Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype

NOTE: This rhesus PBMC transduction protocol requires the use of a gammaretroviral vector. Note that gammaretrovirus can either be produced in the lab or outsourced to a viral production company. In lab production can be carried out with the protocol outlined in step 1 and the virus can be titered as outlined in step 2. Whether the virus is produced on site or by a production company, an MOI (ratio of infectious virions to cells in culture) should be determined, as outlined in step 3, for each newly produced viral preparation and the cells of interest.

CAUTION: Work with gammaretroviral vectors or other retroviral vectors requires proper safety precautions such as use of lab coats and double layers of gloves. All handling of the virus must occur in the biosafety hood. All pipets should be decontaminated in a solution of 10% bleach for 30 min. All liquid infectious waste should be decontaminated with a final concentration of 10% bleach. Secondary containment, with a seal that prevents release of microbiological aerosols, is required for centrifugation

1. Producing the Gammaretroviral Stock

1. Plate 293T cells for transfection. A full virus preparation requires 12 T75 flasks at 4.5 x 10⁶ cells/flask in Dulbecco's modified Eagle medium (DMEM) + 10% fetal bovine serum (FBS) without antibiotics.
2. Allow the cells to grow overnight at 37 °C before transfection.
3. Dilute 60 μL of transfection reagent in 1.5 mL reduced serum media. Prepare one tube for each flask. Incubate for 5 minutes at RT.
4. For each flask, prepare a tube containing 1.5 mL of reduced serum media and the following plasmids: 9.0 μg of a transfer plasmid containing the gene(s) of interest, 3 μg of RD114 and 1.2 μg of VSV-G (envelope plasmids), and 3 µg of gag/pol.
   NOTE: The envelope and gag/pol genes are needed for retroviral packaging. For our retroviral production, we add both the RD114 envelope and, for added stability, a small amount of VSV-G envelope.
5. Add diluted plasmids (step 1.4) to diluted transfection reagent (step 1.3). Mix gently. Incubate for 20 min at room temperature.
6. Feed cells 5 mL of fresh complete media and add 3 mL of transfection reagent/plasmid mixture dropwise per flask and incubate for 5–6 h at 37 °C.
7. Add an additional 5 mL of fresh medium to the flasks and incubate for 42–43 h at 37 °C.
8. After a total transfection time of 48 h, collect media and centrifuge at 1282 x g for 3 min at 4 °C to remove cell debris.
9. Aliquot into appropriate volumes for future use, flash freeze the virus in an ethanol/dry ice bath and store at -80 °C.

2. Titering the Gammaretrovirus

NOTE: Several dilutions of virus may be needed to obtain a valid titer.

1. Plate 293T cells at 600,000 cells/well in a 6-well plate and incubate the plates for 24 h at 37 °C.
2. Remove the medium from the cells and add desired amount of DMEM + 10% FBS to the 293T (2 mL total volume of virus).
3. Add the virus to the corresponding well and swirl gently to mix. For example, add 200 µL, 100 µL, 50 µL and 25 µL respectively to 4 wells. Include a well without virus for flow cytometry (mock sample).
4. Incubate for 48 h at 37 °C.
5. Trypsinize the 293T cells by adding 0.5 mL of Trypsin-EDTA and incubating for 4 min at 37 °C. Stop the trypsin by adding 1.5 mL of DMEM + 10% FBS. Count the cells and determine viability with an automated cell counter or a hemocytometer.
   1. To use the cell counter, add 10 µL of cells to 10 µL of trypan blue, mix, load the chamber slide, and insert into the counter. Push the "capture" button to count the cells.
6. Collect 0.5–1 x 10⁶ cells and evaluate levels of CAR and CXCR5 by flow cytometry (see step 9.5). Expression of CD4 or MBL along with CXCR5 will identify transduced 293T cells co-expressing the CAR and CXCR5. See reactive antibodies in step 9.5.1.
7. Calculate viral titer.
   NOTE: Choose the sample with a transduction level of 20% or less for calculation of titer.
   1. Use the following formula to calculate titer:
      transduction units/mL = (number of cells in culture)(% of cells transduced)/volume of virus added to the culture

3. Determining the Optimal MOI for a Newly Produced Viral Preparation and the Cells of Interest

1. Follow the transduction protocol (sections 4–9) with primary PBMCs and twofold dilutions of the gammaretroviral preparation.
2. Assess the level of expression of the genes of interest by flow cytometry (step 9.5).
3. Choose a concentration of virus that allows maximal transduction without loss of cell viability.

4. Preparing Plates for T Cell Activation by Coating Wells with Anti-CD3 Antibodies

1. Prepare anti-macaque CD3 antibody (FN18) by diluting the stock to 10 µg/mL in phosphate-buffered saline (PBS).
2. Dispense 2 mL/well in 6-well plates. Incubate at 37 °C for 2 h. Alternatively, the plates can be incubated overnight at 4 °C.
3. Aspirate the PBS and unbound antibodies. Rinse wells twice with 2 mL of PBS and use immediately to plate PBMCs.

5. Stimulating Rhesus PBMCs with Plate-bound Anti-CD3 and Soluble Anti-CD28

1. Thaw primary rhesus PBMCs in a 37 °C waterbath, with gentle agitation, until just a small amount of ice remains.
2. In the hood, gently pipette cells into a conical tube. Rinse the vial with 1 mL of basic medium and add it slowly to the cells. Next, slowly add an additional 9 mL of warm basic medium to the cells.
   NOTE: This step can be scaled up but never thaw more than 4 vials at one time.
3. Centrifuge at 600 x g for 5 min at 22 °C to pellet the cells. Aspirate the supernatant and then resuspend the pellet in a small amount of growth medium. Choose a volume such that the concentration of cells will exceed 2 x 10⁶ cells/mL at this point since the final concentration should be 2 x 10⁶ cells/mL.
4. Stain cells with trypan blue and count the cells to determine the number of viable cells.
   NOTE: This can be done with a standard hemocytometer or an automated cell counter. We use an automated cell counter which displays the viability and number of live cells.
   1. To use the counter, add 10 µL of cells to 10 µL trypan blue, mix, load the chamber slide and insert into the counter. Push the "capture" button to count the cells.
5. Calculate the total number of cells by multiplying cell concentration by volume and then dilute the cells to 2 x 10⁶ cells/mL in growth medium. Prior to plating, add anti-CD28 to a final concentration of 5 µg/mL in order to provide a necessary co-stimulatory signal for T cell activation.
6. Pipette cells into anti-CD3 coated plates. Add 3–6 x 10⁶ cells per well (a good target is 4 x 10⁶ cells in 2 mL media per well) and incubate for 2 days at 37 °C in 5% CO₂.

6. Preparing Fibronectin-coated Plates

NOTE: PBMC and T cells express integrin receptors VLA-4 or VLA-5 and are good targets for fibronectin-mediated transduction.

1. Prior to coating the plates, first prepare fibronectin reagent by diluting it 1:100 in sterile PBS.
2. Pipette 2 mL of the sterile fibronectin solution into each well of a 6-well plate. Gently rock plates for 2 h at room temperature.
   NOTE: Non-treated, cell culture-grade tissue culture plates or dishes should be used in this step.
3. Remove the fibronectin solution by aspiration and then block with 1 mL of sterile 2% bovine serum albumin (BSA, Fraction V) in PBS. Rock the plates at room temperature for 30 min.
4. Aspirate the BSA solution and wash the plate with 2 mL of PBS. After aspiration of the PBS, the fibronectin-coated plates can be sealed with seal wrap and stored at 4 °C for up to a week.
   NOTE: We usually prepare the plates one day before transduction.

7. Transduction of activated rhesus PBMCs

CAUTION: Work with viral vectors or with rhesus macaque PBMCs requires utilization of proper safety precautions such as use of lab coats and double layers of gloves. All handling of the virus and the cells must occur in the biosafety hood. All pipets should be decontaminated in a solution of 10% bleach for 30 min. All liquid infectious waste should be decontaminated with a final concentration of 10% bleach. Secondary containment with a seal that prevents release of microbiological aerosols, is required for centrifugation.

1. Attach the gammaretroviral transducing vector to the fibronectin-coated well.
   1. Warm a centrifuge to 32 °C by running at 2000 x g for about 30 min.
   2. Warm both serum free and growth media in a 37 °C waterbath.
   3. Thaw the virus on ice or by gently swirling the vial in a 37 °C waterbath until only small amount of ice remains. To avoid degradation of the viral preparation, do not allow the contents to warm up.
   4. Dilute the retrovirus to a predetermined optimal multiplicity of infection (MOI) in serum-free medium.
      NOTE: With our gammaretrovirus and rhesus PBMC, we have found that an MOI of 0.5 is optimal.
      1. Example dilution: To coat 4 wells with virus and add 1.5 x 10⁶ cells, (4)(1.5 x 10⁶)(0.5) = 3 x 10⁶ TU are needed. If the viral titer is 1.1 TU/mL, then use 2.7 mL virus in 5.3 mL media for a total volume of 8 mL.
   5. Add 2 mL diluted retrovirus to each well of the fibronectin-coated plate.
   6. For negative control mock-transduced cells, add media alone to the fibronectin-coated wells.
   7. Place the plates in the pre-warmed 32 °C centrifuge in microplate carriers with biosafe covers and centrifuge at 2000 x g for 2 h.
      NOTE: Virus coated plates can be used immediately or stored, with virus, at 4 °C overnight.
   8. For immediate use, aspirate the virus preparation from the wells and add 2 mL of growth medium. Keep the virus-coated wells from drying.
2. Plate the stimulated PBMCs.
   1. Check the cells using an inverted microscope. The cells should look healthy, meaning that they are round, bright and refract light. They should also show a clumped appearance indicating stimulation.
   2. Collect the target cells and transfer them to a 50 mL conical tube. Rinse the well once with 1 mL growth media and add to the conical tube. Count the number of living cells with the automated cell counter as in step 5.4.
      NOTE: Stimulated cells will be clumped and this clumping may lead to difficulties in accurately counting the cells.
   3. Pellet the cells in the tubes by centrifuging at 600 x g for 5 min at 32 °C. Aspirate the media from the cell pellet and resuspend the cells in growth medium at a concentration of 1.5 x 10⁶ cells/mL.
   4. To each virus-coated well (prepared in the step 7.1) add 1 mL of cell suspension for a total of 1.5 x 10⁶ cells/3 mL. Note that each well already contains 2 mL growth medium. Perform the same steps for mock-transduced cells but plate them onto fibronectin-coated wells which received no virus.
   5. Centrifuge the plates at 1000 x g for 10 min at 32 °C.
   6. Incubate the plates for 48 h at 37 °C under 5% CO₂.

