of a 5 ml syringe plunger. Rinse with 1-2 ml of R10 medium.
   5. Transfer cells to a 15 ml conical tube and bring the volume up to 14 ml with R10. Centrifuge at 600 x g for 5 min at 4 °C. Discard supernatant by pouring it off with one clean movement to prevent disturbing the cell pellet.
   6. Add 2 ml of cold (4 °C) red cell lysis buffer (Table 1) to each tube. Gently mix for 3.5 min, keeping the tube on ice.
      NOTE: The timing of red cell lysis is critical to prevent death of lymphocytes. Therefore, the exact timing of each batch of Red cell lysis buffer will need to be optimized.
   7. Add ≈12-13 ml of R10 medium. Immediately centrifuge and discard supernatant as in step 2.1.5. Perform the washing steps 2x more with R10 medium to remove any residual red cell lysis buffer from the cells and avoid death of lymphocytes.
   8. Count cells in a hemocytometer diluting 1:20 in trypan blue to determine the yield of viable cells. Expect approximately 8-10 x 10⁷ cells per mouse.
2. Isolation of CD4⁺ T Cells Using Magnetic Beads Kit to Remove CD8⁺, CD11b⁺, CD16/32⁺, CD45R⁺, MHC class II⁺ and Ter-119⁺ Cells.
   1. Aliquot 8-10 x 10⁷ cells from step 2.1.8 per 15 ml conical tube and centrifuge at 600 x g for 5 min at 4 °C. Pour off the supernatant with one clean movement to prevent disturbing the cell pellet. Resuspend the cells in each tube in 100 µl of heat inactivated FBS then add 100 µl of antibody mix from the kit. Incubate for 30 min at 4 °C with gentle mixing on a roller.
   2. While the above cells are incubating, prepare the beads.
      1. Resuspend beads in the vial by vortexing for >30 sec then transfer 1 ml of beads per 8-10 x 10⁷ prepared cells to a 15 ml conical tube. Add 2 ml of R10 medium per ml of beads and gently mix.
      2. Place the tube in the magnet for 1 min then discard the supernatant. Remove tube from the magnet and resuspend beads in 4 ml of R10 medium. This amount of beads is sufficient for two rounds of incubation.
   3. Wash the cells at the end of the antibody incubation (Step 2.2.1) by adding 10 ml of R10 medium per tube. Gently mix well with swirling of the tube and centrifuge cells at 600 x g for 5 min at 4 °C. Pour off the supernatant with one clean movement to prevent disturbing the cell pellet. Resuspend cells in 1 ml of R10 medium.
   4. Add 2 ml of the washed beads from step 2.2.2.2 (½ the amount prepared for each tube) to each tube of antibody treated cells and incubate for 30 min at 4 °C with gentle mixing on a roller.
   5. Resuspend the cell-bead mixture by gently pipetting 5 times with a pipet containing a narrow tip opening. Avoid foaming. Place the tube in the beads magnet for 2 min then transfer the supernatant containing the negatively selected cells to a new tube. Keep these cells, as they are the CD4⁺ population.
   6. Repeat negative selection one more time. Spin cells from step 2.2.5 at 600 x g for 5 min at 4 °C. Pour off the supernatant as in step 2.2.3 and resuspend in 1 ml of R10 medium. Follow steps 2.2.4 and 2.2.5 again. After the final magnetic bead selection, count the cells to determine recovery (typical yields are 15-20 x 10⁶ cells per 10⁸ leukocytes).
3. Isolation of Naïve CD4⁺ T Cells (CD25^– and CD62L^high) Using Cell Separation Columns
   1. Centrifuge CD4⁺ T cells at 600 x g for 5 min at 4° C and resuspend in 150-200 µl of R10 medium per 10⁸ cells. Add 2 µl of stock biotinylated CD25 monoclonal antibody (clone 7D4). Incubate for 30 min at 4° C with gentle mixing on a roller.
   2. Wash cells by adding 10 ml of cell separation column buffer (Table 1). Centrifuge at 600 x g for 5 min at 4 °C. Remove as much supernatant as possible and estimate the volume of buffer/cells remaining. Resuspend to a final volume of approximately 90 µl per 10⁷ cells.
   3. Add 20 µl of Streptavidin beads per 10⁷ cells and mix with gentle flicks. Incubate for 15 min at 4 °C.
   4. While cells are incubating, prepare the medium cell separation (MS) columns for the manual approach. Each MS column retains 10⁷ cells, and since CD25⁺ cells typically represent about 10% of total CD4⁺ cells, use 1 column per 10⁸ CD4⁺ T cells. Add 500 µl of cell separation column buffer to the column and let flow through. Repeat 2 more times.
   5. At end of cell/bead incubation, wash cells by adding 10 ml of cell separation column buffer and centrifuging at 600 x g for 5 min at 4 °C. Resuspend cells in 500 µl of cell separation column buffer per 10⁸ cells, but still use 500 µl if there are fewer cells.
   6. Apply 500 µl of cell/bead suspension to the column and collect the flow through. Pass this over the column 1 more time and collect the flow through once again. Rinse the column 3 times with 500 µl of cell separation column buffer, adding each flow through from these rinses to the collected cells. These are the CD25^– cells. Count cells to determine yield.
   7. If Tregs (CD25⁺ cells) are desired, isolate as follows.
      1. Remove the column from the separator and hold it above an open universal tube taking care to maintain sterility. Quickly pipette 500 µl of cell separation buffer onto the column and firmly flush out the positive fraction, using the plunger supplied with the column.
      2. Repeat the flushing procedure 2 more times. Pass the positive fraction over a fresh MS cell separation column and discard this flow-through. Firmly flush out the positive cells three times, as before and centrifuge cells at 600 x g for 5 min at 4° C. Resuspend cells in 1 ml of R10 medium and count to determine the yield.
   8. To obtain CD25^– CD62L^high T cells, centrifuge the CD25^– T cells isolated above in step 2.3.6 at 600 x g for 5 min at 4° and resuspend in 150-200 µl of R10 medium per 10⁷ cells. Add 5 µl of stock biotinylated CD62L monoclonal antibody (clone MEL-14) and incubate for 30 min at 4° C with gentle mixing on a roller as before.
   9. Perform wash, Streptavidin bead binding, and cell separation column preparation as described for Treg isolation protocol (2.3.7). This time use the large cell separation (LS) column, which has a capacity of 10⁸ cells.
      1. Since CD62L^high T cells typically represent about 60-70% of CD25^– cells, use 1 LS cell separation column per 10⁸ cells. Add cell/bead mixture to LS cell separation column as described in step 2.3.6 – this time discarding the final flow through and column rinses. Collect the CD62L^high T cells as described in step 2.3.7 for the CD25⁺ cell collection.
      2. Centrifuge cells at 600 x g for 5 min at 4° C. Resuspend cells in 1 ml of R10 and count to determine yield. Dilute to 0.75-1 X10⁶ cells per ml in R10 medium.
4. Analyze Purity of Cells by Fluorescent-activated Cell Sorting (FACS).
   1. FACS staining strategy
      1. For cells pre and post all stages of isolation, stain with CD4-FITC, CD8a-PerCP-Cy5.5 (helper and cytotoxic T cells), and MHCII-PE (MHC class II expressing cells).
      2. For cells pre and post CD25 isolation, stain with CD4-FITC and CD25-PE (Tregs versus other helper T cells).
      3. For cells pre and post CD62L isolation stain with CD4-FITC, CD62L-PE and CD44-APC (naïve versus memory and effector helper T cells).
   2. For each analysis place 10⁵ cells in a well of a 96 well U-bottom plate. Centrifuge at 600 x g for 5 min at 4 °C. Pour off the medium and wash the cells once with 200 µl DPBS. Centrifuge as above and pour off DPBS.
   3. Add 50 µl of DPBS per well and 0.5 µl of each antibody (as indicated in 2.4.1) and incubate at RT for 30 min in the dark to avoid bleaching of fluorescent label.
   4. Wash cells 2x with DPBS as in 2.4.2 and immediately analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand.
      NOTE: After the final isolation step of a good preparation, 90-95% of cells will be CD4⁺ CD25^– CD62L^high T cells.

