High-Efficiency Generation of Antigen-Specific Primary Mouse Cytotoxic T Cells for Functional Testing in an Autoimmune Diabetes Model

Mice were maintained under specific pathogen-free conditions at a Transgenic Mouse Facility, and all animal experiments were performed in accordance with protocols approved by the Baylor College of Medicine animal care and use committee.

NOTE: The experiment requires preparing the virus and the T cells in parallel. Table 1 summarizes the protocol. The key reagents and buffers are listed in the Table of Materials. We focus on the generation and expansion of CAR-T cells targeting specific populations of APCs in this protocol.

1. Generation and validation of single chain Fab antibody (scFab)-CARs.

NOTE: CARs typically contain 3 critical domains—an antigen targeting domain, a spacer/transmembrane domain, and a cytoplasmic signaling domain. The precise design of each CAR depends on the intended target, and so, apart from the key features of the construct relevant to the generation of the retrovirus, will not be described in detail in this protocol. The overall design of the CARs used for the studies described below is shown in Figure 1. In brief, the targeting domain comprises the entire light chain and variable and CH1 domain of the heavy chain from the parent monoclonal antibody linked by a semi-rigid linker. The spacer/transmembrane domain is from mouse CD28, and the signaling domain is a fusion containing elements from mouse CD28, CD137 (4-1BB), and CD247 (CD3ζ). These elements are assembled by standard molecular biology procedures such as splice overlap polymerase chain reaction (PCR), or the synthesis of an appropriate “gene block”. Details of the generation of the mAB287 CAR are contained in Zhang et al.⁷. The cDNA sequences can be obtained from the authors upon request.

1. Assembling the CAR construct
   1. Synthesize the targeting single chain Fab antibody (scFab) and combined spacer/signaling domains separately, and use a “3 point” ligation technique¹⁵ to assemble the final construct (Figure 1).
      NOTE: The key requirement for the CAR insert is that it should contain flanking restriction endonuclease sites allowing ligation into the retroviral expression vector pMSCV-IRES-GFP II (pMIG II), or a related derivative¹⁵ are also appropriate.
2. Validation of CAR surface expression
   1. Transduce the hybridoma cells using pMIG II derived retroviral particles generated by a standard protocol (e.g., Holst et al.¹⁶).
   2. Run flow cytometry analysis to detect the expression of GFP from the CAR vector¹⁷.
   3. Stain surface expression of the CAR of the transduced hybridomas using labeled antibodies against the mouse κ chain (e.g., clone RMK-45)¹⁷.
      NOTE: ¹⁸.
3. Validation of CAR specificity
   1. Stimulate the transduced hybridoma cells with appropriate plate-bound or cellular antigens. After overnight co-culture collect supernatants and secreted cytokines, and assayed by enzyme-linked immunosorbent assay (ELISA)⁷.
      NOTE: Ideally, each CAR should be independently validated before being used for transduction. At this step, the experiment may be paused and restarted later.

2. Transfection of viral producer cells (day -4 to day 3)

NOTE: Retrovirus is produced using Phoenix-ECO cells (see the Table of Materials)^19,20. Use appropriate precautions for the generation of potentially infectious agents (preferably including a designated BSL-2 cabinet and separate incubator for culturing transfected/transduced cells).

