Engineering of CAR T cells for research use

Supplement TexMACS™ Medium with MACS® Cytokines Human IL-7, premium grade (40 IU/mL) and Human IL-15, premium grade (40 IU/mL).
▲ Note: Make sure to freshly add the cytokines to the medium for effective T cell expansion.

Prepare buffer by diluting the MACS BSA Stock Solution 1:20 with autoMACS® Rinsing Solution or PBS/EDTA. 
▲ Note: Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column.

Prepare the 10 mM CFSE stock solution in 1 mL of DMSO. Use CFSE at a final concentration of 1 µL by diluting the stock solution 1:10,000 in PBS.

Freshly prepare the following staining cocktails for each sample.
▲ Note: Store staining cocktails in the dark in the refrigerator (2–8 °C) until use. Do not store for extended periods.

• Reconstitute and dilute standard with MACSPlex Buffer or use the same medium as it is used for the dilution of the sample.
• Only use freshly prepared standard solutions. Do not store or reuse reconstituted or diluted standards.
• Use polypropylene or polystyrene reagent tubes. Do not use glass vials. The generation of standard curves requires eight samples: seven samples of the standard, and one blank control. These samples will be measured as duplicates. For details refer to respective data sheet.

1. Thaw one vial containing the lyophilized MACSPlex Cytokine 12 Standard.
2. Open the vial and add 200 μL of MACSPlex Buffer or media to the pellet and mix gently. 
   This is the stock solution (10,000 pg/mL for IL-4, IL-5, IL-17A, IL-23, and 50,000 pg/mL for IL-2, IL-10, IL-12p70, IFN-γ, TNF-α, and GM-CSF).
3. Label six reagent tubes and arrange them in the following order: 
   a. 1:5 (1:5¹; 2,000 pg/mL or 10,000 pg/mL)
   b. 1:25 (1:5²; 400 pg/mL or 2,000 pg/mL)
   c. 1:125 (1:5³, 80 pg/mL or 400 pg/mL)
   d. 1:625 (1:5⁴; 16 pg/mL or 80 pg/mL)
   e. 1:3125 (1:5⁵; 3.2 pg/mL or 16 pg/mL)
   f. 1: 15,625 (1:5⁶; 0.6 pg/mL or 3.2 pg/mL)
4. Pipette 200 μL of MACSPlex Buffer or TexMACS Medium into each tube.
5. Perform a 1:5 dilution by transferring 50 μL from the stock solution to the tube labeled 1:5 and mix thoroughly. Continue making 1:5 serial dilutions by transferring 50 μL from the tube labeled 1:5 to the tube labeled 1:25 and so on to the tube labeled 1:15,625. Mix each dilution before performing the next transfer.
6. Keep 200 μL MACSPlex Buffer or media as blank control (0 pg/mL).

The peripheral blood or buffy coat should not be older than 8 hours and supplemented with anticoagulants (e.g. heparin, EDTA, citrate, ACD-A, or citrate phosphate dextrose (CPD)). PBMCs should be isolated by density gradient centrifugation, e.g., using Ficoll-Paque™. Resuspend cell pellet in an appropriate amount of PBS-EDTA and proceed to magnetic labeling.

•  For optimal performance it is important to obtain a single-cell suspension before magnetic labeling. Pass cell suspension through Pre-Separation Filters to remove cell clumps which may clog the column. Wet filter with buffer before use.
• Refer to user manual for instructions on how to use the autoMACS® Pro Separator.
• All buffer temperatures should be ≥10 °C.
• Place tubes in the following Chill Rack positions: position A = sample, position B = negative fraction, position C = positive fraction.
• For manual separation of CD4⁺ and CD8⁺ T cells, refer to the respective data sheets of the CD4 MicroBeads, human (# 130-045-101) and CD8 MicroBeads, human (# 130-045-201).