8. Expansion of the transduced cells

1. Draw the contents of the wells up and down with a 5 mL pipet in order to resuspend the cells and then transfer to a 50 mL conical tube.
2. Add 1 mL of growth media to each well and draw up and down with a sterile transfer pipet to remove adherent cells.
3. Count cells to determine the total cell number and viability using an automated cell counter as in step 5.4.
4. If desired, remove 1 x 10⁶ cells for flow cytometry to assess gene expression and phenotype. See step 9.5.1 and the Table of Materials for recommended antibodies and gating strategy.
5. Centrifuge the cells at 600 x g for 10 min at 25 °C.
6. Aspirate the media and resuspend the cells in expansion media to a concentration of 1 x 10⁶ cells/mL. Seed each gas-permeable well of a 6-well plate with 5 mL of cells. Carefully layer an additional 25 mL of expansion media per well.
   NOTE: We add the cells in a small volume and then layer remaining media so that the cells remain on the bottom of the well.
7. Incubate the cells undisturbed at 37 °C in 5% CO₂ incubator for 4 days.

9. Collection of expanded cells and evaluating the phenotype of the transduced cell population

1. Collect cells from gas permeable wells by removing and discarding 20 mL media. Caution should be taken to avoid disturbing the cells.
   NOTE: We expand the cells for only 4 days and collect the cells at this point. However, if additional days of culture are desired, remove 20 mL of media without disturbing the cells and replace it with 20 mL of fresh expansion media.
2. Pipet the remaining media up and down to dislodge the cells. The media should be very cloudy if cells have grown well.
3. Using a sterile transfer pipet, rinse each well with 3 mL media to collect any residual cells.
4. Count the cells with the automated counter or a hemocytometer to check for viability and cell number.
5. Perform flow cytometry to determine transduced gene expression and cell phenotype.
   1. In this example, determine rhesus macaque PBMC phenotype using antibodies directed against CD4 (M-T477), a clone that is reactive with both endogenous rhCD4 and the rhCD4-MBL CAR; against CD3 (SP34-2) and CD8 (RPA-T8) which stain total T cells and CD8 T cells respectively; against the death receptor CD95 (DX2) and the co-stimulatory molecule CD28 (CD28.2) which are used to determine the memory phenotype of the cells; and against CXCR5 (MU5UBEE) and MBL (3E7) to detect the CXCR5 and CD4-MBL CAR on the transduced cells.
   2. Assess viability was assessed with a fixable viability dye.
   3. Prepare cells for flow cytometry.
      1. Make up an antibody master mix for the total number of tubes. Bring up to total volume using PBS.
      2. Add the cells to flow tubes. Place 0.5–1 x 10⁶ cells per tube. Include negative controls such as unstained cells as well as stained mock transduced cells.
      3. Add 2 mL of PBS to the cells, centrifuge at 400 x g for 5 min at room temperature (RT) and decant.
      4. Add 100 µL of antibody mix to the tubes and incubate for 30 min at RT.
      5. Add 2 mL of PBS, centrifuge at 400 x g for 5 min at RT and decant.
      6. Add 300 µL of 1% paraformaldehyde and incubate for 15 min.
      7. Centrifuge at 400 x g for 5 min at RT and decant. Add 300 µL of PBS and mix.
   4. On a flow cytometer, acquire 150,000 events for each sample.
   5. Analyze the data with flow analysis software.
      NOTE: The gating strategy was previously published²⁰.
      1. For identification of transduced cells, gate cells on lymphocytes, singlets, live, CD3+, and MBL+CXCR5+.
      2. For identification of the central memory population, gate cells on lymphocytes, singlets, live CD3+, CD8+, CD28+ CD95+.
6. Use cells as needed. Cells can be used for in vitro assays of function, such as a transwell migration assay⁹ or in vitro cell killing assays^5,9. Cells can be used for infusion into animals. Alternatively, freeze any remaining cells in 90% FBS with 10% dimethyl sulfoxide (DMSO) for later use or analysis
   NOTE: While a target dose for infusion of adoptively transferred cells into a rhesus macaque is not yet established, published adoptive transfer studies in non-human primates have used 0.6 to 1.2 x 10⁷ cells/kg²¹, 1 to 5 x 10⁸ cells/kg²² and 1.4 to 8 x 10⁸ cells/kg¹⁰. With this range as a guideline, enough cells for infusion can be produced with this 9-day protocol.