an in vitro assay for studying the cytotoxicity of pre-activated cd8+ t cells against cancer cells

An In Vitro Assay for Studying the Cytotoxicity of Pre-Activated CD8+ T Cells against Cancer Cells

To set up a pre-activated CD8-positive T cell target cell co-culture, first, add 30 microliters of 1 to 100 diluted growth-factor reduced soluble basement membrane matrix to the appropriate wells of a 96-well flat-bottom plate suitable for microscopy. Shake the plate to spread the matrix evenly, and place the plate in the cell culture incubator for at least one hour. While the plate is equilibrating, prepare the target cells. Aspirate the medium, wash with PBS, and add 1 milliliter of 0.05% trypsin-EDTA at room temperature for 1 minute.
Add 9 milliliters of DMEM supplemented with fetal bovine serum by gentle pipetting, and transfer the dissociated cells into tubes. After centrifuging the cells, resuspend the pellet in 500 microliters of EDMEM. Filter the resuspended cells through a cell strainer, and count the live cells. Then, adjust the density to 4 times 10 to the fourth target cells per milliliter in fresh EDMEM on ice.
Next, pipette the pre-activated CD8-positive T cells a few times to resuspend the cells into a single-cell suspension, and transfer the floating T cells into one 1.5-milliliter tube per condition. Collect any remaining cells with 200 microliters of PBS per well, transfer the washes into the appropriate 1.5-milliliter tubes, and centrifuge. Aspirate the medium, and add 1 milliliter of EDMEM to each tube for centrifugation.
After centrifugation, resuspend the pellets in 100 microliters of fresh EDMEM per tube. After counting, adjust the cells in each tube to a density of 1.6 times 10 to the fifth pre-activated CD8-positive T cells per milliliter of medium, and place the cells on ice. When the cells are ready, aspirate the basement membrane matrix from each well of the 96-well microscopy plate, and add 50 microliters of target cells to individual wells within the inner 60 wells of the plate with mixing.
Add 25 microliters of EDMEM supplemented with 4 times 10 to the third units per milliliter of IL-2, and 10 micromolar fluorogenic caspase-3 substrate to the appropriate wells. Then, add 25 microliters of pre-activated CD8-positive T cells to the appropriate wells, and finally, add EDMEM to the appropriate wells to make a total volume of up to 100 microliters. Add 200 microliters of PBS or sterile water into all of the empty wells, shake the plate, and leave the plate on a flat surface for 10 minutes.
To image the cells, set the microscope to acquire images in phase contrast, as well as fluorescent channels suitable for the nuclear-restricted fluorescent protein, and the fluorogenic activated caspase-3 substrate fluorophores used in the experiment. Then, capture images of each experimental well in phase contrast, and the two fluorescent channels every one to three hours for at least 72 hours.