1. Determine cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend cell pellet in 120 μL of buffer per 2×10⁷ total cells.
   ▲ Note: (Optional) To compare purity before and after enrichment, remove a small aliquot (e.g. 50–100 μL) from the fraction and keep it on ice.
   ▲ Note: For manual separation refer to the respective data sheet.
4. Place sample (position A) and collection tubes (position B and C) into the Chill Rack.
   ▲ Note: Refrigerate Chill Rack overnight to ensure samples are kept at 4 °C.
5. Mix CD4 MicroBeads, human and CD8 MicroBeads, human (1:1).
   ▲ Note: Mix at least 45 µL CD4 MicroBeads and 45 µL CD8 MicroBeads.
   ▲ Note: Shelf life of the CD4 and CD8 MicroBead mixture corresponds to the shelf life of the single CD4 or CD8 MicroBeads and can be stored at 2–8 °C until expiration date.
6. Switch on the instrument for automatic initialization.
7. Go to the Reagent menu and press the Read Reagent button. Scan the QR code below. Alternatively, select Enter Reagent in the Reagent menu. Enter ‘130-999-995’ and select Custom_MicroBead_Mix.
   ▲ Note: Software update 2.4.7.0 is required.
8. Place the reagent into the appropriate position on the reagent rack.
9. Go to the Separation menu and select the position for each sample. Select Cust_MB from the Labeling submenu (the correct labeling, separation, and wash protocols will be selected automatically).
10. Enter sample volume (min. 120 µL) into the Volume submenu. Press Enter.
11. Select Run.
12. The positive fraction containing CD4⁺ and CD8⁺ T cells is collected in row C.
13. Remove positive fraction and proceed with section "(Optional) Analysis of purity before and after T cell enrichment".

• Additional antibodies can be included in the analysis according to the respective needs. For additional antibodies refer to www.miltenyibiotec.com/antibodies.

1. Remove a small aliquot from the fraction representing the enriched CD4⁺ and CD8⁺ T cells (e.g. 50–100 μL).
   ▲ Note: For comparative analysis of CD4⁺ and CD8⁺ T cells before and after enrichment use also the aliquot from step 3.3.
2. Determine cell numbers.
3. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
4. For each sample resuspend up to 10⁶ nucleated cells in 100 μL immune cell composition staining cocktail.
   ▲ Note: Refer to section “Reagent and instrument preparation”, table 1 “Staining cocktail for immune cell composition antibody panel”.
5. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
   ▲ Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
6. Wash cells by adding 1−2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
7. Resuspend cell pellet in a suitable amount of buffer (e.g. 500 μL) for analysis by flow cytometry.
   ▲ Note: Cells must be acquired within 1 hour after staining.

• CFSE is added to enriched CD4⁺ and CD8⁺ T cell suspension to assess cell proliferation at any given point in time and for every condition tested. Use an additional separate tube for each condition (e.g. transduced, non-transduced, stimulated, non-stimulated, different CAR constructs,…) because antibodies for flow analysis are conjugated to fluorochromes that exhibit the same spectrum as CFSE 
  (λ_ex 492 nm; λ_em 517 nm). 
  Alternatively, every other cell proliferation dye can be used that does not interfere with the fluorochromes of the antibody panel. 
  ▲ Note: Cell proliferation is usually analyzed on days 3, 6, 8, 10, and 12. However, please feel free to choose time points that are appropriate for the experimental needs. 
  ▲ Note: To assess expansion rates, please also determine cell numbers, e.g., on days 3, 6, 8, and 10 using a Neubauer Chamber or the counting function of the MACSQuant® Analyzer 10.

1. Determine cell numbers.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. Resuspend cells to a final concentration of 2×10⁷ cells/mL in PBS.
4. Add the CFSE stock solution to a final concentration of 1 μM to the cell suspension (e.g. add 1 μL of 10 mM CFSE stock solution to 10 mL cell suspension).
5. Mix well and incubate for 10 minutes at 37 °C.
6. Add one volume of human AB serum, mix gently, and incubate for 5 minutes at room temperature (e.g. add 10 mL human AB serum to 10 mL cell suspension).
7. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
8. Resuspend cells to a final concentration of 2×107 cells/mL in TexMACS™ Medium without supplements.
9. Repeat steps 7 and 8 to wash cells twice. Cells are now ready for activation and expansion.

• For optimal T cell stimulation EDTA must be removed. Ensure removal of EDTA (e.g. over 200-fold reduction).

1. Wash cells twice with TexMACS Medium (without supplements) by filling the tube to the maximum volume and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
2. Resuspend T cells in TexMACS Medium supplemented with Human IL-7, premium grade (40 IU/mL) and Human IL-15, premium grade (40 IU/mL) to a final density of 1×106 cells per mL per cm². 
3. Add T Cell TransAct™, human to a final dilution of 1:100 and carefully resuspend. 
   ▲ Note: T Cell TransAct, human is soluble and applied using an optimized titer and not using bead-to-cell ratio.
4. Add the cell suspension into a suitable cell culture vessel (e.g. 2×10⁶ cells in 2 mL/well of a 24-well plate). For details refer to table 3.
5. Incubate for in total 3 days at 37 °C, 5–10% CO₂.
   ▲ Note: Inspect culture daily and add fresh medium if required.
   ▲ Note: T Cell TransAct, human needs to be washed out very thoroughly after three days (refer to step 8.1).