1. Thawing Phoenix cells (Day -4)
   1. Thaw 2 x 10⁶ Phoenix-Eco cells. Scale up the number of Phoenix cells if multiple transductions are planned.
   2. Plate them in a 10 cm tissue culture dish with 10 mL of medium (Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS)).
2. Passage Phoenix cells (Day -3)
   1. Remove medium, and wash with 5 mL of Dulbecco's phosphate-buffered saline (DPBS).
   2. Add 3 mL of 0.25% trypsin and incubate at 37 °C under 10% CO₂ atmosphere for 3 min.
   3. Harvest the cells then pellet by centrifugation for 3 min at 200 x g. Re-plate cells with 10 mL fresh medium and incubate at 37 °C.
3. Irradiation of Phoenix cells (Day -1 afternoon)
   1. To minimize further cell division, collect Phoenix cells as described in step 2.2, resuspend in 5 mL of medium, and gamma irradiate cells on ice (1000 rad).
      NOTE: Caution should be used for radiation work to avoid personnel exposure.
   2. Centrifuge the irradiated cells, resuspend in fresh medium, plate at 2 x 10⁶ cells (in 10 mL of medium)/plate/CAR, and incubate.
4. Transfection (Day 0 – morning)
   1. Aspirate the supernatant from the Phoenix cells, wash with 5 mL of phosphate-buffered saline (PBS), and carefully add 7 mL of reduced serum medium (e.g., Opti-MEM) dropwise to the sidewall of the plate to avoid disturbing the monolayer. Transfer cells back to incubator.
   2. Take two 14 mL round bottom polypropylene tubes, and add 1.5 mL of reduced serum medium to each. To one tube, add 40 µL of transfection reagent (see the Table of Materials).
   3. To the other tube, add 15 µg of Ab-CAR-plasmid (generated in step 1) and 5 µg of envelope and packaging plasmid (5 µg pCL-Eco). Incubate tubes at room temperature for 5 min.
   4. Add the transfection reagent mixture from step 2.4.2 dropwise to the second tube without contacting the tube sides, and mix by pipetting the solution up and down gently 3 times. Incubate at room temperature for at least 20 min.
   5. Add 3 mL of the mixture dropwise to the Phoenix cells, and place in a tissue culture incubator.
   6. After 4–5 h add 1 mL of FCS. Culture cells overnight at 37 °C.
5. Medium change (Day 1)
   1. Remove the supernatant containing the plasmid/transfection reagent complexes and dispose of them in accordance with institutional procedures for handling infectious material. Add 4 mL of fresh, pre-warmed culture medium to the cells.
6. Harvest virus for Transduction (Day 2)
   1. Collect the virus-containing medium from the Phoenix cells with a sterile syringe, filter (0.45 µm) to remove residual cell debris, and collect in a new tube.
   2. Add rhIL-2 stock to a final concentration of 200 IU/mL. Use the virus immediately for transduction (step 5.3). Add 4 mL of fresh medium to the Phoenix cells and place in the incubator.
7. Repeat virus collection (Day 3)
   1. Repeat step 2.6, but discard Phoenix cells as infectious waste instead of adding fresh medium. This supernatant is used in step 5.4.

3. Primary CD8 T cell isolation and activation (day -1 to day 0)

NOTE: Previously, collect CD8 T cells from female NOD mice at 4–5 weeks, a time point before islet inflammation starts^21,22. Handle all the mice following IACUC approved protocols. CD8 T cells are enriched from splenocytes using a commercial negative selection kit.