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1. Preparation of target cells that express red fluorescent protein in their nuclei
<ol>
	<li>Obtain a target mouse cancer cell line from an appropriate source. 
	NOTE: In this protocol, a highly metastatic derivative of E0771 mouse mammary tumor cells (E0771-LG) is used. Parental E0771 cells originate from C57BL/6 mice.</li>
	<li>Thaw and maintain a vial of E0771-LG cells with Dulbecco&#39;s Modified Eagles Medium (DMEM) including 10% (v/v) fetal bovine serum (FBS) in a cell culture incubator at 37 &#176;C, 95% humidity, and 5% CO2. 
	NOTE: It should be confirmed that cells are negative for mycoplasma. To this end, culture E0771 cells (or cells to be tested) for 2-3 days as described above (in the absence of antibiotics and antimycotics), and collect 500 &#956;L of culture medium. Centrifuge the medium at 12,419 x g for 60 s to eliminate cell debris and transfer the supernatant into new tubes. Determine mycoplasma contamination using a commercially available mycoplasma test kit (refer to Table of Materials) and/or PCR following the manufacturer&#39;s instructions.</li>
	<li>Seed 5 x 103 E0771-LG cells per well into a 12-well plate, and culture the cells with 10% (v/v) FBS-DMEM overnight in an incubator at 37 &#176;C, 95% humidity, and 5% CO2. 
	NOTE: If the proliferation rate of target cells is low (population doubling time greater than 36 h), the number of cells can be increased to 1 x 104.</li>
	<li>Replace the medium with 1 mL of 10% (v/v) FBS-DMEM including 10 &#956;g/mL polybrene and add 25 &#956;L of lentiviral particles (1 x 106 TU/mL) encoding a nuclear restricted red fluorescent protein (mKate2, refer to Table of Materials).</li>
	<li>Culture the cells for 24 h in an incubator at 37 &#176;C, 95% humidity, and 5% CO2.</li>
	<li>Replace the medium with 10% (v/v) FBS-DMEM and culture the cells for 24-48 h in an incubator at 37 &#176;C, 95% humidity, and 5% CO2.</li>
	<li>Replace the medium with 10% (v/v) FBS-DMEM including 1 &#956;g/mL puromycin when cells start to express a red fluorescent protein, and culture the cells until they are 80-90% confluent. 
	NOTE: The concentration of puromycin will be different between target cell types and should be optimized using un-transfected cells.</li>
	<li>Subculture the surviving cells for 1-3 passages with 10% (v/v) FBS-DMEM including 1 &#956;g/mL puromycin, and cryopreserve stocks in a liquid nitrogen vapor phase storage system until use.</li>
</ol>
2. Isolation of suppressor cells from the tumors in mice
NOTE: In this protocol, suppressor cells (i.e., metastasis-associated macrophages (MAMs) and monocytic-myeloid-derived suppressor cells (M-MDSCs)) are isolated from the lung containing metastatic tumors established by E0771-LG cells. Conditions for tissue dissociation and cell sorting should be optimized to isolate the cells from different tissues.
<ol>
	<li>Inject 1 x 106 cancer cells (E0771-LG) into the tail vein of syngeneic (C57BL/6), female, 7-10 week old mice.</li>
	<li>After 14 days, isolate the lung containing metastatic tumors and prepare single-cell suspensions from the perfused lungs via enzymatic digestion as previously described. 
	NOTE: In this protocol, four mice are injected with cancer cells and their metastatic lungs are combined to obtain sufficient suppressor cells.</li>
	<li>Incubate the single cell suspensions with anti-mouse CD16/CD32 antibody for 30 min on ice, and stained with fluorescent antibodies to CD45, F4/80, CD11b, Ly6C, and Ly6G (refer to Table of Materials) for another 30 min.</li>
	<li>Wash the stained cells once with 1 mL of PBS containing 2% (w/v) bovine serum albumin (BSA), and re-suspend the cell pellet with 500&#8211;1000 &#956;L of PBS containing 2% (w/v) BSA.</li>
