Name: Author Spotlight: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory
Uploaded: 2023-10-27T12:30:00
Description: Watch this Scientific Journal Video about High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory at JoVE.com

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.

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)
<ol>
	<li>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 &#176;C w...

Evaluating Quantitative Tumor Cell Killing by CAR-Expressing Jurkat Cells

<ol>
	<li>Mitra, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+bench+to+bedside:+the+history+and+progress+of+CAR+T+cell+therapy.">From bench to bedside: the history and progress of CAR T cell therapy.</a> Front Immunol. 14, 1188049 (2023).</li><li>Labanieh, L., Mackall, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR+immune+cells:+design+principles,+resistance+and+the+next+generation.">CAR immune cells: design principles, resistance and the next generation.</a> Nature. 614, 635-648 (2023).</li><li>Sterner, R. C., Sterner, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR-T+cell+therapy:+current+limitations+and+potential+strategies.">CAR-T cell therapy: current limitations and potential strategies.</a> Blood Cancer J. 11 (4), 69 (2021).</li><li>Lisby, A. N., Carlson, R. D., Baybutt, T. R., Weindorfer, M., Snook, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cell Biology. 167, 81-98 (2022).</li><li>Kiesgen, S., Messinger, J. C., Chintala, N. K., Tano, Z., Adusumilli, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+assays+to+measure+CAR+T-cell-mediated+cytotoxicity.">Comparative analysis of assays to measure CAR T-cell-mediated cytotoxicity.</a> Nat Protoc. 16 (3), 1331-1342 (2021).</li><li>Schneider, U., Schwenk, H. U., Bornkamm, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+EBV-genome+negative+&amp;#34;null&amp;#34;+and+&amp;#34;T&amp;#34;+cell+lines+derived+from+children+with+acute+lymphoblastic+leukemia+and+leukemic+transformed+non-Hodgkin+lymphoma.">Characterization of EBV-genome negative &amp;#34;null&amp;#34; and &amp;#34;T&amp;#34; cell lines derived from children with acute lymphoblastic leukemia and leukemic transformed non-Hodgkin lymphoma.</a> Int J Cancer. 19 (5), 621-626 (1977).</li><li>Abraham, R. T., Weiss, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jurkat+T+cells+and+development+of+the+T-cell+receptor+signalling+paradigm.">Jurkat T cells and development of the T-cell receptor signalling paradigm.</a> Nat Rev Immunol. 4 (4), 301-308 (2004).</li><li>Bloemberg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+characterizing+novel+chimeric+antigen+receptors+in+Jurkat+cells.">A high-throughput method for characterizing novel chimeric antigen receptors in Jurkat cells.</a> Mol Ther Methods Clin Dev. 16, 238-254 (2020).</li><li>Lipowska-Bhalla, G., Gilham, D. E., Hawkins, R. E., Rothwell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+tumor+antigen-specific+single-chain+variable+fragments+using+a+chimeric+antigen+receptor+bicistronic+retroviral+vector+in+a+Mammalian+screening+protocol.">Isolation of tumor antigen-specific single-chain variable fragments using a chimeric antigen receptor bicistronic retroviral vector in a Mammalian screening protocol.</a> Hum Gene Ther Methods. 24 (6), 381-391 (2013).</li><li>Alonso-Camino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CARbodies:+Human+antibodies+against+cell+surface+tumor+antigens+selected+from+repertoires+displayed+on+T+cell+chimeric+antigen+receptors.">CARbodies: Human antibodies against cell surface tumor antigens selected from repertoires displayed on T cell chimeric antigen receptors.</a> Mol Ther Nucleic Acids. 2 (5), e93 (2013).</li><li>Jahan, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+Jurkat+reporter+T+cell+line+for+evaluating+the+functionality+of+novel+chimeric+antigen+receptors.">Using the Jurkat reporter T cell line for evaluating the functionality of novel chimeric antigen receptors.</a> Front Mol Med. 3, 1070384 (2023).</li><li>Subham, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+as+a+potent+CAR+T+target+in+triple+negative+breast+cancer+brain+metastases.">EGFR as a potent CAR T target in triple negative breast cancer brain metastases.</a> Breast Cancer Res Treat. 197 (1), 57-69 (2023).</li><li>Guedan, S., Calderon, H., Posey, A. D., Maus, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+Design+of+Chimeric+Antigen+Receptors.">Engineering and Design of Chimeric Antigen Receptors.</a> Mol Ther Methods Clin Dev. 12, 145-156 (2019).</li><li>Zaritskaya, L., Shurin, M. R., Sayers, T. J., Malyguine, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+flow+cytometric+assays+for+monitoring+cell-mediated+cytotoxicity.">New flow cytometric assays for monitoring cell-mediated cytotoxicity.</a> Expert Rev Vaccines. 9 (6), 601-616 (2010).</li><li>Shan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+PTEN+in+Jurkat+T+cells+causes+constitutive+localization+of+Itk+to+the+plasma+membrane+and+hyperresponsiveness+to+CD3+stimulation.">Deficiency of PTEN in Jurkat T cells causes constitutive localization of Itk to the plasma membrane and hyperresponsiveness to CD3 stimulation.</a> Mol Cell Biol. 20 (18), 6945-6957 (2000).</li><li>Wu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+signaling+roles+of+CD3&#949;+and+its+application+in+CAR-T+cell+therapy.">Multiple signaling roles of CD3&#949; and its application in CAR-T cell therapy.</a> Cell. 182 (4), 855-871 (2020).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma-targeted+CD4++CAR+T+cells+mediate+superior+antitumor+activity.">Glioblastoma-targeted CD4+ CAR T cells mediate superior antitumor activity.</a> JCI Insight. 3 (10), e99048 (2018).</li><li>Haggerty, T. J., Dunn, I. S., Rose, L. B., Newton, E. E., Kurnick, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+screening+assay+to+identify+agents+that+enhance+T-cell+recognition+of+human+melanomas.">A screening assay to identify agents that enhance T-cell recognition of human melanomas.</a> Assay Drug Dev Technol. 10 (2), 187-201 (2012).</li><li>Spencer, V. A., Kumar, S., Paszkiet, B., Fein, J., Zmuda, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+media+for+fluorescence+imaging:+Striking+the+right+balance+between+signal+strength+and+long-term+cell+health.">Cell culture media for fluorescence imaging: Striking the right balance between signal strength and long-term cell health.</a> Genetic Engineer Biotech News. 34 (10), 16-18 (2014).</li><li>. Immune Cell Killing Assays for Live-Cell Analysis Available from: <a target="_blank" href="https://www.sartorius.com/en/applications/life-science-research/cell-analysis/live-cell-assays/cell-function/immune-cell-killing">https://www.sartorius.com/en/applications/life-science-research/cell-analysis/live-cell-assays/cell-function/immune-cell-killing</a> (2023)</li><li>Kessel, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+3D+tumor+spheroid+screening+method+for+cancer+drug+discovery+using+celigo+image+cytometry.">High-throughput 3D tumor spheroid screening method for cancer drug discovery using celigo image cytometry.</a> SLAS Technol. 22 (4), 454-465 (2017).</li></ol>

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-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL Conical Tube (Sterile)</td>
			<td>Midwest Scientific</td>
			<td>#C15B</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>50 mL Conical Tube (Sterile)</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Black/Clear 96 well plate</td>
			<td>Falcon</td>
			<td>353219</td>
			<td>Celligo has a list of compatible plates</td>
		</tr>
		<tr>
			<td>Celigo 4 Channel Imaging Cytomenter</td>
			<td>Nexcelcom Bioscience</td>
			<td>200-BFFL-5C</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Celigo Software</td>
			<td>Nexcelcom Bioscience</td>
			<td>Version 5.3.0.0</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Cell Culture Incubator</td>
			<td>Thermo Scientific</td>
			<td>HeraCell 160i</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Cell Culture Treated Flasks (T75, various sizes, Sterile)</td>
			<td>TPP</td>
			<td>90076</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>CFSE</td>
			<td>Tonbo</td>
			<td>13-0850-U500</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Cytek Muse Cell Analyzer</td>
			<td>Cytek</td>
			<td>0500-3115</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-040</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini bio-products</td>
			<td>900-108</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>Cytek, BD, etc</td>
			<td>Aurora, LSR II, etc</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>FlowJo Sortware</td>
			<td>Becton Dickinson &#38; Company&#160;</td>
			<td>Version 10.7.1</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Fluorobrite DMEM</td>
			<td>Gibco</td>
			<td>A18967-01</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>GraphPad Software</td>
			<td>GraphPad</td>
			<td>Version 9.3.1 (471)</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Multichanel Pipette</td>
			<td>Thermo Scientific</td>
			<td>Finnpipette F2</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010-031</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>PenStrep</td>
			<td>Gibco</td>
			<td>15070-063</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Pipette tips (Sterile, filtered, 1 mL, Various sizes)</td>
			<td>Pr1ma</td>
			<td>PR-1250RK-FL, etc</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Pipettors&#160;</td>
			<td>Thermo Scientific</td>
			<td>Finnpipette F2</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>P1304MP</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Corning</td>
			<td>10-041-cv</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Serological Pipette Aid</td>
			<td>Drummond Scientific</td>
			<td>4-000-105</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Serological Pipettes (Sterile, various sizes)</td>
			<td>Pr1ma</td>
			<td>PR-SERO-25, etc</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Corning</td>
			<td>25-000-CI</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Sterile Reservoirs</td>
			<td>Midwest Scientific</td>
			<td>RESE-2000</td>
			<td>Any similar will work</td>
		</tr>
		<tr>
			<td>Table top centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Any similar will work</td>
		</tr>
	</tbody>
</table>