• When transducing T cells with a lentiviral vector the multiplicity of infection (MOI) should be calculated.
• In general, conditions for the viral transduction must be defined for each virus independently. The given step-by-step instruction is an example and must be adapted to the given conditions.
• Include mock-transduced T cells as negative control.

1. On day two (24 hours after seeding and application of T Cell TransAct™, human to enrich CD4⁺ and CD8⁺ T cells), thaw lentiviral vector on ice following manufacturer’s recommendations and mix gently by pipetting up and down.
2. Calculate the volume of lentiviral vector for transduction with a known T cell number and MOI. 
   Refer to example below:
   - Number of T cells to be transduced: 10⁶ cells
   - Viral titer: 2×10⁸ TU/mL
   - MOI: 2
   Example:
   (Number of cells/viral titer) × MOI = Volume of viral vector 
   (1×10⁶ cells/2×10⁸ TU/mL) × 2 = 10 μL
3. Add the required amount of lentiviral vector (e.g. CD19 CAR construct) to the T cell culture.
4. Mix gently and incubate T cells for 48 hours (until day 4) at 37 °C, 5–10% CO2.
5. On day 4, (three days after activation), centrifuge transduced cells at 300×g for 10 min. Replace 1.8 mL of activation medium by TexMACS™ Medium supplemented with Human IL-7, premium grade (40 IU/mL) and Human IL-15, premium grade (40 IU/mL).

• Inspect culture daily. Depending on the expansion rate, split transduced cells at least every third day or when the cell confluence exceeds 70%.
• A 9-day expansion period should be adequate to obtain sufficient numbers of expanded lentiviral-transduced cells.
• Upon stimulation with T Cell TransAct, human, expression of CD3 will be transiently down-regulated. Thus, the antibody staining of CD3 on the cell surface of activated cells might be affected.

1. On day 4 (three days after activation), remove T Cell TransAct, human thoroughly. Centrifuge cell suspension at 300×g for 10 min and remove supernatant.
2. Resuspend cell pellet in 1.8 mL of pre-warmed TexMACS Medium supplemented with Human IL-7, premium grade (40 IU/mL) and Human IL-15, premium grade (40 IU/mL).
3. When media is exhausted, split cell suspension into two equal parts and add TexMACS Medium supplemented with Human IL-7, premium grade (40 IU/mL) and Human IL-15, premium grade (40 IU/mL). Incubate at 37 °C, 5–10% CO₂.
4. Repeat step 3 when media is exhausted (e.g. every 2–3 days) or when cells reach 70% confluency.
5. Incubate for 9 days (until day 12) at 37 °C, 5–10% CO². 
   ▲ Note: The ideal cell density for T cell expansion until next splitting is 1–2×10⁶ T cells per mL.

• On day 9 of expansion remove a small aliquot from the fraction representing the transduced CD19 CAR-expressing CD4⁺ and CD8⁺ T cells (e.g. 50–100 μL).
  ▲ Note: Transduction efficiency can also be determined before and within the CAR T cell expansion period.

1. Determine the cell number.
2. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
3. For each sample resuspend up to 10⁶ nucleated cells in 100 μL transduction efficiency staining cocktail.
   ▲ Note: Refer to section “Reagent and instrument preparation”, table 2 “Staining cocktail for transduction efficiency antibody panel”.
4. Mix well and incubate for 10 minutes in the dark in the refrigerator (2−8 °C).
   ▲ Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
5. Wash cells by adding 1−2 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
6. Resuspend cell pellet in a suitable amount of buffer (e.g. 500 μL) for flow cytometry analysis.
   ▲ Note: Cells must be acquired within 1 hour after staining.

1. For monitoring T cell proliferation rates samples of 25–50 μL are taken at appropriate points in time (e.g. day 3, 6, 8, 10, and 12) and diluted 1:2 with buffer. CFSE dilution is measured on the MACSQuant® Analyzer 10. At least 20,000 events are recorded.
2. For determination of the T cell expansion rates samples of 25–50 µL are taken at appropriate points in time (e.g. days 3, 6, 8, and 10) and the count of viable cells is measured on the MACSQuant Analyzer 10. For dead cell exclusion propidium iodide is added prior to flow cytometric acquisition. Expansion rate is calculated afterwards.
   ▲ Note: Points in time can be adapted.

For CAR T cell phenotyping, e.g., to assess activation, differentiation, proliferation, and exhaustion of CAR T cells, contact macstec@miltenyi.com. All CAR T cell phenotyping panels are compatible with MACSQuant® Express Modes.

