Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging

Summary

A protocol to evaluate quantitative tumor cell killing by Jurkat cells expressing chimeric antigen receptor (CAR) targeting single tumor antigen. This protocol can be used as a screening platform for rapid optimization of CAR hinge constructs prior to confirmation in peripheral blood-derived T cells.

Abstract

Chimeric antigen receptor (CAR) T cells are at the forefront of oncology. A CAR is constructed of a targeting domain (usually a single chain variable fragment, scFv), with an accompanying intra-chain linker, followed by a hinge, transmembrane, and costimulatory domain. Modification of the intra-chain linker and hinge domain can have a significant effect on CAR-mediated killing. Considering the many different options for each part of a CAR construct, there are large numbers of permutations. Making CAR-T cells is a time-consuming and expensive process, and making and testing many constructs is a heavy time and material investment. This protocol describes a platform to rapidly evaluate hinge-optimized CAR constructs in Jurkat cells (CAR-J). Jurkat cells are an immortalized T cell line with high lentivirus uptake, allowing for efficient CAR transduction. Here, we present a platform to rapidly evaluate CAR-J using a fluorescent imager, followed by confirmation of cytolysis in PBMC-derived T cells.

Introduction

CAR-T cell therapy has shown great promise in hematological malignancies, evident from the 6 FDA-approved CAR-T products since 2017, as reported by the National Cancer Institute¹. There are numerous CAR-T cells in clinical trials for targeting solid tumors. Engineering novel CAR targets and optimizing the CAR construct is vital to the efficacy of a CAR-T cell. Choosing the ideal CAR construct for each application is essential for accurate targeting of tumor associated antigens (TAA) while avoiding low levels of TAA expression in normal tissues².

A CAR construct is primarily made of five compartments: (1) extracellular single-chain variable fragment (scFv) domain targeting tumor antigen; (2) hinge domain; (3) transmembrane domain; (4) intracellular cytoplasmic T cell costimulatory domain; and (5) signaling domain. Modifying each of these domains affects the precision of the CAR-T cell engaging with its target cell³. Hence, evaluating the cytotoxicity and cross-reactivity of these CAR constructs in vitro is critical to choose the right construct for progressing toward in vivo experiments. Current methods of evaluating cytolysis by T cells include ⁵¹Cr release assay, lactate dehydrogenase release assay, bioluminescent imaging assay, real-time impedance-based cell analysis, and cell-based flow cytometry assay^4,5. The fluorescent imaging-based platform described here identifies the number of live vs. dead cells, which is a direct quantification of T cell cytolysis as opposed to an indirect method of evaluating the cytolysis by T cells.

Here is an easy, cost-efficient, rapid, and high throughput technique with minimal intervention to evaluate the cytotoxicity of Jurkat cells expressing epidermal growth factor receptor (EGFR) CAR against MDA-MB-231 triple-negative breast cancer (TNBC) cells and EGFR CRISPR knock out MDA-MB-231 cells. Jurkat cells are immortalized human T Lymphocyte Cells⁶ that have been widely used for studying T cell activation and signaling mechanisms⁷. Furthermore, Jurkat cells have been used for in vitro CAR testing in multiple studies^8,9,10,11. Jurkat cells are easily transduced by lentivirus and have sustained proliferation, and this system was leveraged to optimize the hinge domain of various EGFR CAR constructs.

This assay can be used for screening multiple CAR constructs targeting various tumor antigens and used against multiple adherent tumor cell lines and in various effector to tumor (E:T) ratios. Additionally, multiple time points can be evaluated, and number of replicates can be modified to identify best killing among the various CAR constructs. The best constructs need to be confirmed using peripheral blood mononuclear cells (PBMCs) derived CD3 T cells. The overall goal behind developing this method is to rapidly optimize CAR hinge geometry in a high throughput manner overcoming barriers such as low transduction efficiency, followed by confirmation in PBMC derived T cells.