	<li>Add 3 &#956;M of DAPI, and sort M-MDSCs (DAPI&#8211;CD45+F4/80+Ly6G&#8211;CD11bhighLy6Chigh) and MAMs (DAPI&#8211;CD45+F4/80+Ly6G&#8211;CD11bhighLy6Clow) using a cell sorter (Figure 1). 
	NOTE: The threshold of Ly6C level to distinguish MAMs (Ly6Clow) and M-MDSCs (Ly6Chigh) is based on that of resident alveolar macrophages (RMAC). The purity of the sorted cells is measured via flow cytometry with an expected purity of more than 90%.</li>
	<li>Resuspend the sorted cells with 400 &#956;L of DMEM containing 20% (v/v) FBS, 1% (v/v) penicillin/streptomycin, 2 mM L-glutamine, 1% (v/v) non-essential amino acid, 1 mM sodium pyruvate, and 50 nM 2-mercaptoethanol (called enriched-DMEM, E-DMEM).</li>
	<li>Count the number of live cells using the Trypan blue exclusion method and adjust to 2 x 106 cells/mL with E-DMEM.</li>
	<li>Keep the cells on ice until use.</li>
</ol>
3. Isolation of CD8+ T cells from the spleen of mice
<ol>
	<li>Isolate the spleen from a mouse that is syngeneic to the target cancer cell line (i.e., C57BL/6 mice in this protocol) as follows:
	<ol>
		<li>Euthanize the animal by CO2 inhalation.</li>
		<li>Place the animal on a clean dissection board and wipe the skin with 70% (v/v) ethanol.</li>
		<li>Cut the abdominal skin using scissors to expose the spleen</li>
		<li>Isolate the spleen, which is located inferior to the stomach, and place it into a tube containing 5 mL of ice-cold PBS.</li>
	</ol>
	</li>
	<li>Using the inner plunger of a sterile 5 mL syringe, grind the spleen on a 100 &#956;m cell strainer set on a 50 mL tube.</li>
	<li>Pass the cells through the filter using a total of 10 mL of PBS.</li>
	<li>Centrifuge the cell suspension at 337 x g for 5 min, and aspirate the supernatant.</li>
	<li>Resuspend the cell pellet in 1 mL of PBS containing 2 mM ethylenediaminetetraacetic acid (EDTA) and 0.5% (w/v) BSA (running buffer) and filter through a 40 &#956;m cell strainer.</li>
	<li>Count the live cell number and adjust to 1 x 108 cells/mL using the running buffer. 
	NOTE: Keep a small aliquot of cells as a pre-enrichment sample for a purity check.</li>
	<li>Enrich for CD8+ T cells using a negative selection kit and a magnetic sorter (refer to Table of Materials).
	<ol>
		<li>Transfer 1 x 108 (1 mL) of the splenocyte cells to a 5 mL polystyrene round-bottom tube.</li>
		<li>Add 50 &#956;L of biotinylated antibodies, and incubate at room temperature for 10 min.</li>
		<li>Add 125 &#956;L of streptavidin-conjugated magnetic beads, and incubate at room temperature for 5 min.</li>
		<li>Add 1.325 mL of running buffer, and gently mix by pipetting.</li>
		<li>Place the tube into the magnet, and incubate at room temperature for 2.5 min.</li>
		<li>Pick up the magnet, and pour the enriched cell suspension into a new tube.</li>
	</ol>
	</li>
	<li>Resuspend the enriched cells with 200 &#956;L of E-DMEM (i.e., DMEM containing 20% (v/v) FBS, 1% (v/v) penicillin/streptomycin, 2 mM L-glutamine, 1% (v/v) non-essential amino acid, 1 mM sodium pyruvate, and 50 nM 2-mercaptoethanol).</li>
	<li>Count the number of live cells and adjust to 2 x 106 cells/mL with E-DMEM. Keep the cells at 37 &#176;C in a CO2 incubator until use. 
	NOTE: Keep a small aliquot of cells as a post-enrichment sample for a purity check.</li>
	<li>Determine the purity of CD8+ T cells by flow cytometry as follows:
	<ol>
		<li>Take 1 x 104 cells from pre-enrichment (step 3.6) or post-enrichment (step 3.9) samples and adjust the total volume of each to 100 &#956;L using a running buffer.</li>
		<li>Incubate the single cell suspensions with anti-mouse CD16/CD32 antibody for 30 min on ice, and stain with fluorescent antibodies to CD45, CD3, CD4, and CD8 (refer to Table of Materials) for another 30 min.</li>
		<li>Wash the stained cells with 500 &#956;L of PBS containing 2% (w/v) BSA, and re-suspend the cell pellet with 500&#8211;1000 &#956;L of PBS containing 2% (w/v) BSA.</li>
	</ol>
	</li>
	<li>Add 3 &#956;M of 4&#39;,6-diamidino-2-phenylindole (DAPI), and determine the percentage of CD3+CD4&#8211;CD8+ cells in the total CD45+ cell population.</li>
</ol>
4. Activation and expansion of the isolated CD8+ T cells
<ol>
	<li>Aliquot 1 x 105 cells per 50 &#956;L CD8+ T cells (prepared in step 3.9) into wells of a U-bottom 96-well plate.</li>
	<li>Add 1 x 105 cells per 50 &#956;L suppressor cells (prepared in step 2.7) or 50 &#956;L of E-DMEM into the wells.</li>
	<li>Prepare activation medium that consists of E-DMEM, 4 x 104 U/mL colony-stimulating factor 1 (CSF-1), 240 U/mL interleukin-2 (IL-2), 8 &#956;g/mL anti-mouse CD3&#949; antibody, and 16 &#956;g/mL anti-mouse CD28 antibody. 
	NOTE: CSF-1 is not required for T cell activation, but is essential for survival of suppressor cells in this protocol (i.e., MAMs and M-MDSCs). Thus, it is retained in the co-culture of T cells with target cancer cells to maintain consistency in culture conditions. Since CSF-1 is found in nano-molar concentrations in all tissues and is required for monocyte/macrophage viability in vivo, this is a physiological context for these cells.</li>
	<li>Add 50 &#956;L of activation medium (step 4.3) and 50 &#956;L of E-DMEM with or without reagents to be tested.</li>
	<li>Place the plate into an incubator at 37 &#176;C, 95% humidity, and 5% CO2, and culture the cells for 4 days.</li>
</ol>
5. Setup of co-culture of target cells with pre-activated CD8+ T cells
<ol>
	<li>Add 30 &#956;L of 1:100 diluted growth factor-reduced soluble basement membrane matrix (Table of Materials) into the wells of flat bottom 96-well plate suitable for microscopy, and incubate at 37 &#176;C in a CO2 incubator for at least 1 h.</li>
	<li>Prepare target cells (i.e., E0771-LG cells expressing nuclear-restricted red fluorescent protein).
	<ol>
		<li>Incubate the target cells with 1 mL of 0.05% trypsin/EDTA at room temperature for 1 min, and harvest the cells by gentle pipetting.</li>
		<li>Add 9 mL of DMEM including 10% (v/v) FBS, and centrifuge the cell suspension at 337 x g for 5 min.</li>
		<li>Re-suspend the cells with 500 &#956;L of E-DMEM, and count the number of live cells. 
		NOTE: Before counting the target cells, filter the cells through a 40&#956;m cell strainer to produce a single-cell suspension.</li>
		<li>Adjust the density to 2 x 104 cells/mL (= 1 x 103 cells per 50 &#956;L) by adding E-DMEM, and keep the cells on ice.</li>
	</ol>
	</li>
	<li>Prepare effector cells (i.e., pre-activated CD8+ T cells).
	<ol>
		<li>Resuspend the cells in a well of a 96-well plate (step 4.5) thoroughly by pipetting, and transfer the floating CD8+ T cells into a new 1.5 mL tube. 
		NOTE: MAMs and M-MDSCs tightly adhere to the well and are not detached by the pipetting.</li>
		<li>To collect the remaining cells, add 200 &#956;L of PBS into the well and transfer it into the tube in step 5.3.1.</li>
		<li>Wash the cells with 1 mL of E-DMEM once (centrifuge at 337 x g) for 5 min, aspirate and discard supernatant), and resuspend them with 100 &#956;L of E-DMEM.</li>
		<li>Count the number of live cells (using the trypan blue exclusion method), adjust the density to 1.6 x 105 cells/mL (= 4 x 103 cells/25 &#956;L), and keep the cells on ice.</li>
	</ol>
	</li>
	<li>Aspirate the basement membrane matrix from each well of the plate in step 5.1.</li>
	<li>Add 1 x 103 cells/50 &#956;L of target cells (step 5.2.4) into each well and mix well. 
	NOTE: To avoid edge effects due to evaporation of the medium, only the inner 60 wells of the 96-well plate should be used for analysis.</li>
	<li>Add 25 &#956;L of E-DMEM including 4 x 103 U/mL IL-2 and 10 &#956;M fluorogenic caspase-3 substrate (refer to Table of Materials).</li>