rapid in vitro cytotoxicity evaluation of jurkat expressing chimeric antigen receptor using fluorescent imaging

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.

Author Spotlight: High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory 

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

CAR T-cells are being constantly improved to achieve better responses in human clinical trials. We are identifying ways to evaluate this in our lab using an unbiased high throughput method, such as using fluorescent imaging to screen combinations that work best. There are several combinations of constructs that can be designed for any single chain variable fragment by modifying the geometry of the extracellular and hinge domains.We have designed this protocol to identify the combinations with the best efficacy in eliminating target cells using Jurkat cells. This is a rapid, high throughput protocol to screen CAR constructs based on its target cell cytotoxicity using Jurkat cells, which are then validated with conventional T-cells. A dozen different CAR constructs can be tested in a 96-well plate simultaneously using this technique.Here in Akhavan Lab, we quantify in absolute numbers the direct cyclosis of target cells by the CAR expressing Jurkat cells in a high throughput manner to screen multiple CAR constructs. Our laboratory's primary aim is to improve the clinical translation of CAR T-cell therapy against high grade brain tumors. We leverage high throughput drug screens as one modality to improve CAR T-cell cytotoxicity, as well as targeting the tumor microenvironment.

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...

Watch this Scientific Journal Video about High-Throughput Screening of CAR T-Cell Constructs for Enhanced Cytotoxicity and Immunologic Memory at JoVE.com

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.

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 ...

rapid-vitro-cytotoxicity-evaluation-jurkat-expressing-chimeric

Research

JoVE Journal

Cancer Research

Plating CFSE-Labelled Tumor Cells and Co-Culturing CAR-Expressing Jurkats for In Vitro Cytotoxicity Evaluation

