Name: isolation and expansion of cytotoxic cytokine-induced killer t cells for cancer treatment
Uploaded: 2020-01-24T14:00:00
Description: Watch this Scientific Journal Video about Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment at JoVE.com

This study was supported by China Medical University Hospital (DMR-Cell-1809).

The clinical protocol was performed and approved in accordance with the guidelines of the Institutional Review Board of the China Medical University and Hospital Research Ethics Committee. Peripheral blood specimens were harvested from healthy volunteers with their informed consent.
1. Preparation of materials
<ol>
	<li>Store reagents, antibodies, and chemicals as shown in the Material Safety Data Sheet (MSDS). Dissolve the drugs or cytokines in solvents as stock solutions and then aliquot f...

<ol>
	<li>Cappuzzello, E., 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=Cytokines+for+the+induction+of+antitumor+effectors:+The+paradigm+of+Cytokine-Induced+Killer+(CIK)+cells.">Cytokines for the induction of antitumor effectors: The paradigm of Cytokine-Induced Killer (CIK) cells.</a> Cytokine &amp; Growth Factor Reviews. 36, 99-105 (2017).</li><li>Schmidt-Wolf, R. 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=Propagation+of+large+numbers+of+T+cells+with+natural+killer+cell+markers.">Propagation of large numbers of T cells with natural killer cell markers.</a> British Journal of Haematology. 87 (3), 453-458 (1994).</li><li>Grainger, 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=Wnt+Signaling+in+Hematological+Malignancies.">Wnt Signaling in Hematological Malignancies.</a> Progress in Molecular Biology and Translational Science. 153, 321-341 (2018).</li><li>Dai, C., 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=Implication+of+combined+PD-L1/PD-1+blockade+with+cytokine-induced+killer+cells+as+a+synergistic+immunotherapy+for+gastrointestinal+cancer.">Implication of combined PD-L1/PD-1 blockade with cytokine-induced killer cells as a synergistic immunotherapy for gastrointestinal cancer.</a> Oncotarget. 7 (9), 10332-10344 (2016).</li><li>Schmidt-Wolf, I. G., 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=Use+of+a+SCID+mouse/human+lymphoma+model+to+evaluate+cytokine-induced+killer+cells+with+potent+antitumor+cell+activity.">Use of a SCID mouse/human lymphoma model to evaluate cytokine-induced killer cells with potent antitumor cell activity.</a> TheJournal of Experimental Medicine. 174 (1), 139-149 (1991).</li><li>Introna, M., 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=Rapid+and+massive+expansion+of+cord+blood-derived+cytokine-induced+killer+cells:+an+innovative+proposal+for+the+treatment+of+leukemia+relapse+after+cord+blood+transplantation.">Rapid and massive expansion of cord blood-derived cytokine-induced killer cells: an innovative proposal for the treatment of leukemia relapse after cord blood transplantation.</a> Bone Marrow Transplantation. 38 (9), 621-627 (2006).</li><li>Schmeel, L. C., 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=Cytokine-induced+killer+(CIK)+cells+in+cancer+immunotherapy:+report+of+the+international+registry+on+CIK+cells+(IRCC).">Cytokine-induced killer (CIK) cells in cancer immunotherapy: report of the international registry on CIK cells (IRCC).</a> Journal of Cancer Research and Clinical Oncology. 141 (5), 839-849 (2015).</li><li>Rutella, 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=Adoptive+immunotherapy+with+cytokine-induced+killer+cells+generated+with+a+new+good+manufacturing+practice-grade+protocol.">Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol.</a> Cytotherapy. 14 (7), 841-850 (2012).</li><li>Nausch, N., 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=NKG2D+ligands+in+tumor+immunity.">NKG2D ligands in tumor immunity.</a> Oncogene. 27 (45), 5944-5958 (2008).</li><li>Gammaitoni, L., 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=Effective+activity+of+cytokine-induced+killer+cells+against+autologous+metastatic+melanoma+including+cells+with+stemness+features.">Effective activity of cytokine-induced killer cells against autologous metastatic melanoma including cells with stemness features.</a> Clinical Cancer Research. 19 (16), 4347-4358 (2013).