	<li>Add 4 x 103 cells/25 &#956;L of CD8+ T cells (step 5.3.4) into appropriate wells and mix well. 
	NOTE: The presence of too many cells in a well makes the analysis more difficult. In this model, a 4:1 effector: target ratio (total cell number 5 x 103 cells/well) was optimal, but an 8:1 ratio was suboptimal. Wells containing the following four controls are necessary to aid with data analysis: target cells at the density used for the co-culture wells (1 x 103 cells/well) in medium with and without caspase-3 substrate, effector cells (1 x 103 cells/well) in medium with and without caspase-3 substrate.</li>
	<li>Add 200 &#956;L of PBS or sterile water into all empty wells (particularly wells on the periphery of the plate) to reduce evaporation of medium from experimental wells.</li>
	<li>Set the plate into a time-lapse fluorescence microscope that is maintained at 37 &#176;C, 95% humidity, and 5% CO2. 
	NOTE: Shake the plate in a cross-pattern to distribute all cells evenly, and then allow the plate to remain at room temperature on a flat surface for 10-20 min before transferring to the incubator.</li>
</ol>
6. Imaging of the cells
NOTE: Detailed image acquisition settings will vary with the microscope and fluorophores used; the following general acquisition parameters should be employed for optimal results.
<ol>
	<li>Using an appropriate autofocus routine on the microscope, acquire images covering at least 25% of the total surface area in each experimental well of the 96-well plate.</li>
	<li>Set the microscope to acquire images in phase contrast as well as a fluorescent channel suitable for the nuclear-restricted red fluorescent protein (mKate2) and a fluorescent channel suitable for the green fluorogenic activated caspase-3 substrate (with excitation at 488 nm) (Figure 2).</li>
	<li>Capture images in experimental wells in phase contrast and the 2 fluorescent channels every 1 to 3 h for at least 72 h.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin EDTA (1X)</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>12-Well Cell Culture Plate</td>
			<td>Freiner Bio-One</td>
			<td>665-180</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptethanol</td>
			<td>Sigma</td>
			<td>M6250-10ML</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL Plystyrene Round-Bottom Tube</td>
			<td>FALCON</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>96-Well Cell Culture Plate (Round Bottom )</td>
			<td>Costar</td>
			<td>3799</td>
			<td>Co-culture of CD8+T cells with sorted myeloid cells</td>
		</tr>
		<tr>
			<td>96-well plate (Flat bottom)</td>
			<td>Nunc</td>
			<td>165305</td>
			<td>Co-culture of CD8+T cells with target cells for cytotoxicity assay</td>
		</tr>
		<tr>
			<td>AF647 anti-mouse F4/80 Antibody</td>
			<td>BIO-RAD</td>
			<td>MCA497A647</td>
			<td>Clone: CIA3-1, Lot#: 1707, 2 &#956;L/1x10^6 cells</td>
		</tr>
		<tr>
			<td>AlexaFluor700 anti-mouse CD8 Antibody</td>
			<td>Biolegend</td>
			<td>100730</td>
			<td>Clone: 53-6.7, Lot#: B205738, 0.5 &#956;L/1x10^6 cells</td>
		</tr>
		<tr>
			<td>anti-mouse CD28 Antibody</td>
			<td>Biolegend</td>
			<td>102111</td>
			<td>Activation of isolated CD8+ T cells, Clone: 37.51, Lot#: B256340</td>
		</tr>
		<tr>
			<td>anti-mouse CD3e Antibody</td>
			<td>Biolegend</td>
			<td>100314</td>
			<td>Activation of isolated CD8+ T cells, Clone: 145-2C11, Lot#: B233720</td>
		</tr>
		<tr>
			<td>APC anti-mouse CD3 Antibody</td>
			<td>Biolegend</td>
			<td>100236</td>
			<td>Clone: 17A2, Lot#: B198730, 0.5 &#956;L/1x10^6 cells</td>
		</tr>
		<tr>
			<td>APC/Cy7 anti-mouse Ly6C Antibody</td>
			<td>Biolegend</td>
			<td>128026</td>
			<td>Clone: HK1.4, Lot#: B248351, 1 &#956;L/1x10^6 cells</td>
		</tr>
		<tr>
			<td>Bovine Serum Albmin</td>
