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JoVE Journal Cancer Research

Cancer Research

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

Published: October 27, 2023 doi: 10.3791/65560
Automatic Translation
*1,2,3, *1, 4, 2, 1, 4,5,6, 1,2,3
1Department of Radiation Oncology, University of Kansas Cancer Center, 2Department of Cancer Biology, University of Kansas Cancer Center, 3BioEngineering Program, University of Kansas, 4Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, 5University of Kansas Cancer Center, 6Kansas Institute for Precision Medicine, University of Kansas Medical Center
* These authors contributed equally

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 assay4,5. 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 studies8,9,10,11. 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.

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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.

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Representative 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 rapid cytotoxicity evaluation of multiple CAR constructs. EGFR targeting CAR constructs with the hinge domain modified were designed (Table 1). The 4 different hinges used are IgG long, IgG medium, IgG short and CD8a as described here13. These 3 constructs were expressed on Jurkat cells and CAR positivity determined by evaluating the percent of Flag tagged cells by flow cytometry (Figure 2A-D) as described here12. Un-transduced Jurkat cells were used as control group to determine CAR expression and the gating strategy is shown in (Figure 2E). The cytotoxicity of these 4 CAR constructs expressed on Jurkat cells was evaluated against EGFR expressing MDA-MB-231 cells and CRISPR EGFR KO MDA-MB-231 cells. Antigen specific killing was observed by all the constructs (Figure 3A) whereas there was no significant killing observed against EGFR KO cells (Figure 3B) suggesting that killing was specifically mediated through the scFv only. There was no killing by the un-transduced Jurkat cells. Representative images of tumor killing are also shown where the overlap of CFSE labeled tumor cells with PI-stained dead nucleus make them appear yellow (Figure 4). Data is representative of 3 independent experiments with 6 technical replicates per group.

This was further confirmed by expressing these CAR constructs on PBMC derived CD3 T cells. There is low expression of CAR on CD3 T cells which are then enriched by sterile flow sorting (Figure 5). However, there was no expression of CD8 hinge containing EGFR CAR. Hence the other 3 CAR constructs containing IgG long, IgG medium and IgG short were evaluated for their cytotoxic potential against MDA-MB-231 cells. There is a similar trend of killing with IgG short having the least cytotoxic potency compared to the other 2 constructs (Figure 6). To identify the best construct among IgG long and IgG medium CAR-T cells, their activation with and without exposure to TNBC tumor cells was evaluated by intracellular staining of cytokines as described previously12. MDA-MB-436 has low levels of EGFR and MDA-MB-468 has the highest EGFR protein expression based on a western blot with a panel of 13 TNBC cell lines12. IgG long CAR-T cells had the least basal activation levels (TNFa, 4-1BB) and low release of cytotoxic granules (perforin and granzyme B)14 without any exposure to tumor cells (Figure 7). Upon exposure to high EGFR expressing MDA-MB-468 cells, IgG long CAR-T cells had the highest activation based on TNFa and 4-1BB.

Figure 1
Figure 1: EGFR CAR-J1 cytotoxic evaluation along a range of E:T. CFSE labelled MDA-MB-231 tumor cells were plated with un-transduced Jurkat cells or EGFR targeting CAR-J1 at E:T ratio of 1:8, 1:4, 1:2, 1:1, 2:1, 4:1 and 8:1 showing increasing cytotoxicity with higher effector cells. Data are represented as mean ± SD and statistical significance was evaluated using Student's t-test. 1:8 E:T was non-significant, 1:4 E:T had ***p<0.001, and rest ****p<0.0001. Please click here to view a larger version of this figure.

