an in vitro assay to evaluate the anti-tumor activity of tumor-specific car t cells

An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

First, seed 10,000 syngeneic target CD19-positive tumor cells with or without luciferase expression, and 100 microliters of TCM into each well of a 96-well U-bottom tissue culture plate. Add 10,000 CD19 CAR T cells and 100 microliters of TCM to each well.
Set up the controls as outlined in the text protocol. Co-culture the cells at 37 degrees Celsius with 5% carbon dioxide for 16 to 24 hours. After this, centrifuge the plate at 500 times g for five minutes. Collect the supernatant for further interferon-gamma and interleukin-12 p70 ELISA analysis.
Resuspend the cell pellets in 100 microliters of PBS containing luciferin, and incubate at 37 degrees Celsius for 10 minutes. Then, use a luminometer to measure the luminescence from each well.

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1. In vitro Validation of CAR T cell Activity
<ol>
	<li>Seed syngeneic target CD19+ tumor cells with or without luciferase expression at a density of 1 x 104 cells in 100 &#956;L complete T cell medium (TCM)/well in a 96-well U-bottom tissue culture plate.</li>
	<li>Add 1 x 104 CD19 CAR T cells/well in a volume of 100 &#956;L/well to achieve an effector to target (E:T) ratio of 1:1. 
	NOTE: E:T ratios should be established for each CAR construct and target cell line.</li>
	<li>Use T cells alone and tumor cells alone as negative controls and T cells stimulated by phorbol-myristate-acetate (PMA) (50 ng/mL) and ionomycin (1 &#956;g/mL) as positive control for Interferon-gamma (IFN&#947;) release. Co-culture cells at 37 &#176;C, 5% CO2 for 16-24 h.</li>
	<li>Following co-culture, centrifuge the plates at 500 x g for 5 min and collect the supernatant for further IFN&#947; and IL-12p70 ELISA analysis. 
	NOTE: This can be stored at -80 &#176;C.</li>
	<li>Re-suspend cell pellets in 100 &#956;L of PBS containing luciferin (final concentration of 1.5 mg/mL). Incubate the plates for 10 min at 37 &#176;C. Then measure the luminescence from each well with a suitable luminometer. 
	NOTE: Exposure times must be optimized for cell lines and density. Representative results are shown in Figure 1a. Ex-vivo cytotoxicity of CAR T cells can be modified to express luciferin by co-culture with cell lines expressing target antigen. As CAR T cells kill target cells, luciferin is released, therefore a reduction in luminometry signal is correlated with cell kill. Non-transduced cells can often have an effect on target cell viability, particularly over long incubation periods. Measure the concentration of murine IFN&#947; and IL-12p70 in the supernatant according to the manufacturer&#39;s ELISA protocols. Representative results are shown in (Figure 1b and 1c). Ex-vivo activation of CAR T cells by co-culture with cell lines expressing target antigen can be assayed by analyzing supernatant contents using ELISA. The ratio of CAR T cell to target cells and length of co-culture period must be optimized for each CAR construct, target cell line and analyte. PMA and ionomycin treatment can be used as a positive control to confirm quality of T cells and their ability to respond.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100X penicillin-streptomycin-glutamine (PSG)</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer - LSR Fortessa x20</td>
			<td>BD Biosciences</td>
			<td>658222R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Foetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10270</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-12 p70 Mouse Uncoated ELISA Kit</td>
			<td>Invitrogen</td>
			<td>88-7121-76</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin Calcium Salt</td>
			<td>Sigma- Aldrich</td>
			<td>I0634</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer - Lumistart Omega</td>
			<td>BMG Labtech</td>
			<td>415-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine IFN-&#947; ELISA kit</td>
			<td>Diaclone</td>
			<td>861.050.010</td>
			<td></td>
		</tr>
		<tr>
			<td>Phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma- Aldrich</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium</td>
			<td>Lonza</td>
			<td>BE12-167F</td>
			<td></td>
		</tr>
		<tr>
			<td>XenoLight D-Luciferin</td>
			<td>Perkin Elmer</td>
			<td>122799</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Kueberuwa, G., et al. <a href="https://app.jove.com/v/58492">A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells.</a> J. Vis. Exp. (2018).
This video demonstrates an in vitro assay to validate chimeric antigen receptor (CAR) T cell activity. B-cell lymphoma cells expressing CD19 antigen and cytoplasmic luciferase are co-cultured with CD19-specific CAR T cells. CAR binding to CD19 on a tumor induces the CAR T cells to release cytokines and toxins that cause tumor cell cytotoxicity. The CAR T cell activity is evaluated by quantifying the released cytokine and decreased luciferase-mediated bioluminescence in the tumor cells.