			<td>Sigma</td>
			<td>A1470-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer (100&#956;m Nylon)</td>
			<td>FALCON</td>
			<td>352360</td>
			<td>To smash the spleen</td>
		</tr>
		<tr>
			<td>Cell Strainer (40&#956;m Nylon)</td>
			<td>FALCON</td>
			<td>352340</td>
			<td>To filter the lung digestion</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Biolegend</td>
			<td>422801</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8242;s Modified Eagle&#8242;s Medium</td>
			<td>Gibco</td>
			<td>41966-029</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse CD8+ T Cell Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19853</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC anti-mouse CD4 Antibody</td>
			<td>Biolegend</td>
			<td>100406</td>
			<td>Clone: GK1.5, Lot#: B179194, 0.5 &#956;L/1x10^6 cells</td>
		</tr>
		<tr>
			<td>Geltrex Ready-to-Use</td>
			<td>Gibco</td>
			<td>A1596-01</td>
			<td>Coating the 96-well plates for cytotoxicity assay</td>
		</tr>
		<tr>
			<td>IncuCyte NucLight Red Lentivirus Reagent</td>
			<td>Essen BioScience</td>
			<td>4476</td>
			<td>Lenti viral particules encoding mKate2</td>
		</tr>
		<tr>
			<td>IncuCyte ZOOM</td>
			<td>Essen BioScience</td>
			<td></td>
			<td>Detector (fluorescence microscope)</td>
		</tr>
		<tr>
			<td>IncuCyte ZOOM 2018A</td>
			<td>Essen BioScience</td>
			<td></td>
			<td>Analysis software</td>
		</tr>
		<tr>
			<td>L-Glutamine (100X)</td>
			<td>Gibco</td>
			<td>A2916801</td>
			<td></td>
		</tr>
		<tr>
			<td>Lung Dissociation Kit</td>
			<td>Miltenyi</td>
			<td>130-095-927</td>
			<td>Preparation of single cell suspension from the tumor-bearing lung</td>
		</tr>
		<tr>
			<td>MycoAlert Mycoplasma Detection Kit</td>
			<td>Lonza</td>
			<td>LT07-318</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acid (100X)</td>
			<td>Gibco</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>NucView488</td>
			<td>Biotium</td>
			<td>10403</td>
			<td>Fluoregenic caspase-3 substrate</td>
		</tr>
		<tr>
			<td>PE anti-mouse Ly6G Antibody</td>
			<td>Biolegend</td>
			<td>127607</td>
			<td>Clone: 1A8, Lot#: B258704, 0.5 &#956;L/1x10^6 cells</td>
		</tr>
		<tr>
			<td>PE/Cy7 anti-mouse CD11b Antibody</td>
			<td>Biolegend</td>
			<td>101216</td>
			<td>Clone: M1/70, Lot#: B249268, 0.5 &#956;L/1x10^6 cells</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>Penicillin Streptomycin for primary culture of cells</td>
		</tr>
		<tr>
			<td>PerCP/Cy5.5 anti-mouse CD45 Antibody</td>
			<td>Biolegend</td>
			<td>103132</td>
			<td>Clone: 30-F11, Lot#: B249564, 0.5 &#956;L/1x10^6 cells</td>
		</tr>
		<tr>
			<td>Polybrene (Hexadimethrine bromide)</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Gibco</td>
			<td>A11138-03</td>
			<td></td>
		</tr>
		<tr>
			<td>RBC Lysis Buffer (10X)</td>
			<td>Biolegend</td>
			<td>420301</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine IL-2</td>
			<td>Peprotech</td>
			<td>212-12</td>
			<td>Activation of isolated CD8+ T cells</td>
		</tr>
		<tr>
			<td>Sodium pyruvate (100X)</td>
			<td>Gibco</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>TruStain fcX (anti-mouse CD16/32) Antibody</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kitamura, T., et al. <a href="https://app.jove.com/v/58841">Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells.</a> J. Vis. Exp. (2019)
This video showcases an apoptosis assay in cancer cells induced by pre-activated effector T cells. By co-culturing these cells with cancer cells marked with fluorescent nuclei, a caspase-cleavable fluorogenic substrate is added. Apoptotic cancer cells will display a different color in their nuclei, leading to dual fluorescence. This dual fluorescence is indicative of cancer cells undergoing apoptosis.