3. Retroviral Transduction of Activated CD4⁺ T Cells and their Differentiation into Specific T Helper Subsets

1. Retroviral Transduction
   NOTE: Retroviral integration and expression of genes requires cell division. Therefore, T cells must be activated O/N.
   1. While isolating the naïve T cells, coat a 24 well tissue culture plate with anti-CD3 and anti-CD28 antibodies diluted in DPBS. Add 250 µl per well. Use anti anti-CD3 at 1µg/ml. Use anti-CD28 at 2 µg/ml if cells will ultimately be differentiated under Th0, Th1, Th2 and iTregs conditions. Use anti-CD28 at 10 µg/ml for Th9 and Th17 conditions. Incubate ~2 hr at 37 °C in a tissue culture incubator.
   2. Just prior to culturing the cells, remove the antibody solution and wash the plate with ~250 µl of DPBS per well to remove unbound antibody. Be careful not to let the plate dry out so process a maximum of 6 wells at a time. Remove DPBS wash and add 1 ml of naïve CD4⁺ T cells in R10 medium at 0.75-1 x 10⁶ cells per ml. Activate cells O/N (14-16 hr).
   3. The following day, centrifuge cells in the plate at 900 x g for 5 min at 30° C to attach cells to the bottom of the wells.
   4. Collect and save the medium being careful not to displace the cells, and replace with 1 ml virus culture supernatant prepared from the virus production protocol. Do not let the cells dry out so process a maximum of 4 wells at a time.
      1. To each well add 1 µl of 8 mg/ml polybrene and 10 µl of 1M HEPES pH 7.5 to aid virus uptake and prevent significant alkalization in the ambient CO₂ during the following centrifugation. Centrifuge cells in plate at 900 x g for 90 min at 30° C.
2. Differentiation of Cells into Specific Subsets
   1. Carefully remove the virus culture supernatant and replace with 1 ml of the medium collected above in step 3.1.4, as it contains IL-2 and other T cell growth factors. Add reagents indicated in Table 2 and culture cells for 3-4 days to differentiate cells into specific subsets.
3. Analysis of Cells
   1. For intracellular staining of cytokines, treat cells with 1 µg/ml each of Phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 hr. During the last 2 hr of stimulation, add 1 µg/ml of brefeldin A.
   2. Resuspend cells from the bottom of each well by pipetting up and down several times. Transfer approximately 10⁵ cells to a 96 well U bottom plate (usually 100 µl from the 1 ml of culture of Th cells) for fixation and staining.
   3. For GFP staining, fix cells with 100 µl of 2% paraformaldehyde at RT for exactly 5 min, as a longer incubation time will bleach the GFP and interfere with Foxp3 staining. Wash cells by adding 100 µl of DPBS to each well and centrifuge at 600 x g at 22 °C for 5 min. Pour off the supernatant.
      Caution: Paraformaldehyde is toxic. Handle it in a fume hood with skin and eye protection.
   4. Fix the cells again using 100 µl 1x Fixation buffer made from the Foxp3 staining kit (1 volume of Fixation buffer to 3 volumes of diluent). Incubate the cells for 45 min at RT or alternatively incubate at 4 °C for 16 hr.
   5. After fixation, permeabilize cells by adding 100 µl of permeabilization buffer from the same kit. Incubate at RT for 30-45 min then centrifuge at 600 x g at 22 °C for 5 min.
   6. For antibody staining, remove fixation/permeabilization buffer and add 50 µl of fresh permeabilization buffer. Add the required antibody diluted in permeabilization buffer at 0.5 µl per 50 µl buffer per sample. Incubate for 30-45 min at RT in dark to avoid bleaching of fluorescent label.
   7. Add 150 µl permeabilization buffer and centrifuge at 600 x g at 22 °C for 5 min. Discard the permeabilization buffer and add 200 µl DPBS. Analyze on a flow cytometer using protocols and guidelines specific to the instrument at hand.
      NOTE: Be sure to include proper controls for cytokine staining such as staining with isotype control antibodies, analyzing non-activated or Th0 activated cells, etc.