</li><li>Rettinger, E., 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=The+cytotoxic+potential+of+interleukin-15-stimulated+cytokine-induced+killer+cells+against+leukemia+cells.">The cytotoxic potential of interleukin-15-stimulated cytokine-induced killer cells against leukemia cells.</a> Cytotherapy. 14 (1), 91-103 (2012).</li><li>Narayan, R., 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=Donor-derived+cytokine-induced+killer+cell+infusion+as+consolidation+after+nonmyeloablative+allogeneic+transplantation+for+myeloid+neoplasms.">Donor-derived cytokine-induced killer cell infusion as consolidation after nonmyeloablative allogeneic transplantation for myeloid neoplasms.</a> Biology of Blood and Marrow Transplantation. 19, 1083 (2019).</li><li>Castiglia, 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=Cytokines+induced+killer+cells+produced+in+good+manufacturing+practices+conditions:+identification+of+the+most+advantageous+and+safest+expansion+method+in+terms+of+viability,+cellular+growth+and+identity.">Cytokines induced killer cells produced in good manufacturing practices conditions: identification of the most advantageous and safest expansion method in terms of viability, cellular growth and identity.</a> Journal of Translational Medicine. 16 (1), 237 (2018).</li><li>Bonanno, G., 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=Thymoglobulin,+interferon-&#947;+and+interleukin-2+efficiently+expand+cytokine-induced+killer+(CIK)+cells+in+clinical-grade+cultures.">Thymoglobulin, interferon-&#947; and interleukin-2 efficiently expand cytokine-induced killer (CIK) cells in clinical-grade cultures.</a> Journal of Translational Medicine. 8, 129 (2010).</li><li>Iudicone, P., 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=Interleukin-15+enhances+cytokine+induced+killer+(CIK)+cytotoxic+potential+against+epithelial+cancer+cell+lines+via+an+innate+pathway.">Interleukin-15 enhances cytokine induced killer (CIK) cytotoxic potential against epithelial cancer cell lines via an innate pathway.</a> Human Immunology. 77 (12), 1239-1247 (2016).</li><li>Liu, J., 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=Phenotypic+characterization+and+anticancer+capacity+of+CD8++cytokine-induced+killer+cells+after+antigen-induced+expansion.">Phenotypic characterization and anticancer capacity of CD8+ cytokine-induced killer cells after antigen-induced expansion.</a> PLoS One. 12 (4), 0175704 (2017).</li><li>Chen, 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=Cytokine-induced+killer+cells+as+a+feasible+adoptive+immunotherapy+for+the+treatment+of+lung+cancer.">Cytokine-induced killer cells as a feasible adoptive immunotherapy for the treatment of lung cancer.</a> Cell Death &amp; Disease. 9 (3), 366 (2018).</li><li>Tario, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+cell+proliferation+by+dye+dilution:+considerations+for+probe+selection.">Monitoring cell proliferation by dye dilution: considerations for probe selection.</a> Methods in Molecular Biology. 1678, 249-299 (2018).</li><li>Last'ovicka, J., 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=Assessment+of+lymphocyte+proliferation:+CFSE+kills+dividing+cells+and+modulates+expression+of+activation+markers.">Assessment of lymphocyte proliferation: CFSE kills dividing cells and modulates expression of activation markers.</a> Cellular Immunology. 256 (1-2), 79-85 (2009).</li><li>Yoshida, T., 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=Characterization+of+natural+killer+cells+in+tamarins:+a+technical+basis+for+studies+of+innate+immunity.">Characterization of natural killer cells in tamarins: a technical basis for studies of innate immunity.</a> Frontiers in Microbiology. 1, 1-9 (2010).</li></ol>

The described method is a fast, convenient, and reliable protocol for the isolation and expansion of cytotoxic cytokine-induced killer (CIK) T cells from whole blood samples of healthy donors. It also shows the cytotoxic effect of CIK against leukemia (K562) and ovarian cancer cells (OC-3) using a flow cytometry setup and tracking (CS &#38; T) system. CIK cells can be induced and expanded in good manufactory practices (GMP) conditions by using GMP-grade cytokines and serum-free medium for further clinical infusion<sup cl...

Cytotoxic T lymphocytes are a specific immune effector cell population that mediates immune responses against cancer. Several effector cell populations including lymphokine-activated killer (LAK) cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, &#947;&#948; T cells, and cytokine-induced killer (CIK) cells have been developed for adoptive T cell therapy (ACT) purposes1. There is a growing interest in CIK cells, because they represent a mixture of cytokine-induced cytotoxic cell populations expanded from autologous peripheral blood mononuclear cells (PBMCs)2.
The uncontrolled growth of lymphoid progenitor cells, myeloblasts, and lymphoblasts leads to three main types of blood cancers (i.e., leukemia, lymphoma, and myeloma), solid tumors, including carcinomas (e.g., lung cancer, gastric cancer, cervical cancer), and sarcomas, among other cancers3. CIK cells are a mixture of cell populations that exhibit a wide range of major histocompatibility complex (MHC)-unrestricted antitumor activity and thus hold promise for the treatment of hematological and advanced tumors4,5,6,7. CIK cells comprise a combination of cells, including T cells (CD3+CD56&#8722;), NK-T cells (CD3+CD56+), and NK cells (CD3&#8211;CD56+). Optimization of the CIK induction protocol by use of a fixed schedule for the addition of IFN-&#947;, anti-CD3 antibody, and IL-2, results in the expansion of CIK cells8. The cytotoxic capability of CIK cells against cancer cells mainly depends on the engagement of NK group 2 member D (a member of the C-type lectin-like receptor family) NKG2D ligands on tumor cells, and on perforin-mediated pathways9. The results of a preclinical study revealed that IL-15-stimulated CIK cells induced potent cytotoxicity against primary and acute myeloid leukemia cell lines in vitro and exhibited a lower alloreactivity against normal PBMCs and fibroblasts9. Recently, the outcome of one-time healthy donor-derived CIK (1&#8239;x&#8239;108/kg CD3+ cells) infusion as consolidation following nonmyeloablative allogeneic transplantation for myeloid neoplasms treatment in a phase II clinical study was published10.
In the present study, we developed an optimized cell culture formula composed of IFN-&#947;, IL-1&#945;, anti-CD3 antibody, and IL-2 added to the hematopoietic cell medium to increase CIK production, and investigated the cytotoxic effect of CIK cells against human chronic myeloid leukemia (K562) cells and ovarian cancer (OC-3) cells.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>7-Amino Actinomycin D</td>
			<td>BD</td>
			<td>559925</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse Anti-Human CD56 antibody</td>
			<td>BD</td>
			<td>555518</td>
			<td>B159</td>
			<td></td>
		</tr>
		<tr>
			<td>APC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555751</td>
			<td>MOPC-21</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACSCanto II Flow Cytometer</td>
			<td>BD</td>
			<td>338962</td>
			<td></td>
			<td>SN: R33896202856</td>
		</tr>
		<tr>
			<td>Carboxyfluorescein diacetate succinimidyl ester (CFSE)</td>
			<td>BD</td>
			<td>565082</td>
			<td></td>
			<td>Reconstritution of CFSE dye (500 mg) with 90 mL of DMSO</td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution</td>
			<td>SIGMA</td>
			<td>G8644</td>
			<td></td>
			<td>For K562 cell culture. Add 12.5 mL to 500 mL of complete medium</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium/F12</td>
			<td>HyClone</td>
			<td>SH30023.02</td>
			<td></td>
			<td>Basal medium for OC-3 cell culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
			<td>For K562 and OC-3 cell culture. Complete medium contains 10% of FBS</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare Life Sciences</td>
			<td>71101700-EK</td>
			<td></td>
			<td>Density gradient solution</td>
		</tr>
		<tr>
			<td>FITC Mouse Anti-Human CD3 antibody</td>
			<td>BD</td>
			<td>555332</td>
			<td>UCHT1</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC Mouse IgG1, &#954; Isotype Control</td>
			<td>BD</td>
			<td>555748</td>
			<td>MOPC-21</td>
			<td></td>
		</tr>
		<tr>
			<td>Human anti-CD3 mAb</td>
			<td>TaKaRa</td>
			<td>T210</td>
			<td>OKT3</td>
			<td>Add 2.5 mL of stock (1 mg/1 mL) to 50 mL of Induction medium. Storage stock at -80 &#176;C</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
			<td>Add 5 mL of stock (10,000 U/mL) to 500 mL of complete medium. Storage stock at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Proleukin</td>
			<td>NOVARTIS</td>
			<td></td>
			<td></td>
			<td>Reconstitution of Proleukin Powder (22x106 IU) with 1.2 mL of sterile water and add 2.7 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human Interferon-gamma</td>
			<td>CellGenix</td>
			<td>1425-050</td>
			<td></td>
			<td>Reconstitution of rh IFN-g (5x105 IU/50 &#181;g) with 200 &#181;L of sterile water and add 20 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Recombinant Human Interleukin-1 alpha</td>
			<td>PEPROTECH</td>
			<td>200-01A</td>
			<td></td>
			<td>Reconstitution rh IL-1&#945; (10 &#181;g) with 1 mL of sterile water and add 5 mL to 50 mL of Induction medium. Storage stock at -20 &#176;C</td>
		</tr>
		<tr>
			<td>RPMI1640 medium</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td></td>
			<td>Basal medium for K562 cell culture. Storage stock at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Sigma 3-18K Centrifuge</td>
			<td>Sigma</td>
			<td>10295</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express Enzyme</td>
			<td>Gibco</td>
			<td>12605028</td>
			<td></td>
			<td>Cell dissociation enzyme; For deattachment of adheren cells. Storage at room temperature</td>
		</tr>
		<tr>
			<td>X-VIVO 15 medium</td>
			<td>Lonza</td>
			<td>04-418Q</td>
			<td></td>
			<td>Basal medium for PBMC and CIK cells. Storage at 4 &#176;C</td>
		</tr>
	</tbody>
</table>

isolation and expansion of cytotoxic cytokine-induced killer t cells for cancer treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

The purpose of the present protocol is to isolate and expand cytokine-induced killer (CIK) T cells from peripheral blood monocytes and evaluate the cytotoxic effect of CIK against hematological malignancy and solid cancer cells, respectively. The induction of CIK was identified by the CD3/CD56 recognition. Figure 1A shows the protocol for CIK induction and expansion. The representative results of the gating strategy for analyzing the subpopulation of CD3+CD56...

Watch this Scientific Journal Video about Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment at JoVE.com

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Research

JoVE Journal

Cancer Research

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

A Syngeneic Mouse B-Cell Lymphoma Model for Pre-Clinical Evaluation of CD19 CAR T Cells

The astonishing clinical success of CD19 chimeric antigen receptor (CAR) T-cell therapy has led to the approval of two second generation chimeric antigen ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

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

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 ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

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

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 ...

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from SÃƒÆ’Ã‚Â©zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Chimeric antigen receptor (CAR)-modified immune cell therapy has become an emerging treatment for cancers and infectious diseases. NK-based immunotherapy, ...

Expansion and Enrichment of Gamma-Delta (&#947;&#948;) T Cells from Apheresed Human Product

Expansion and Enrichment of Gamma-Delta ÃƒÅ½Ã‚Â³ÃƒÅ½Ã‚Â´ T Cells from Apheresed Human Product

Although V&#947;9V&#948;2 T cells are a minor subset of T lymphocytes, this population is sought after for its ability to recognize antigens in a major ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...