<img alt="Figure 1" src="/files/ftp_upload/21904/21904_Figure1.jpg" /> 
Figure 1. Gating strategy to isolate suppressor cells from the metastatic lung. (A) Representative dot plots to isolate monocytic myeloid-derived suppressor cells (M-MDSCs) and metastasis-associated macrophages (MAMs). The threshold of Ly6C level to distinguish MAMs (Ly6Clow) and M-MDSCs (Ly6Chigh) is based on that of resident alveolar macrophages (RMAC). (<...

1. Preparation of target cells that express red fluorescent protein in their nuclei

	Obtain a target mouse cancer cell line from an appropriate source.
	...

Take two CD8+ T cell populations: effector&#160; CD8+ T cells pre-activated through antibody-mediated engagement of T cell and co-stimulatory receptors and suppressed CD8+ T cells inhibited by myeloid-derived suppressor cells.
Add these T cells to basement-membrane-coated wells with cancer cells expressing a nuclear-restricted fluorescent protein.
Pipette a fluorogenic&#160; caspase-3 substrate &#8212; a DNA-binding fluorogenic dye conjugated to a caspase-3 recognition site-containing peptide.
Incubate. Effector T cell receptors bind to the class I MHC-peptide complex on the cancer cells, triggering the release of cytotoxic granules containing perforins and granzymes.
Perforins create cancer cell membrane pores, allowing granzyme entry and caspase activation, initiating apoptosis.
Activated caspases additionally cleave the fluorogenic caspase substrate, releasing the dye that binds to DNA and fluoresces.
Under a fluorescence microscope, cancer cells co-cultured with effector T cells exhibit dual nuclear fluorescence, indicating cancer cell apoptosis.
In contrast, cancer cells co-cultured with suppressed T cells display only nuclear protein fluorescence, suggesting non-apoptotic cells.

62 조회수. 암세포에 대한 사전 활성화된 CD8+ T 세포의 세포 독성을 연구하기 위한 체외 분석

비디오: 암세포에 대한 사전 활성화된 CD8+ T 세포의 세포 독성을 연구하기 위한 체외 분석 - 실험

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

종양 침투 T 세포 독성의 정형 외 췌장 종양 및 Ex 생체 특성의 생성

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

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JoVE Journal

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Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

3D 배양에서 암 세포 침략 및 T 세포 세포 독성 을 감시

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte (CTL) responses elicited against tumor- and pathogen-derived peptides. They offer several advantages over traditional killing assays. First, they permit the monitoring of CTL-mediated cytotoxicity within architecturally intact secondary lymphoid organs, typically in the spleen. Second, they allow for mechanistic studies during the priming, effector and recall phases of CTL responses. Third, they provide useful platforms for vaccine/drug efficacy testing in a truly in vivo setting. Here, we provide an optimized protocol for the examination of concomitant CTL responses against more than one peptide epitope of a model tumor antigen (Ag), namely, simian virus 40 (SV40)-encoded large T Ag (T Ag). Like most other clinically relevant tumor proteins, T Ag harbors many potentially immunogenic peptides. However, only four such peptides induce detectable CTL responses in C57BL/6 mice. These responses are consistently arranged in a hierarchical order based on their magnitude, which forms the basis for TCD8 &#8220;immunodominance&#8221; in this powerful system. Accordingly, the bulk of the T Ag-specific TCD8 response is focused against a single immunodominant epitope while the other three epitopes are recognized and responded to only weakly. Immunodominance compromises the breadth of antitumor TCD8 responses and is, as such, considered by many as an impediment to successful vaccination against cancer. Therefore, it is important to understand the cellular and molecular factors and mechanisms that dictate or shape TCD8 immunodominance. The protocol we describe here is tailored to the investigation of this phenomenon in the T Ag immunization model, but can be readily modified and extended to similar studies in other tumor models. We provide examples of how the impact of experimental immunotherapeutic interventions can be measured using in vivo cytotoxicity assays.

Tailoring In Vivo Cytotoxicity Assays to Study Immunodominance in Tumor-specific CD8+ T Cell Responses

We describe here a flow cytometry-based in vivo killing assay that enables examination of immunodominance in cytotoxic T lymphocyte (CTL) responses to a model tumor antigen. We provide examples of how this elegant assay may be employed for mechanistic studies and for drug efficacy testing.

종양 특이 CD8+ T 세포 반응에서 면역 우세를 연구 하기 위한 생체 내 세포 독성 분석 법

Carboxyfluorescein succinimidyl ester (CFSE)-based in vivo cytotoxicity assays enable sensitive and accurate quantitation of CD8+ cytolytic T lymphocyte ...

An In Vitro Assay for Studying the Cytotoxicity of Pre-Activated CD8⁺ T Cells against Cancer Cells

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