Protocol

NOTE: All cell culture work is done in a biosafety cabinet with a lab coat, gloves, and following standard aseptic techniques.

1. Generating CAR expressing Jurkats (CAR-J)

1. Purchase Jurkat cells, clone E6-1 from ATCC. Thaw 1 x 10⁶ cells in a T-75 flask and culture them in T-75 flasks. Maintain them in suspension at 0.6 x 10⁶ cells per mL using Roswell Park Memorial Institute (RPMI) media supplemented with 10% FBS in an incubator at 37 °C with 5% CO₂.
2. Plate 1 x 10⁵ Jurkat cells per well of a tissue culture treated 24 well plate in 500 µL of RPMI growth media containing 4 µg/mL polybrene which enhances lentiviral efficiency. Count cells using a laser-based fluorescent detection bench top cell analyzer. The number of wells depends on the number of constructs to be evaluated. This example will be using 4 constructs and an un-transduced control. The CAR construct design is shown in Table 1.
3. Add 10 µL of lentivirus/well of each CAR construct. CAR construct design and lentivirus production was done as described previously¹².
4. Next day add 1 mL of growth RPMI media to each well and continue culturing in the incubator at 37 °C with 5% CO₂.
5. Collect cells 2 days later (total 72 h after Jurkat transduction) and count them.
6. Take 1 x 10⁴ cells for running flow to confirm CAR expression on the Jurkat cells. Briefly, wash the cells 2x with FACS staining solution (FSS) before labelling the CAR-J with antibodies targeting Flag tag used to detect CAR positivity for 30 min in the dark at 4 °C. Wash 2x again with FSS and run cells through a flow cytometer as previously described¹². Use antibody concentration as recommended by the manufacturer.
7. Jurkat cells are easily transduced and almost always show >90% CAR expression. Produce CAR-J long in advance of the co-culture cytotoxicity assay and freeze down for later use.

2. Plating CFSE labelled tumor cells

NOTE: MDA-MB-231 (from ATCC, HTB-26) cells were a gift from a collaborator, and EGFR KO MDA-MB-231 were created as previously described¹².

1. Thaw 1 x 10⁶ cells in a T-75 flask and culture them in T-75 flasks. Maintain MDA-MB-231 tumor cells and CRISPR EGFR KO MDA-MB-231 tumor cells in 14 mL of Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in an incubator at 37 °C with 5% CO₂ and split when approximately 70% confluent.
2. Observe adherent tumor cells under regular brightfield microscope to ensure nearly 70% confluence.
3. Remove growth media from the flask using a serological pipette. Add 3 mL of trypsin to the T75 flask and place in an incubator at 37 °C with 5% CO₂ for 3-5 min to detach the cells from the flask.
4. Neutralize the trypsin using equal amount (3 mL) of growth media. Collect the cell suspension in a centrifuge tube and spin down the cells at 400 x g for 4 min to pellet down the cells.
5. Discard the supernatant using a pipette and resuspend the cells in 2 mL of phosphate-buffered saline (PBS). Determine the cell concentration using a cell counter.
6. Transfer 8 x 10⁵ cells into another 15 mL tube and add PBS to make it to a volume of 1 mL.
7. Add 2 µL of carboxyfluorescein succinimidyl ester (CFSE; 5 µM stock concentration) into each tube and mix well using a 1 mL pipette.
8. Incubate the cells with CFSE for 20 min in the incubator at 37 °C with 5% CO₂. Remove the tubes from the incubator after 20 min and add 5 mL of growth media to the tube.
9. Centrifuge the tubes at 400 x g for 4 min to pellet down the CFSE labelled cells. Discard the supernatant using a pipette and add 1 mL of fresh media to resuspend the cells.
10. Revaluate the cell concentration using a cell counter. Transfer 4 x 10⁵ cells into a 25 mL reagent reservoir and add media for a total volume of 8 mL.
    1. To ensure sufficient volume to seed 5000 tumor cells/well in 100 µL of media, volume needed to be able to pipette using the multichannel pipette, and to compensate for the loss of a few cells during step 1.7, prepare 10%-20% extra cells and media volume.
11. Mix the cell suspension using a 5 mL serological pipette thoroughly. Using a 100 µL multichannel pipette, pipette 100 µL of the cell suspension into each row of the clear flat bottom black 96 well plate on the left half. A sample plating strategy can be found in Table 2.
12. Pipette EGFR KO cells similarly on the right half of the plate. Once the whole plate is plated, drag the plate back and forth and side to side on the tissue culture hood platform to ensure uniform distribution of the tumor cells in the wells.
13. Incubate the plate for 4 h in the incubator at 37 °C with 5% CO₂ for the tumor cells to attach.