Figure 2
Figure 2: EGFR CAR Jurkat production for in vitro cytotoxic evaluation. (A-D) Nearly 90%-100% CAR expression of 4 different constructs CAR IgG long, CAR IgG medium, CAR IgG short, CAR CD8a. (E) Gating strategy for CAR positivity determination: Debris excluded in SSC-A vs FSC-A plot; Single Cells selected in FSC-H vs FSC-A plot; and Live cells selected by viability dye DAPI negative gating. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Cytotoxicity evaluation in co-culture of CAR-J and CFSE labelled tumor cells. (A) CFSE labelled MDA MB 231 cells were plated with CAR-J at 4:1 E:T ratio for 48 h showing varying efficacy in tumor cell killing. (B) CFSE labelled CRISPR EGFR KO MDA MB 231 cells did not experience any killing by any of the CAR-J cells. Data are represented as mean ± SD and statistical significance was evaluated using one-way ANOVA. **p<0.01; ****p<0.0001; ns: not significant. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Visual representation of images after 48 h of co-culture. (A) Tumor only group with CFSE (green) labelled tumor cells. (B) Addition of un-transduced Jurkat cells with red cells being the dead Jurkat cells and (C) overlay of green and red indicating dead tumor cells (yellow) as shown by arrows. The number of green cells that are not yellow are quantified. Please click here to view a larger version of this figure.

Figure 5
Figure 5: EGFR CD3 CAR-T cell enrichment. (A-B) CAR expression evaluated by determining percent of Flag positive and IgG positive cells before enrichment and (C) post enrichment by sterile flow sort. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Cytotoxicity evaluation in co-culture of CD3 CAR-T and CFSE labelled tumor cells. CFSE labelled MDA MB 231 cells were plated with CAR-J at 4:1 E:T ratio for 48 h showing varying efficacy in tumor cell killing. Data are represented as mean ± SD and statistical significance was evaluated using one-way ANOVA. **p<0.01; ****p<0.0001; ns: not significant. Please click here to view a larger version of this figure.

Figure 7
Figure 7: EGFR CD3 CAR-T cell Activation. CAR-T cells were exposed to tumor cells and intracellular cytokine levels for(A) TNFa, (B) 4-1BB, (C) perforin, and (D) granzyme B were evaluated. Data are represented as mean ± SD and statistical significance was evaluated using one-way ANOVA *p<0.05; **p<0.01; **p<0.001; ***p<0.0001; ns: not significant. Please click here to view a larger version of this figure.

No. of PA EGFR806 scFv Vh-Vl Linker Hinge/Spacer TM Cytoplasmic
CAR IgG Long Whitlow (18 aa) IgG4 EQ CH2 CH3 (229 aa) CD4 4-1BB / CD3z
CAR IgG Medium Whitlow IgG4 CH3 (129 aa) CD28 4-1BB / CD3z
CAR IgG Short Whitlow IgG short (12 aa) CD4 4-1BB / CD3z
CAR CD8a Whitlow CD8 (45 aa) CD8a 4-1BB / CD3z

Table 1: CAR construct designs. Abbreviations: aa = amino acids; TM = Transmembrane.

1 2 3 4 5 6 7 8 9 10 11 12
A Triton-X MDA MB 231 Only Triton-X MDA MB 231 EGFR KO Only
B MB231 + Mock 4:1 MB231 EGFR KO + Mock 4:1
C MB231 + EGFR IgG Long 4:1 MB231 EGFR KO + EGFR IgG Long 4:1
D MB231 + EGFR IgG Medium 4:1 MB231 EGFR KO + EGFR IgG Medium 4:1
E MB231 + EGFR IgG Short 4:1 MB231 EGFR KO + EGFR IgG Short 4:1
F MB231 + EGFR CD8a 4:1 MB231 EGFR KO + EGFR CD8a 4:1
G
H

Table 2: Sample plating strategy.

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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 CAR-J. Cytotoxic potential of a range of E:T ratios can be easily determined identifying the least number of effector cells required for eliminating tumor cells at specific time points. Multiple cell lines from same or different cancer type and normal cells can be also used to evaluate the specificity of these constructs. The most potent candidates identified by CAR-J platform can then be further characterized by expressing CAR on PBMC derived CD3 T cells and performing similar killing assays and evaluating exhaustion and activation upon exposure to antigen positive and negative tumor cells.

It must be noted that Jurkat cells are CD4+ cells with an altered T cell receptor (TCR) signaling pathway15. Hence, intracellular signaling domain optimization of CAR constructs should ideally be done with CD3 T cells16 and may not work best with Jurkat cells. Interestingly, CD4 T cells showcase cytotoxicity as demonstrated in a previous publication where EGFR targeting CD4 CAR-T cells with IgG long hinge are able to completely eradicate TNBC tumors in an intracranial tumor model12. Additionally, CD4 CAR T cells show long term potency and better killing as compared to mixture of CD4 and CD8 T cells or CD8 T cells alone in GBM tumor model making them clinically important T cells subset for effective CAR-T therapy17.