1. Coating plates with CD3/CD28 antibodies (Day -1)
   1. Add 1 mL of a mixture of anti-mouse CD3 and CD28 antibodies (both at 1 µg/mL in PBS) to each well of a 24-well plate, and incubate at 4 °C overnight.
   2. The next day, wash the plates with 1 mL of sterile PBS 3 times before adding the murine CD8 T cells (step 4.1).
      NOTE: The number of wells to be coated will vary for each experiment, depending on the total number of activated CD8 T cells required.
2. Collection of splenocytes (Day 0)
   1. Euthanize two NOD female mice aged 4–5 weeks using CO₂ inhalation followed by decapitation. Harvest the spleens and put them onto a cell strainer soaking in 10 mL PBS in a cell culture dish on the ice.
   2. In a cell culture hood, cut each spleen into 3–5 pieces, press tissues with a sterile plunger of a 3 or 5 mL syringe to force spleen fragments apart and allow cells to pass through the wire mesh.
   3. Gently remove red blood cells by resuspending splenocytes in 1:4 diluted red cell lysis buffer (1 mL of lysis buffer in 3 mL of PBS for one spleen), and incubating for 5 min at room temperature.
   4. Then, dilute 10 μL of the cell suspension with trypan blue dye solution for counting cells with a hemocytometer, and pellet the rest of the cells by centrifugation at 350 x g for 7 min.
3. Enrichment of CD8 T cells (Day 0)
   1. Enrich CD8 T cells by negative selection using a mouse CD8 T cell isolation kit, following the manufacturer’s instructions.
      NOTE: To ensure high purity always round up the cell numbers when calculating the volume of biotinylated-antibody to be added (e.g., use the volume of reagents suggested for 10⁸ cells for a calculated 9.1 x 10⁷ cells).
   2. Suspend cell pellets in 400 µL of buffer and 100 µL of biotin-antibody cocktail per 1 x 10⁸ cells, mix well and incubate for 5 min in the refrigerator (4 °C) to allow antibody binding.
   3. Add 300 µL of labeling buffer and 200 µL of anti-biotin micro-beads per 1 x 10⁸ cells, mix well and incubate for 10 min at 4 °C.
   4. While waiting for the micro-bead binding, set up the separation column onto the separator. Wash column by rinsing with 3 mL of labeling buffer.
   5. Pass 1000 µL of bead/cell mixture through a 40 µm cell strainer before loading onto the separation column to remove cell aggregates. Collect the column flow-through into a pre-chilled 15 mL tube.
   6. Wash the column as instructed by the manufacturer, collecting all the effluent into the same tube. Determine the cell number (same as step 3.2.4) and collect by centrifugation at 350 x g for 5 min. Wash the cells by resuspending in 2 mL of complete T cell medium (RPMI-1640 containing FCS, 2-mercaptoethanol, rhIL-2 (200 U/mL), mIL-7 (0.5 ng/mL), ITS, HEPES and penicillin-streptomycin) and centrifuging at 350 x g for 5 min.
   7. Resuspend the cells in pre-warmed (37 °C) complete T cell medium at a concentration of 0.25–0.5 x 10⁶/mL.

4. T cell activation (Day 0 to 2)

1. Add 2 mL of the cell suspension (0.25–0.5 x 10⁶/mL) to each coated well of the CD3/CD28 antibody coated 24-well plate from step 3.1.2. Use a swirling motion to dispense the cells evenly.
   NOTE: Add the cells using a swirling motion to distribute them evenly and minimize edge effects. If the cells cluster along the edge of the wells, both the transduction rate and cell viability will be decreased.
2. As a control, plate the same number of CD8 T cells into a single non-coated well of the plate. Incubate the cells at 37 °C using a 10% CO₂ gassed incubator for 48 h.
   NOTE: After 48 h, activation can be confirmed using a microscope; the activated cells will be larger than the cells that did not encounter anti-CD3/CD28 antibodies.

[place figure 2 here]

5. Transduction of activated CD8 T cells (days 1 to 3)

NOTE: This protocol uses a spin-transduction method. A centrifuge with a swing-out rotor and tissue culture plate adaptors that is capable of maintaining an internal temperature of 37 °C is required. To ensure maximum efficiency, on the day of transduction pre-warm the centrifuge to 37 °C before collecting the virus.