3. Co-culturing CAR expressing Jurkats with CFSE labelled tumor cells

1. Using the counts of un-transduced and CAR expressing Jurkat cells, transfer 4 x 10⁵ cells of each CAR-J into a 25 mL reservoir. Add DMEM growth media for a total volume of 2 mL.
2. For E:T of 4:1, 2 x 10⁴ CAR-J were added per well in 100 µL of media using a multichannel pipette gently along the side of each well so as not to disturb the tumor cells attached.
3. Add another 100 µL of growth media using a multichannel pipette to the side of wells containing tumor cells and Jurkat cells. The tumor only groups get 200 µL of media to make it a total of 300 µL of media in all wells.
4. Drag the plate along the platform in back and forth and side to side motion to ensure uniform distribution of the Jurkat cells on the tumor cells.
5. Allow co culture in the incubator at 37 °C with 5% CO₂ for 48 h.

4. Preparation of plate for imaging

1. Make a solution of propidium iodide (PI) at 1 µg/mL in low background fluorescence media based on the number of wells and each getting 100 µL of media.
2. For 84 wells prepare 9 mL of media containing PI. Mix the media thoroughly using a pipette.
3. Make 10 mL of 20% Triton-X solution by diluting Triton-X with deionized water. After 48 h of co culture, discard the supernatant containing CAR-J by a single inversion of the plate and tapping on paper towel.
4. Now add 100 µL of above prepared media (step 4.2) containing PI into each well using a multichannel pipette gently so as not to disturb the adhered tumor cells.
5. Add 20 µL of 20% Triton-X solution to the first well of each tumor type which functions as a total dead control.
6. Leave the plate in the incubator for 20 min. Image the plate using the fluorescent imaging cytometer. Data is stored on the computer and can be analyzed at a later time.

5. Analyzing fluorescent images

1. Use one of the tumor only well of cells to set the green fluorescent channel.
2. Decrease the well mask to 98% to remove the cells on the edge of the well as the signal is not perfect on the edge.
3. Identify CFSE labelled tumor cells on the green, fluorescent CFSE channel. Modify fluorescence intensity threshold value to pick up all the cells on the well.
4. Set the minimum cell diameter to 25 µm to remove any debris detected on the CFSE channel. This varies depending on the cell type that is analyzed.
5. Enable Separate Touching Objects to identify individual cells when they are in contact with each other.
6. Select CFSE on the image display and look at graphic overlay to figure out what's being picked by the system.
7. Once CFSE labelled cells are being properly picked up, set gates to define live vs dead cell population.
8. Selecting the CFSE labelled cells, generate a histogram of counts on the y-axis vs mean PI intensity on the x-axis.
9. Based on the well where we added Triton-X (step 4.5), draw a splitter to differentiate low PI-stained vs high PI-stained cells. This well should have most of the cells in a high PI-stained region.
   NOTE: PI shows basal signal on live cells. Hence, adding Triton-X kills all the cells and they stain bright in the red fluorescent channel. This facilitates drawing gates to separate dead cells from live cells.
10. Adjust the x-axis value to be better able to view the cells. Now label the low PI-stained cells as Live cells.
11. Selecting the Live cells, set another histogram with area on the x-axis and counts on the y-axis.
12. Draw another splitter using tumor only well to capture cells and not debris. Jurkat treated wells will start accumulating debris that will be CFSE stained and need to be removed from the counts. The remaining non-debris are labelled as “Big cells".
13. Run the analysis on the entire plate and export the spreadsheet containing the numbers.
14. Plot the Big counts to identify the number of live CFSE labelled tumor cells remaining in the well after exposure to CAR-J. Determine statistics using one-way ANOVA.

