Generation of Memory T Cells by Viral Gene Delivery of Pathogen-Targeting Receptors

Resumo

Source: Pampusch, M. S., Skinner, P. J. Transduction and Expansion of Primary T Cells in Nine Days with Maintenance of Central Memory Phenotype. J. Vis. Exp. (2020)

The video demonstrates retroviral transduction of activated PBMCs on fibronectin-coated wells. Vectors encoding CAR and CXCR5 are immobilized and aligned with PBMCs by centrifugation. Incubation allows viral fusion, reverse transcription, and formation of the pre-integration complex. During cell division, the complex enters the nucleus for stable integration, yielding CAR/CXCR5-expressing T cells with a central memory phenotype for targeted immunotherapy.

Protocolo

NOTE: This rhesus peripheral blood mononuclear cell (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, 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 vesicular stomatitis virus glycoprotein, or 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-Ethylenediaminetetraacetic acid (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 chimeric antigen receptor (CAR) and C-X-C motif chemokine receptor 5 (CXCR5) by flow cytometry. Expression of cluster of differentiation markers (CD4) or mannose-binding lectin (MBL) along with CXCR5 will identify transduced 293T cells co-expressing the CAR and CXCR5.
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. 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.

4. 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.

5. 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.

6. 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 transduction units (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 4.4.
      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 6.1) 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.

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
Gammaretrovirus preparation
0.025% Trypsin, 0.01% EDTA	Gibco	R-001-100
293T cells	ATCC	CRL-3216
6 well plates, treated	CytoOne	CC7682-7506
DMEM	Gibco	10569-010
Heat-inactivated FBS	Hyclone	Sh30088.03
Lipofectamine	Invitrogen	11668019	transfection reagent
Opti-Mem	Invitrogen	31985070	reduced serum media
pBS-CMV-gagpol	Addgene	35614	A gift from Dr. Patrick Salmon
pMSGV1 containing CAR P2A CXCR5	custom order from GenScript
RD114			A gift from Dr. Ed Berger
T75 flasks	CytoOne	CC7682-4875
VSV-G		pMD.G	A gift from Dr. Scott McIvor
T cell stimulation
6 well plates, untreated	CytoOne	CC7672-7506
Anti-CD28	NHP Reagent Resource	Clone: CD28.2
Anti-macaque CD3	NHP Reagent Resource	Clone: FN18
Phosphate buffered saline	Gibco	14190-144
Rhesus macaque PBMC or CD8 T cells	WNPRC		Primary cells
For Fibronectin coating
6 well plates, untreated	CytoOne	CC7672-7506
BSA (Fraction V)	HyClone	SH 30574.02
RetroNectin (1 mg/ml)	TaKaRa	T100A
For T cell Expansion
G-Rex 6 Well Plate	Wilson Wolf	P/N 80240M	Plates with gas permeable wells
Media Components
b mercaptoethanol	Gibco	21985-023
Heat-inactivated FBS	Hyclone	Sh30088.03
IL-2	NCI Preclinical Repository
Penicillin/Streptomycin/Glutamine	Gibco	10378-016
X-Vivo-15 medium	Lonza	04-418Q
Variations of Media used
Basic medium:	X-Vivo 15 medium, 10% heat-inactivated FBS, 1 x Penicillin/Streptomycin/L-Glutamine
Expansion medium:	Growth medium + 50 mM b mercaptoethanol
Growth medium:	Basic medium + 50 IU/ml IL-2		Completion of media by addition of anti-CD28, IL-2 or b-mercaptoethanol should occur on the day of use.