1. Preparation of human fibronectin fragment coated plates (Day 1 to day 2)
   1. On day 1, add 0.5 mL of fibronectin (50 µg/mL in PBS) to the wells of a 24-well plate, and incubate overnight at 4 °C.
      NOTE: Typically, two fibronectin-coated wells are required per plate of transfected Phoenix cells.
   2. On day 2 remove the fibronectin solution, and replace with 1 mL of 2% bovine serum albumin (BSA) in PBS. Incubate at room temperature for 30 min to “block” non-specific binding sites.
   3. Wash the treated wells with 1 mL of sterile PBS. After removing the wash solution, the plate is ready for use; or, can be sealed and stored at 4 °C for up to one week.
2. Collection of activated CD8 T cells (Day 2)
   1. Harvest the activated CD8 T cells, count and calculate cell viability using trypan blue or a suitable automated instrument.
   2. Collect cells by centrifugation and resuspend at 5 x 10⁶ viable cells/mL for transduction. Maintain a small aliquot of cells in culture in the complete T cell medium in the CO₂ incubator to provide a control for subsequent fluorescence activated cell sorting of the transduced cells (step 6).
      NOTE: After activation for 48 h, the total number of cells should have increased by approximately 1.5 fold, and have a viability greater than 95%.
3. Transduction (Day 2)
   1. Add 100 µL of activated CD8 cell suspension per well (0.5 x 10⁶ cells) to the fibronectin coated plate. Then add 1.5–2 mL of virus-containing medium (from step 2.6) to each well. Mix using a swirling motion to dispense the cells evenly (Figure 2).
   2. Place the plate in a zip-lock plastic bag and seal (to provide secondary containment). Centrifuge at 2000 x g for 90 min at 37 °C.
   3. Remove the plate from the centrifuge. In the biological safety, cabinet carefully remove the plastic bag and ensure that the outside of the plate is not contaminated with any medium.
   4. Then transfer the plate to the dedicated 37 °C CO₂ incubator. After 4 h, remove 1 mL of the medium from each well and replace with 1 mL of pre-warmed complete T cell medium. Replace the plate in the CO₂ incubator.
      NOTE: Handle all media from the transduced cells as infectious waste.
4. Second transduction (Day 3)
   1. In the dedicated biological safety cabinet, incline the plate containing the transduced cells by resting on the lid and carefully remove most of the medium (leaving 100–200 µL) making sure not to contact the cells at the bottom of the well.
   2. Add the virus-containing medium collected in step 2.7, and repeat steps 5.3.2–5.3.4.
      NOTE: In our experience, a third transduction rarely improves overall efficiency. In addition, the cell viability will likely drop significantly if a third transduction is used. If cells are plated at a higher concentration than 0.5 x 10⁶/well, the T cells may reach confluence after the overnight incubation following the second transduction step. In this event, split cells after the 4 h incubation on day 3.
5. Wash cells (Day 4)
   1. Remove 1 mL of medium from each well, resuspend the cells in the remaining medium and transfer to a 15 mL tube. Wash the wells with 1 mL of complete T cell medium and add to the tube containing the pooled cells from each transduction.
   2. Centrifuge at 350 x g for 7 min, then wash twice by resuspending in 2 mL of complete T cell medium and pelleting. Finally resuspend in 2 mL of medium and determine the cell number.
      NOTE: If 1 x 10⁶ cells were originally transduced, the yield at this stage should be ~3 x 10⁶.
6. Transfer
   1. Transfer aliquots of 0.5–1 x 10⁶ cells in 2 mL of complete T cell medium to the wells of a new 24-well plate and incubate at 37 °C. Approximately 48–72 h post-transduction the cells are ready for CAR expression analysis and cell sorting.
      NOTE: The number of CAR-T cells usually doubles each 24 h at this stage. It is critical to never let them overgrow. Split the cells immediately if density is higher than 2 x 10⁶/mL (or if the medium ever becomes bright yellow). In our experience the CAR-T cells proliferate more robustly in 24-well and 12-well plates than if transferred to a larger vessel.

6. Purification of transduced cells by fluorescence-activated cell-sorting (FACS) (day 5 or day 6)

1. Collect the cells.
   1. Resuspend the cells by pipetting up and down multiple times (taking care not to cause frothing), transfer to 15 mL tubes and centrifuge at 350 x g for 5 min.
   2. Resuspend in sorting buffer (2% BSA in sterile PBS containing gentamicin) at 1 x 10⁶ cells/mL. Also harvest the control (un-transduced) CD8 T cells from step 5.2).
      NOTE: From 1 x 10⁶ cells at day 2, a yield of ~2 x 10⁷ transduced cells is expected at this time point.
2. Wash the cells once with sorting buffer by centrifuging at 350 x g for 5 min, and resuspend at 1 x 10⁷ cells/mL in sorting buffer. Remove a small aliquot for the Foxp3^GFP compensation control (to be used in step 6.4.1), and stain the remainder with labeled anti-mouse CD8 (clone 53-6.7; 0.2 µg of antibody/5 x 10⁶ cells) by incubating for 20 min at 4 °C.
   1. Similarly, stain an aliquot of the non-transduced CD8 T cells to provide a compensation control for the fluorophore labeling the anti-CD8 antibody.
      NOTE: Avoid adding sodium azide to any buffer, as this is toxic to the cells.
3. Wash the labeled cells twice with sorting buffer, resuspend in cold sorting buffer at 1 x 10⁷ cells/mL.
4. Sort the cells.
   1. Sort CD8 GFP⁺ positive cells into pre-chilled complete T cell medium (Figure 3B). Take a small aliquot for post-sorting analysis to determine the purity.
      NOTE: To maximize the purity of the sorted cells tight gates should be used. Use a 100 µm nozzle to ensure high cell viability. Minimize the amount of time that the sorted cells are kept on ice. T cells that have been kept on ice for more than 3 h take much longer to recover than cells chilled for less than 2 h. Thus, if 3 transduced cell lines need to be sort, collect and label the second line while the first is being sorted and so forth rather than having the second and third lines spend an extended time at 0 °C. The expression of other T cell markers such as CD28 and CD3 can also be monitored (Figure 3C) but is not essential for sorting purposes.
   2. (Alternative sorting strategy) Before staining the bulk population, analyze the CD8 expression of a small population of the transduced T cells. If the purity is >99% then the bulk population can be safely sorted solely on the basis of GFP expression.