Results

A range of E:T ratio between 1:8 and 8:1 for CAR-J1 was evaluated at 72 h which targeted EGFR on TNBC MDA-MB-231 cells. Jurkat cells were transduced with CAR lentivirus with polybrene to generate CAR-J cells as described in step 2. Cytotoxicity of CAR-J1 significantly increased with higher E:T ratio with no difference in killing at 1:8 ratio (Figure 1). More than 50% killing was observed at 4:1 E:T over 72 h. This E:T was used for subsequent experiments with duration reduced to 48 h for rapi...

Discussion

Here we have proposed a rapid method to efficiently evaluate the target-specific cytolytic activity induced by CAR expression in Jurkat cells. All CAR constructs have the same scFv but different hinge and transmembrane domains which have been shown to affect CAR-T cells potency¹³. Further evaluation of non-specific killing by these CAR-J was done by culturing them with antigen knock out (KO) cells. This demonstrates that the killing is tumor antigen specific and not due to basal activation by the ...

Materials

Name	Company	Catalog Number	Comments
15 mL Conical Tube (Sterile)	Midwest Scientific	#C15B	Any similar will work
50 mL Conical Tube (Sterile)	Thermo Scientific	339652	Any similar will work
Black/Clear 96 well plate	Falcon	353219	Celligo has a list of compatible plates
Celigo 4 Channel Imaging Cytomenter	Nexcelcom Bioscience	200-BFFL-5C	Any similar will work
Celigo Software	Nexcelcom Bioscience	Version 5.3.0.0	Any similar will work
Cell Culture Incubator	Thermo Scientific	HeraCell 160i	Any similar will work
Cell Culture Treated Flasks (T75, various sizes, Sterile)	TPP	90076	Any similar will work
CFSE	Tonbo	13-0850-U500	Any similar will work
Cytek Muse Cell Analyzer	Cytek	0500-3115	Any similar will work
DMEM	Gibco	11995-040	Any similar will work
FBS	Gemini bio-products	900-108	Any similar will work
Flow Cytometer	Cytek, BD, etc	Aurora, LSR II, etc	Any similar will work
FlowJo Sortware	Becton Dickinson & Company	Version 10.7.1	Any similar will work
Fluorobrite DMEM	Gibco	A18967-01	Any similar will work
GraphPad Software	GraphPad	Version 9.3.1 (471)	Any similar will work
Multichanel Pipette	Thermo Scientific	Finnpipette F2	Any similar will work
PBS	Gibco	10010-031	Any similar will work
PenStrep	Gibco	15070-063	Any similar will work
Pipette tips (Sterile, filtered, 1 mL, Various sizes)	Pr1ma	PR-1250RK-FL, etc	Any similar will work
Pipettors	Thermo Scientific	Finnpipette F2	Any similar will work
Propidium Iodide	Invitrogen	P1304MP	Any similar will work
RPMI	Corning	10-041-cv	Any similar will work
Serological Pipette Aid	Drummond Scientific	4-000-105	Any similar will work
Serological Pipettes (Sterile, various sizes)	Pr1ma	PR-SERO-25, etc	Any similar will work
Sodium Pyruvate	Corning	25-000-CI	Any similar will work
Sterile Reservoirs	Midwest Scientific	RESE-2000	Any similar will work
Table top centrifuge	Eppendorf	5810R	Any similar will work