• Antigen-expressing target cells must be chosen upon the respective CAR construct utilized for the viral transduction (e.g. co-culture of GFP⁺CD19⁺ JeKo-1 B cell lymphoma target cell line with CD19 CAR T cells).
• If not working with GFP⁺ target cells: target cell labeling can be performed using CellTrace™ Violet Cell Proliferation Kit. For detailed information refer to end of this section “(Optional) Target cell labeling for killing assay”.

1. On day 12 set CD19 CAR T cells cytokine-free: Wash the cells twice with TexMACS™ Medium by filling the tube to the maximum volume and centrifuge at 300×g for 10 minutes. Completely aspirate supernatant and resuspend in plain TexMACS Medium without any additional Human IL-7, premium grade and Human IL-15, premium grade.
2. Seed target cells (e.g. JeKo-1 GFP⁺ CD19 surface protein-expressing target cells) and control cells (e.g. JeKo-1 GFP⁺ CD19 knockout cells), respectively, in a 96-well plate.
3. After 24 hours of cytokine-free cultivation determine CAR T cell number.
4. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
5. Resuspend cells to a final concentration of 2×10⁷ cells/mL in PBS.
6. Add CAR T cells (effector cells (E)) to each well plate containing target cells (T) or control cells at different E:T ratios (e.g. 10:1, 5:1, 1:1, 0.2:1).
7. Co-culture CAR T cells with respective target cells for 24 hours.
8. Remove 96-well plate from the incubator and incubate for 20 minutes at 2–8 °C.
   ▲ Note: (Optional) Supernatant of the E:T 1:1 condition can be used to determine secreted cytokines using MACSplex Assays (please refer to section 10.4).
9. (Optional) Take out 100 µL of the supernatant of each well of the 96-well plate for the cytokine analysis (section 10.4) and fill up with 100 µL of fresh TexMACS Medium without supplements.
10. The killing rate is determined by analyzing the GFP fluorescence intensity of the target cells using the MACSQuant® Analyzer. Make sure that for measurement sufficient running buffer and washing solution are in the containers of the MACSQuant Analyzer. Refill if the levels are low. Additionally, the waste container must be empty.
11. Make sure that propidium iodide is automatically added by the MACSQuant Analyzer using the autolabel function.
12. Define the sample volume (200 µL) and the uptake volume (100 µL). Choose the Acquisition Mode “High” and activate mixing.
13. Start the acquisition of all samples. For results refer to figure 6 in section “Results”.

(Optional) Target cell labeling for killing assay

• If not working with GFP⁺ target cells: target cell labeling can be performed using CellTrace™ Violet Cell Proliferation Kit.

1. Dilute CellTrace Violet dye stock solution to 1 μM using sterile 1× standard buffer.
2. Determine the cell number.
3. Calculate the volume to adjust the cells to a final concentration of 5×10⁶ cells/mL.
4. Centrifuge cells at 300×g for 10 minutes.
5. Discard supernatant and resuspend the cell pellet in the calculated volume of CellTrace Violet dye staining solution, mix well, and incubate for 5 minutes at 37 °C, 5% CO₂.
6. After incubation add 5 mL of FCS and 5 mL of TexMACS Medium (without supplements).
7. Centrifuge the cells at 300×g for 10 minutes.
8. Discard supernatant and resuspend the cell pellet in 5 mL TexMACS™ Medium.
9. Determine the cell number.
10. Calculate the volume to adjust the cells to a concentration of 1×10⁵ cells/mL.
11. Centrifuge the cells at 300×g for 10 minutes.
12. Discard supernatant and resuspend the cell pellet in the calculated volume of TexMACS Medium.

MACSPlex Assays are designed for determining concentrations of soluble analytes in a single sample. The analysis is based on MACSPlex Capture Beads, which display defined fluorescence properties and can be identified using standard flow cytometry techniques (figure 2).

The MACSPlex Cytokine 12 Kit, human is used to detect GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and TNF-α.

• Run the assay at room temperature. Work fast and keep samples protected from light, for example, cover plate or tubes with aluminum foil, especially during incubation steps.
• Unknown samples should be run in replicates, for example, in duplicates or triplicates and in different dilutions to make sure the fluorescence values are within the dynamic range of the standard curve.
• Additional cytokines can be included in the analysis according to the respective needs. For additional cytokines refer to www.miltenyibiotec.com/cytokines.