Jurkat cells produce IL2 and upregulate CD69 upon activation, though they do not secrete all the cytokines of primary T cell activation8,18. However, evaluating IL2 and CD69 levels informs regarding the Jurkat cells getting activated and not about the direct cytolysis of target cells which are being quantified in absolute tumor cell numbers by this approach. However, evaluation of cytotoxicity and activation is important to fully understand the effects of novel CAR molecules.

The duration of co-culture in this experiment can be modified based on the constructs being used. CFSE labeling on tumor cells was found to be detectable until 4 days after plating. Hence, to attain a high signal to noise ratio, experiments were performed within 72 h. Since CFSE intensity decreases on division of tumor cells, the amount of CFSE used and the duration may need to change as per the tumor cells used. Since this is a high throughput experiment to analyze multiple constructs at a time, the seeding density of target cells in a 96 well plate also needs to be optimized. Target cells must be maintained so that they do not grow overconfluent or have competitive growth restriction by the end of the assay without the need to change media to minimize any intervention. Larger well plates may be used to accommodate more cells and increase the longevity of the experiment. However, the images need to be stitched to get a full picture of the well and each well has to be worked on individually, which may not make it quick and high throughput.

Another approach of long-term fluorescent labeling tumor cells is by lentiviral transduction with green fluorescent protein (GFP) or red fluorescent protein (RFP). Proper clonal selection must be done to select highly enriched (nearly 100%) brightest colonies for the best signal to noise ratio. The viability dye needs to be appropriately selected based on the fluorescent detectors in the imaging instrument.

Background fluorescence depends on the media being used and hence must be checked to avoid loss of signal while acquiring images. Regular media contain components that emit significant fluorescence when excited. It is recommend to use a low background media19. PBS may be used but it may affect the cells during image acquiring as it takes roughly 20 min to acquire images depending on the settings set for capturing images.

One of the limitations of this assay is not to be able to take images at multiple time points of the same 96 well plate after adding a viability dye. The effect of addition of PI over long time was not evaluated for this publication. However, it would be ideal to be able to detect live and dead/dying target cells over a period using the same plate. It may be difficult to segregate individual cells if cells become over confluent. The area of cell monolayer and fluorescence intensity may be used to compare percent of cells living/dead as described20.

Alternate methods used to detect cytolysis by CAR-T cells do not directly measure the absolute number of dead and live cells, rather evaluate potency by indirect methods which are described in detail elsewhere4,5. So, this method gives an absolute account of effector cells and may be used in co-culture of 3 cell types as well depending on the capacity of the imaging instrument. There are very few cells required (5000 per well) which may change along with the freedom to choose a time point which are huge advantages of using this assay. Target cells in suspension and in 3-dimensional spheroid culture can also be used in culture with effector cells and cytolysis determined as described elsewhere21. Furthermore, this platform can be used to screen large libraries of small molecules and compounds to delineate the most effective molecules and multiple dosing can be done at multiple time points.

Besides the speed and the flexibility of the system, the final benefit is that of cost. The fluorescent imaging machine, plates, and reagents are all common and affordable to purchase, especially when compared to devices that are more sophisticated and may even use precious metals in their single use plates.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

MDA-MB-231 were a kind gift from Dr. Shane Stecklein. The authors acknowledge funding from the University of Kansas Cancer Center to conduct this research.