To begin using a serological pipette, remove the growth media from the T75 flask containing 70%confluent MDA-MB-231 culture. Add three milliliters of trypsin to the flask and incubate at 37 degrees with 5%carbon dioxide for three to five minutes to detach the cells from the flask. Then to neutralize the trypsin, add three milliliters of DMEM into the flask.Collect the cell suspension in a centrifuge tube and spin at 400 G for four minutes. Discard the supernatant using a pipette and resuspend the pellet in two milliliters of PBS. Using a cell counter, determine the cell concentration.Transfer eight times 10 to the fifth cells into a 15 milliliter tube and add PBS to make the volume to one milliliter. Then add two microliters of CFSE into the tube and mix the cell suspension thoroughly using a one milliliter pipette. Place the tube at 37 degrees Celsius and 5%carbon dioxide incubator for 20 minutes.Next, add five milliliters of DMEM to the tube and centrifuge to pellet down the CFSE labeled cells. Discard the supernatant and resuspend the pellet in one milliliter of DMEM. After reevaluating the cell concentration, transfer four times 10 to the fifth cells into a 25 milliliter reagent reservoir.Add DMEM to make the volume eight milliliters and mix the cell suspension with a five milliliter serological pipette. Using a multichannel pipette, transfer 100 microliters of cell suspension into each row on the left half of a black 96-well plate. Similarly, add EGFR knockout cell suspension on the right half of the plate.To ensure uniform cell distribution, move the plate back and forth and side to side on the platform. Incubate the plate for four hours at 37 degrees Celsius and 5%carbon dioxide for the tumor cells to attach. Based on counts of untransduced and CAR-expressing Jurkat cells, transfer four times 10 to the fifth CAR-J cells into a 25 milliliter reservoir.Add DMEM to the reservoir to a final volume of two milliliters. For an effector to tumor ratio of 4:1, add two times 10 to the fourth CAR-J cells per well in 100 microliters of medium along the side of the well without disturbing the attached tumor cells. Then add 100 microliters of DMEM to the side of the wells containing tumor and Jurkat cells to get 300 microliters of volume per well.Move the plate as demonstrated to ensure uniform distribution of the Jurkat cells on the tumor cells. Allow the co-culture to establish at 37 degrees Celsius with 5%carbon dioxide for 48 hours.

Preparation of Plate for Fluorescent Imaging and Cell Viability Assessment in CAR-Jurkat Co-Culture with CFSE-Labeled Tumor Cells

After co-culturing CAR expressing jurkats with CFSE-labeled tumor cells, gently invert the plate on a paper towel and tap to discard the supernatants containing CAR-J. Using a multichannel pipette, add 100 microliters of FluoroBrite DMEM media containing propidium iodine, or PI, into the wells without disturbing the adhered tumor cells. Then add 20 microliters of 20%Triton X to the first well of each tumor type, serving as a total dead control.Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 20 minutes before imaging. After fluorescent imaging, use one of the tumor-only wells of cells to calibrate the green fluorescent channel. To exclude cells at the edge of the wells decrease the well mask to 98%Identify CFSE-labeled tumor cells on the green fluorescent channel and adjust the fluorescence intensity threshold value to capture all the cells in the well.Set the minimum cell diameter to 25 micrometers to exclude debris detected in the CFSE channel. Then enable separate touching objects to identify individual cells. Next set gates to define live versus dead cell populations.Select the CFSE-labeled cells and generate a histogram of counts on the y-axis versus mean PI intensity on the x-axis. Adjust the x-axis value to better visualize the cells and draw a splitter to differentiate between low PI stained cells and high PI stained cells. Label the low PI stained cells as live cells.Next, set another histogram on live cells with area on the x-axis and counts on the y-axis. Label the non-debris cells as big live cells. Then draw another splitter using the tumor only well to capture cells and filter out debris.After running the analysis on the entire plate, export the spreadsheet containing the numbers. Plot the counts of big cells to determine the number of live CFSE-labeled tumor cells remaining in the well after exposure to CAR-J. Finally perform statistical analysis using one-way ANOVA.The cytotoxicity of CAR-J1 increased with a higher effector to tumor ratio and more than 50%killing was observed at an effector to tumor ratio of four to one. EGFR-targeting CAR constructs with modified hinge domains showed antigen-specific killing against EGFR expressing MDA-MB-231 cells, and no significant killing was observed against EGFR knockout cells. CAR constructs were also expressed on PBMC derived CD3 T cells.However, there was no expression of CD8 hinge-containing EGFR-CAR. The IgG short showed the least cytotoxic potency against MDA-MB-231 cells compared to the IgG long and IgG medium.