Setup of the assay in a 96-well plate

Design the assay using two columns of the MACSPlex Filter Plate for the standards. Add each of the seven standard samples in duplicates next to each other. Standards should be run in order from the lowest concentration (blank control: 0 pg/mL) to the highest concentration (stock solution: 10,000 pg/mL or 50,000 pg/mL). Start with the unknown sample in the next column of the plate. For more details, refer to the respective data sheet, section ”Protocol for the assay using the MACSPlex Filter Plate”.

1. Pre-wet required wells of the MACSPlex Filter Plate with 200 μL of MACSPlex Buffer per well and aspirate off using a vacuum manifold designed to accommodate the filter plate (max. -300 mbar) until the wells are drained.
2. Place the filter plate briefly on a tissue to remove any residual liquid.
3. Add 50 μL of MACSPlex Buffer or media as a blank control, 50 μL of each dilution, and the stock solution of the MACSPlex Cytokine 12 Standard to the corresponding wells of the filter plate.
4. Add 50 μL of each unknown sample per well.
5. Resuspend MACSPlex Cytokine 12 Capture Beads by vortexing for at least 30 seconds and transfer 20 μL of MACSPlex Capture Beads to each well.
6. Incubate filter plate for 2 hours protected from light on an orbital shaker (450 rpm).
7. Apply the filter plate to the vacuum manifold and aspirate until wells are drained. Place the filter plate briefly on a tissue to remove any residual liquid.
8. Add 200 μL MACSPlex Buffer to each well and apply the filter plate to the vacuum manifold and aspirate off until wells are drained. Place the filter plate briefly on a tissue to remove residual liquid.
9. Repeat step 8.
10. Add 80 μL of MACSPlex Buffer to each well.
11. Add 20 μL of MACSPlex Cytokine 12 Detection Reagent to each well.
12. Incubate filter plate for 1 hour protected from light on an orbital shaker (450 rpm).
13. Repeat wash steps 7 and 8.
14. Add 200 μL of MACSPlex Buffer to each well.
15. For sample acquisition using MACSQuant® Instruments and the Express Mode place the filter plate onto the Chill 96 Rack. To prevent liquid transfer from the wells, ensure that residual drops under the plate are completely removed by placing the plate briefly on a tissue.
    ▲ Note: Perform the flow cytometric acquisition on the same day, as prolonged storage of samples can result in increased background and reduced sensitivity.
    ▲ Note: Keep samples protected from light by using the protection lid during the flow cytometric acquisition with the MACSQuant Instrument.

Flow cytometer setup

The kit includes MACSPlex Setup Beads for flow cytometer setup. MACSPlex Setup Beads are not required when using the MACSQuant Analyzer or MACSQuant Analyzer 10 but for all other flow instruments that can detect FITC, PE, and APC. 
▲ Note: The kits are not suitable for use with the MACSQuant VYB.

Setup of the MACSQuant® Instrument

Calibrate the MACSQuant® Instrument using MACSQuant Calibration Beads (# 130-093-607). For details, refer to the data sheet of the MACSQuant Calibration Beads. After successfully completing the calibration, the MACSQuant Instrument is ready for measurement. No further steps are required as all necessary setup steps are performed automatically during calibration.

Setup of other flow cytometers

The analysis of MACSPlex Cytokine 12 Kit, human requires a flow cytometer with a blue (e.g. 488 nm) and a red (e.g. 635 nm) laser, which can detect FITC, PE, and APC. For setting up these cytometers use MACSPlex Setup Beads (included in the kit). For instructions on the setup procedures of other flow cytometers, refer to the application note "General instructions for MACSPlex Cytokine Kits" also available on the product page at www.miltenyibiotec.com/130-099-169 in the "Resources" section.

Flow cytometric acquisition and data analysis using the MACSQuant® Express Mode

To perform the acquisition and data analysis of the MACSPlex Cytokine 12 Kit, human with the MACSQuant® Instrument it is recommended to use the Express Modes MACSPlex_Standard and MACSPlex_Sample to achieve automated measurement and data analysis. For details refer to the special protocol “Data acquisition and analysis of the MACSPlex Cytokine Kits using the MACSQuant® Analyzer Express Modes” available at www.miltenyibiotec.com/130-099-169 in the "Resources" section. The minimum version number of the Express Mode package needed to run the assay on the MACSQuant Instrument can be found there as well. To check the version number of the Express Mode package available on the MACSQuant Instrument please select Help> Info> expressModes within the MACSQuantify™ Software. The version number of the Express Mode package is increasing with each Express Mode updates. Make sure the MACSQuant Instrument contains an Express Mode package with at least the same or higher version number than the special protocol is marked with. 
For results refer to figure 7 in section “Results”.