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

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References

  1. Mitra, A. From bench to bedside: the history and progress of CAR T cell therapy. Front Immunol. 14, 1188049 (2023).
  2. Labanieh, L., Mackall, C. L. CAR immune cells: design principles, resistance and the next generation. Nature. 614, 635-648 (2023).
  3. Sterner, R. C., Sterner, R. M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11 (4), 69 (2021).
  4. Lisby, A. N., Carlson, R. D., Baybutt, T. R., Weindorfer, M., Snook, A. E. Methods in Cell Biology. 167, Academic Press. 81-98 (2022).
  5. Kiesgen, S., Messinger, J. C., Chintala, N. K., Tano, Z., Adusumilli, P. S. Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity. Nat Protoc. 16 (3), 1331-1342 (2021).
  6. Schneider, U., Schwenk, H. U., Bornkamm, G. Characterization of EBV-genome negative "null" and "T" cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma. Int J Cancer. 19 (5), 621-626 (1977).
  7. Abraham, R. T., Weiss, A. Jurkat T cells and development of the T-cell receptor signalling paradigm. Nat Rev Immunol. 4 (4), 301-308 (2004).
  8. Bloemberg, D., et al. A high-throughput method for characterizing novel chimeric antigen receptors in Jurkat cells. Mol Ther Methods Clin Dev. 16, 238-254 (2020).
  9. Lipowska-Bhalla, G., Gilham, D. E., Hawkins, R. E., Rothwell, D. G. Isolation of tumor antigen-specific single-chain variable fragments using a chimeric antigen receptor bicistronic retroviral vector in a Mammalian screening protocol. Hum Gene Ther Methods. 24 (6), 381-391 (2013).
  10. Alonso-Camino, V., et al. CARbodies: Human antibodies against cell surface tumor antigens selected from repertoires displayed on T cell chimeric antigen receptors. Mol Ther Nucleic Acids. 2 (5), e93 (2013).
  11. Jahan, F., et al. Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors. Front Mol Med. 3, 1070384 (2023).
  12. Subham, S., et al. EGFR as a potent CAR T target in triple negative breast cancer brain metastases. Breast Cancer Res Treat. 197 (1), 57-69 (2023).
  13. Guedan, S., Calderon, H., Posey, A. D. Jr, Maus, M. V. Engineering and Design of Chimeric Antigen Receptors. Mol Ther Methods Clin Dev. 12, 145-156 (2019).
  14. Zaritskaya, L., Shurin, M. R., Sayers, T. J., Malyguine, A. M. New flow cytometric assays for monitoring cell-mediated cytotoxicity. Expert Rev Vaccines. 9 (6), 601-616 (2010).
  15. Shan, X., et al. Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation. Mol Cell Biol. 20 (18), 6945-6957 (2000).
  16. Wu, W., et al. Multiple signaling roles of CD3ε and its application in CAR-T cell therapy. Cell. 182 (4), 855-871 (2020).
  17. Wang, D. Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity. JCI Insight. 3 (10), e99048 (2018).
  18. Haggerty, T. J., Dunn, I. S., Rose, L. B., Newton, E. E., Kurnick, J. T. A screening assay to identify agents that enhance T-cell recognition of human melanomas. Assay Drug Dev Technol. 10 (2), 187-201 (2012).
  19. Spencer, V. A., Kumar, S., Paszkiet, B., Fein, J., Zmuda, J. F. Cell culture media for fluorescence imaging: Striking the right balance between signal strength and long-term cell health. Genetic Engineer Biotech News. 34 (10), 16-18 (2014).
  20. Immune Cell Killing Assays for Live-Cell Analysis. , https://www.sartorius.com/en/applications/life-science-research/cell-analysis/live-cell-assays/cell-function/immune-cell-killing (2023).
  21. Kessel, S., et al. High-throughput 3D tumor spheroid screening method for cancer drug discovery using celigo image cytometry. SLAS Technol. 22 (4), 454-465 (2017).

Tags

Rapid In Vitro Cytotoxicity Evaluation Jurkat Chimeric Antigen Receptor Fluorescent Imaging CAR T Cells Targeting Domain Single Chain Variable Fragment Intra-chain Linker Hinge Domain Transmembrane Domain Costimulatory Domain CAR Construct CAR-mediated Killing CAR-T Cells Time-consuming Process Expensive Process Testing Constructs Time And Material Investment Platform Hinge-optimized CAR Constructs Jurkat Cells Lentivirus Uptake CAR Transduction Fluorescent Imager Cytolysis PBMC-derived T Cells
Rapid <em>In Vitro</em> Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging
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Subham, S., Jeppson, J. D., Gibbs,… More

Subham, S., Jeppson, J. D., Gibbs, B. K., Babai, J., Alker, R., Godwin, A. K., Akhavan, D. Rapid In Vitro Cytotoxicity Evaluation of Jurkat Expressing Chimeric Antigen Receptor using Fluorescent Imaging. J. Vis. Exp. (200), e65560, doi:10.3791/65560 (2023).

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