7. Expansion of sorted CAR-T cells (Day 5 to 10)

1. CAR-T cell expansion
   1. Wash the sorted CAR-T cells once, then resuspend in pre-warmed complete T cell medium at 2.5–5 x 10⁵ cells/mL, and plate 2 mL aliquots in 24-well plates.
   2. Count and split the cells every 1–2 days. Usually the cell number doubles every day until ~day 10, with viability remaining above 95%.
      NOTE: Without re-stimulation, the CAR-T cells will stop proliferating around day 10 and eventually die. Thus, T cell functional assays and adoptive transfers should be scheduled accordingly.
2. Alternative expansion strategy
   1. After sorting, culture the CAR-T cells in complete T cell medium containing rhIL-2 at 100 U/mL rather than 200 U/mL.
      NOTE: The CAR-T cells proliferate at a slightly slower rate in this medium. However, they will often continue to proliferate until days 11 to 13 without re-stimulation. Thus, although this alternative expansion strategy does not generate a higher number of cells it provides a slightly longer time window for downstream assays to be performed.

8. Verification of the antigen specificity and functionality of the CAR T cells.

NOTE: The binding specificity of CAR T cells targeting peptide/MHC complexes can be verified by tetramer staining^7,23. Similarly, their functionality can be confirmed by measuring cytokine secretion or cytotoxicity following stimulation by their cognate ligands. The NIH Tetramer Core Facility (TCF) at Emory University is a recommended source of “tetramers” and relevant staining protocols.

1. Peptide-MHC Tetramer staining.
   1. Label aliquots of 2 x 10⁵ transduced CAR-T cells in 100 µL of sorting buffer by incubating with ~0.6 µg of fluorescently labeled antigen-specific and control tetramers at 37 °C for 2 h.
   2. Pellet the cells by centrifugation for 5 min at 350 x g, then wash twice by resuspending in 0.5 mL of sorting buffer and re-centrifuging. Finally, resuspend the cells in 300 µL of sorting buffer and analyze by flow cytometry (Figure 4).
      NOTE: For these studies, we typically use BV421-labeled IA^g7-B:9-23(RE) (test) and IA^g7-HEL (control) tetramers. However, any fluorophore/tetramer combination that is appropriate for the CAR(s) under investigation can be used instead. In this case, the concentration and staining time should be optimized for each tetramer used. Both sorted and un-sorted CAR-T cells can be used for tetramer staining.
2. Specificity measurement by ligand stimulation.
   1. Incubate 2 x 10⁵ sorted CAR-T cells in 200 µL of cytokine-free T cell medium with appropriate plate-bound or cellular ligands.
   2. After 6–24 h measure cytokine production by ELISA or intracellular staining using manufacturer’s protocols.
      NOTE: For our studies of IA^g7-B:9-23 redirected T cells, we culture the cells overnight with M12C3 murine B-cell lymphoma cells expressing IA^g7-B:R3 or “empty” IA^{g7 24,25}, then collect the supernatants and measure secreted mouse interferon gamma (IFN-γ) by ELISA²⁶ (Figure 5).