<img alt="Figure 1" src="/files/ftp_upload/22258/22258_Figure1.jpg" /> 
Figure 1. Validation of CAR T-cell activity. &#945;mCD19 CAR T cells were co-cultured with A20 lymphoma cells modified to express luciferase (1 x 104:1 x 104) for 16 h in a U-bottom 96-well plate. After co-culture, cells were pelleted, and supernatant was collected. A) Cells were re-suspended in PBS and luminometry was used to assess the viability of the tar...

1. In vitro Validation of CAR T cell Activity

	Seed syngeneic target CD19+ tumor cells with or without luciferase expression at a density of 1 x 104 ...

Take a multiwell plate containing B cell lymphoma tumor cells expressing a B cell-specific surface antigen and a cytoplasmic luciferase enzyme. 
Introduce CAR T cells into selected wells and incubate. The cells express chimeric antigen receptors, or CARs, comprising an extracellular domain targeting the tumor antigen and intracellular signaling domains.
CAR binds to the tumor antigen, inducing activation of the CAR T cells.
The activated cells release cytokines that promote the cells&#39; anti-tumor activity, triggering the release of toxins.
The toxins enter the tumor cells and induce cell death. 
Spin down the cells and isolate the supernatant. Quantify the cytokines to assess CAR T cell activity. 
Resuspend the cells in a buffer containing luciferin, a bioluminescent substrate.
Upon entry, luciferin is oxidized by luciferase in living tumor cells, emitting light.
Measure the bioluminescence to identify a decreased signal in the treated tumor cells, confirming CAR T cell-mediated cytotoxicity.

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A Spheroid Killing Assay by CAR T Cells

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric antigen receptor (CAR) redirected T cells obtained the most spectacular results, in particular with pediatric B-acute lymphoblastic leukemia (B-ALL). Classical validation methods of CAR T cells rely on the use of specificity and functionality assays of the CAR T cells against target cells in suspension and in xenograft models. Unfortunately, observations made in vitro are often decoupled from results obtained in vivo and a lot of effort and animals could be spared by adding another step: the use of 3D culture. The production of spheroids out of potential target cells that mimic the 3D structure of the tumor cells when they are engrafted into the animal model represents an ideal alternative. Here, we report an affordable, reliable and easy method to produce spheroids from a transduced colorectal cell line as a validation tool for adoptive cell therapy (exemplified here by CD19 CAR T cells). This method is coupled with an advanced live imaging system that can follow spheroid growth, effector cells cytotoxicity and tumor cell apoptosis.

This protocol is designed to assess immunotherapeutic redirected T-cell (CAR T-cell) cytotoxicity against 3D structured cancerous cells (spheroids) in real time.

一种用汽车 t 细胞进行的切杀检测

Immunotherapy has become a field of growing interest in the fight against cancer otherwise untreatable. Among all immunotherapeutic methods, chimeric ...

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Manufacturing Chimeric Antigen Receptor (CAR) T Cells for Adoptive Immunotherapy

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells with chimeric antigen receptor (CAR) and expand their progeny ex vivo. We include assays to measure CAR expression as well as differentiation, proliferative capacity and cytolytic activity. We describe assays to measure effector cytokine production and inflammatory cytokine secretion in CAR T cells. Our approach provides a reliable and comprehensive method to culture CAR T cells for preclinical models of adoptive immunotherapy.

Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy

We describe an approach to reliably generate chimeric antigen receptor (CAR) T cells and test their differentiation and function in vitro and in vivo.

制造用于采用免疫疗法的奇美抗原受体（CAR）T细胞

Adoptive immunotherapy holds promise for the treatment of cancer and infectious disease. We describe a simple approach to transduce primary human T cells ...

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

Here, we present a protocol for gene editing in primary human T cells using CRISPR Cas Technology to modify CAR-T cells.

An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

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An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells