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 for storage at -20 &#176;C or -80 &#176;C. 
	NOTE: Detailed information for material preparation is noted in the Table of Materials.</li>
</ol>
2. PBMC isolation
<ol>
	<li>Warm the density gradient solution (Table of Materials) to 18&#8211;20 &#176;C before use. Invert the solution bottle several times to ensure thorough mixing.</li>
	<li>Collect 3&#8722;5 mL of human venous blood sample in a heparinized vial and mix well by gently inverting the tube several times.</li>
	<li>Prepare 4 mL of density gradient solution in a 15 mL sterile tube.</li>
	<li>Carefully layer 1 mL of the blood sample onto the density gradient solution.</li>
	<li>Centrifuge at 400 x g for 30 min at 18&#8722;20 &#176;C (turn off the break).</li>
	<li>Carefully and immediately aspirate the buffy coat layer of mononuclear cells (about 1 mL) at once to avoid disturbing the layers to a sterile 15 mL tube using a 1 mL sterile pipette.</li>
	<li>Add at least 3 volumes (~3 mL) of phosphate-buffered saline (PBS) to the buffy coat in the centrifuge tube. Suspend the cells by gently pipetting them up and down at least 3x with a sterile pipette.</li>
	<li>Centrifuge at 400 x g for 10 min at 18&#8722;20 &#176;C. Aspirate the supernatant.</li>
	<li>Suspend the cell pellet with 5 mL of basal medium (Table of Materials) and transfer into a flask. Culture the cells in a cell culture incubator at 37 &#176;C and 5% CO2.</li>
</ol>
3. CIK induction and expansion
<ol>
	<li>On Day 0, culture the PBMCs (1 x 106) in fresh basal medium containing 1,000 IU/mL of IFN-&#947; for 24 h in a humidified cell culture incubator at 37 &#176;C and 5% CO2.</li>
	<li>On Day 1, refresh the medium with fresh basal medium containing 50 ng/mL of anti-CD3 antibody, 1 ng/mL of rh IL-1&#945;, and 1,000 U/mL of rh IL-2. Refresh the medium every 3 days.</li>
	<li>On Day 7, refresh the medium with fresh basal medium containing 1,000 U/mL of rh IL-2. Refresh the medium every 3 days until the end of cell expansion (Day 14).</li>
</ol>
4. Immunophenotyping for assessment of CIK cells
<ol>
	<li>Wash the CIK cells with 10 mL of sterile PBS. Centrifuge for 10 min at 300 x g and 18&#8722;20 &#176;C, aspirate the supernatant, and resuspend the cells with 10 mL of PBS. Count the cell number and test cell viability using the trypan blue exclusion assay.</li>
	<li>Aliquot the CIK cells into six sterile 1.5 mL tubes at a density of ~5&#8211;10 x 105 cells/mL PBS. Label and treat as follows: Tube 1, Blank (no antibody); Tube 2, add 20 &#181;L of isotype IgG1-FITC; Tube 3, add 20 &#181;L of isotype IgG1-APC mAbs; Tube 4, add 20 &#181;L of CD3-FITC; Tube 5, add 20 &#181;L of CD56-APC mAbs; and Tube 6, add 20 &#181;L of CD3-FITC and 20 &#181;L of CD56-APC mAbs.</li>
	<li>Gently mix the CIK cells with the antibodies by gently pipetting them up and down at least 3x with a 1 mL sterile pipette, and then incubate for 30 min at room temperature in the dark.</li>
	<li>Centrifuge the tubes for 10 min at 300 x g and 18&#8722;20 &#176;C. Aspirate the supernatant and suspend the cell pellet once with 1 mL of PBS. Gently pipet them up and down at least 3x with a 1 mL sterile pipette.</li>
	<li>Repeat step 4.4.</li>
	<li>Leave the tubes in the dark before flow cytometric analysis.</li>
</ol>
5. CD marker recognition
<ol>
	<li>Transfer the cell suspension to a sterile 5 mL polystyrene round bottom tube with a cell strainer cap (100 &#956;m mesh) by gently pipetting through the cap. Put the tubes on the carousel in order.</li>
	<li>Open the flow cytometry analysis software and create an experimental folder. Click the New Specimen button to add a specimen and tube to the experiment and name the tubes as follows: Tube 1, Blank; Tube 2, Isotype IgG1-CD3; Tube 3, Isotype IgG1-CD56; Tube 4, CD3; Tube 5, CD56; Tube 6, CD3CD56.</li>
	<li>Create a scatter gating system for the CIK cell populations (Figure 2A).
	<ol>
		<li>Select Tube 1 (Blank) and click on the Dot Plot button to create an FSC-A/SSC-A plot. Draw a rectangle gate over the entire cell population with an FSC-A threshold &#62;5 x 104 to exclude cell debris.</li>
		<li>Select the SSC-A/SSC-H parameter for the new dot plot and draw a polygon gate around all single cells. Select the Count/FITC (CD3) and Count/APC (CD56) parameter for the new histogram plot, respectively. Select the FITC (CD3)/APC (CD56) parameter for the new dot plot and draw a four quadrant gate to define the four subpopulations.</li>
		<li>Record the data from 20,000 single cells in each specimen. Click the Load Sample button to analyze the Blank control sample first. Identify the whole CIK cell population by using the CD56 and CD3 channel parameters.</li>
	</ol>
	</li>
	<li>Repeat step 5.3 for the investigation of all specimens.</li>
	<li>Open the files containing the statistical values of the individual specimen to analyze CIK cell populations and reprint them into analysis files.</li>
</ol>
6. Culturing and staining of human chronic myeloid leukemia K562 cells and ovarian cancer OC-3 cells
<ol>
	<li>K562 cells
	<ol>
		<li>Culture K562 cells in complete media (RPMI basal medium containing 10% fetal bovine serum [FBS] and 50 U/mL antibiotics and adjust glucose to 4.5 g/L) at a density of 0.5&#8722;1 x 106 cells/mL in a cell culture flask and incubate in a humidified incubator at 37 &#176;C and 5% CO2.</li>
		<li>Transfer the culture media containing the K562 cells into 50 mL sterile tubes and pellet the cells at 300 x g for 10 min at 18&#8722;20 &#176;C on the day of the experiment.</li>
		<li>Aspirate the supernatant, resuspend the cells in 5 mL of sterile PBS, and mix well gently.</li>
		<li>Pellet the cells at 300 x g for 10 min. Aspirate the supernatant, resuspend the cells in PBS, and adjust the K562 cells to a concentration of 0.5-1 x 106 cells/mL.</li>
		<li>Add 0.5 &#181;L of CFSE dye to the 1 mL of K562 cell suspension in a 15 mL sterile tube at a final concentration of 5 &#956;M. Gently mix the suspension by pipetting up and down at least 3x.</li>
		<li>Leave the tube in a cell culture incubator at 37 &#176;C and 5% CO2 for 10&#8211;15 min.</li>
		<li>Add 9 mL of PBS to the tube and pellet the cells at 300 x g for 10 min. Decant the supernatant and then suspend the cell pellet in 10 mL of complete media. Transfer the cell suspension to a cell culture flask and place in the incubator.</li>
	</ol>
	</li>
	<li>OC-3 cells
	<ol>
		<li>Culture OC-3 cells in complete media (DMEM/F12 medium containing 10% FBS and 50 U/mL antibiotics) at a density of 0.5&#8211;1 x 106 cells in a cell culture flask at 37 &#176;C and 5% CO2.</li>
		<li>Aspirate the culture media and wash the cells with PBS 1 day before the experiment.</li>
		<li>Detach the cells by adding 1 mL of cell dissociation enzyme solution (Table of Materials) and incubate for 5 min at 37 &#176;C.</li>
		<li>Suspend the cells by adding 5 mL of PBS and mix well gently. Pellet the cells at 300 x g for 10 min and aspirate the supernatant. Resuspend the cells in PBS and adjust the cells to a concentration of 0.5&#8211;1 x 106 cells/mL.</li>
		<li>Add 0.5 &#181;L of CFSE dye to 1 mL of the OC-3 cell suspension in a 15 mL sterile tube at a final concentration of 5 &#956;M. Gently mix the suspension by pipetting up and down at least 3x.</li>
		<li>Leave the tube in a cell culture incubator at 37 &#176;C and 5% CO2 for 10&#8211;15 min.</li>
		<li>Add 9 mL of PBS to the tube and pellet the cells at 300 x g for 10 min. Decant the supernatant and then suspend the cell pellet with complete media. Seed 5 x 105 cells/well into a 6 well plate and incubate in a humidified incubator at 37 &#176;C and 5% CO2 overnight.</li>
	</ol>
	</li>
</ol>
7. Cytotoxic assay
<ol>
	<li>Coculture of CIK and K562 cells (CIK-K562)
	<ol>
		<li>Count the K562 cells from step 6.1.7 and test the cell viability by trypan blue exclusion assay. Add 1 mL of K562 cells to each well in a 6 well plate at a density of 5 x 105/mL.</li>
		<li>Add 1 mL of basal medium with or without CIK cells from step 3.4 to the 6 well plate from step 7.1.1 as follows: Well 1 = Blank, K562 cells alone (5 x 105); Well 2 = CFSE-stained K562 cells alone (5 x 105); Well 3 = CIK cells (E [effector], 25 x 105) + CFSE-stained K562 cells (T [target], 5 x 105); Well 4 = CIK cells (E, 50 x 105) + CFSE-stained K562 cells (T, 5 x 105).</li>
		<li>Mix the cell suspensions by gently pipetting them up and down at least 3x. Place the plate in the incubator for 24 h.</li>
	</ol>
	</li>
	<li>Coculture of CIK and OC-3 cells (CIK-OC-3)
	<ol>
		<li>Add 1 mL of basal medium with or without CIK cells from step 3.4 to the 6 well plate from step 6.2.7 as follows: Well 1 = Blank, OC-3 cells alone (5 x 105); Well 2 = CFSE-stained OC-3 cells alone (5 x 105); Well 3 = CIK cells (E, 25 x 105) + CFSE-stained OC-3 cells (T, 5 x 105); Well 4 = CIK cells (E, 50 x 105) + CFSE-stained OC-3 cells (T, 5 x 105).</li>
		<li>Mix the cell suspensions by gently pipetting them up and down at least 3x. Put the plate in the incubator for 24 h.</li>
	</ol>
	</li>
	<li>7-Aminoactinomycin D (7-AAD) dye staining
	<ol>
		<li>Harvest the CIK-K562 cell suspension from step 7.1.3 directly into a 15 mL sterile tube.</li>
		<li>Harvest both the suspension and adherent cells from the CIK-OC-3 groups from step 7.2.2.
		<ol>
			<li>Transfer the cell suspension to a 15 mL sterile tube. Wash the well with 1 mL of sterile PBS, collect the PBS, and add to the tube. Add 0.5 mL of cell dissociation enzyme solution, and incubate for 5 min at 37 &#176;C.</li>
			<li>Add 1 mL of the solution from the same tube to the corresponding well and gently mix the cells by pipetting them up and down at least 3x with a 1 mL sterile pipette. Collect all the cells in the same tube.</li>
		</ol>
		</li>
		<li>Centrifuge at 300 x g for 10 min, aspirate the supernatant, and resuspend the cells in 1 mL of sterile PBS. Pellet the cells at 300 x g for 10 min, aspirate the supernatant, and resuspend cells in 100 &#181;L of sterile PBS.</li>
		<li>Add 5 &#181;L of 7-AAD dye (50 ng/&#181;L stock) to the cell suspension. Gently mix the cells by pipetting them up and down at least 3x with a 1 mL sterile pipette. Incubate for 10 min and leave in the dark before analysis.</li>
	</ol>
	</li>
	<li>Cytolytic capability assay
	<ol>
		<li>Mix the cell suspension from step 7.3.4 and repeat steps 5.1 and 5.2 once.</li>
		<li>Click the New Specimen button to add a specimen and tube to the experiment and name the tubes as follows: Tube 1, K562 (or OC-3) cells only; Tube 2, CFSE-stained K562 (or OC-3) cells only; Tube 3, E:T = 5:1; Tube 4, E:T = 10:1.</li>
		<li>Create a Scatter Gating System for the cytolytic assay (Figure 3A).
		<ol>
			<li>Select Tube 1 and click on the Dot Plot button to create an FSC-A/SSC-A plot. Draw a rectangle gate over all events with an FSC-A threshold &#62;5 x 104 to exclude cell debris.</li>
			<li>Select the SSC-A/CFSE parameter for the new dot plot. Select the 7-AAD/CFSE parameter for the new dot plot and draw a four-quadrant gate to define the four subpopulations.</li>
			<li>Click the Load Sample button to analyze the blank control sample first.</li>
			<li>Adjust the voltage of SSC-A and FSC-A. Identify the dead cell population by using the CFSE and 7-AAD channel parameters. Record the data from &#62;20,000 CFSE+ cells in each specimen.</li>
		</ol>
		</li>
		<li>Repeat section 7.4.6 for the investigation of all specimens.</li>
		<li>Open the files containing the statistical values of each individual specimen to analyze the non-viable cell populations and export the data into analysis files.</li>
	</ol>
	</li>
</ol>

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The authors declare no competing conflicts of financial interest.

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><tbody><tr><td>7-Amino Actinomycin D</td><td>BD</td><td>559925</td><td></td></tr><tr><td>APC Mouse Anti-Human CD56 antibody</td><td>BD</td><td>555518</td><td>B159</td></tr><tr><td>APC Mouse IgG1, &amp;kappa; Isotype Control</td><td>BD</td><td>555751</td><td>MOPC-21</td></tr><tr><td>BD FACSCanto II Flow Cytometer</td><td>BD</td><td>338962</td><td></td></tr><tr><td>Carboxyfluorescein diacetate succinimidyl ester (CFSE)</td><td>BD</td><td>565082</td><td></td></tr><tr><td>D-(+)-Glucose solution</td><td>SIGMA</td><td>G8644</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium/F12</td><td>HyClone</td><td>SH30023.02</td><td></td></tr><tr><td>Fetal bovine serum</td><td>HyClone</td><td>SH30084.03</td><td></td></tr><tr><td>Ficoll-Paque Plus</td><td>GE Healthcare Life Sciences</td><td>71101700-EK</td><td></td></tr><tr><td>FITC Mouse Anti-Human CD3 antibody</td><td>BD</td><td>555332</td><td>UCHT1</td></tr><tr><td>FITC Mouse IgG1, &amp;kappa; Isotype Control</td><td>BD</td><td>555748</td><td>MOPC-21</td></tr><tr><td>Human anti-CD3 mAb</td><td>TaKaRa</td><td>T210</td><td>OKT3</td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Proleukin</td><td>NOVARTIS</td><td></td><td></td></tr><tr><td>Recombinant Human Interferon-gamma</td><td>CellGenix</td><td>1425-050</td><td></td></tr><tr><td>Recombinant Human Interleukin-1 alpha</td><td>PEPROTECH</td><td>200-01A</td><td></td></tr><tr><td>RPMI1640 medium</td><td>Gibco</td><td>11875-085</td><td></td></tr><tr><td>Sigma 3-18K Centrifuge</td><td>Sigma</td><td>10295</td><td></td></tr><tr><td>TrypLE Express Enzyme</td><td>Gibco</td><td>12605028</td><td></td></tr><tr><td>X-VIVO 15 medium</td><td>Lonza</td><td>04-418Q</td><td></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 ...

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JoVE Journal

Cancer Research

10.8K Views.  Wan Fang Hospital. We hebben een hooggekwalificeerde en klinisch toepasbare methode geoptimaliseerd voor de uitbreiding van pbmc-afgeleide cytokine-geïnduceerde killer T-cellen en voor de evaluatie van hun cytotoxiciteit tegen kanker. Het voordeel van deze techniek is om een standaard procedure voor technici en clinici om de potentie van cytokine-geïnduceerde killer cellen kwantificeren in zowel wetenschappelijk onderzoek en klinische evaluatie. Begin deze procedure met een preparaat van CIK-cellen zoals besc...

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

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide complete protection against the development or recurrence of breast cancer. To overcome this problem by increasing the frequency of tumor-reactive T cells, adoptive immunotherapy has been employed. A variety of protocols have been used for the expansion of tumor-specific T cells. These protocols, however, are restricted to the use of tumor antigens ex vivo for the activation of antigen-specific T cells. Very recently, common gamma chain cytokines such as IL-2, IL-7, IL-15, and IL-21 have been used alone or in combination for the enhancement of anti-tumor immune responses3. However, it is not clear what formulation would work best for the expansion of tumor-reactive T cells. Here we present a protocol for the selective activation and expansion of tumor-reactive T cells from the FVBN202 transgenic mouse model of HER-2/neu positive breast carcinoma for use in adoptive T cell therapy of breast cancer. The protocol includes activation of T cells with bryostatin-1/ionomycin (B/I) and IL-2 in the absence of tumor antigens for 16 hours. B/I activation mimics intracellular signals that result in T cell activation by increasing protein kinase C activity and intracellular calcium, respectively4. This protocol specifically activates tumor-specific T cells while killing irrelevant T cells. The B/I-activated T cells are cultured with IL-7 and IL-15 for 24 hours and then pulsed with IL-2. After 24 hours, T cells are washed, split, and cultured with IL-7 + IL-15 for additional 4 days. Tumor-specificity and anti-tumor efficacy of the ex vivo expanded T cells is determined.

Ex vivo Expansion of Tumor-reactive T Cells by Means of Bryostatin 1/Ionomycin and the Common Gamma Chain Cytokines Formulation

An efficient protocol for the ex vivo expansion of tumor-reactive T cells from tumor-draining lymph nodes or other secondary lymphoid tissues of tumor-bearing hosts is described. This protocol selectively expands tumor-specific T cells for use in adoptive immunotherapy of breast cancer.

It was reported that breast cancer patients have pre-existing immune responses against their tumors1,2. However, such immune responses fail to provide ...

Immunology and Infection

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells 

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

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.

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

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

Adoptive Therapy Using Tumor-Infiltrating Lymphocytes in Pancreatic Cancer Models

Flow Cytometry-Based Isolation and Therapeutic Evaluation of Tumor-Infiltrating Lymphocytes in a Mouse Model of Pancreatic Cancer

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and activating a patient&#39;s own immune cells, such as tumor-infiltrating lymphocytes (TILs), before re-infusing them, represents a promising experimental approach. However, techniques for adoptive cell transfer in preclinical pancreatic cancer models are not well established. Here, we describe a detailed protocol for adoptive cell therapy using TILs from a syngeneic pancreatic cancer mouse model. The procedure involves implanting live or irradiated mouse pancreatic cancer cells in fluorescence-labeled reporter mice to initiate immune cell influx, then isolating lymphocytes from primary tumors via flow cytometry sorting and/or activating and expanding tumor-reactive T cells ex vivo, and adoptively transferring these activated T cells intraperitoneally into tumor-bearing mice, followed by interleukin-2 administration.&#160;Bioluminescent tumor imaging allows for longitudinal monitoring of orthotopic tumor growth and response to therapy, especially evaluating the tumor-specific cytotoxic effects. This approach recapitulates the logistics involved in developing adoptive cell transfer therapies for pancreatic cancer patients. The results demonstrate enhanced antitumor efficacy of adoptively transferred tumor-reactive T cells compared to irrelevant lymphocyte controls. This versatile methodology enables the in vivo study of adoptive immunotherapy in pancreatic cancer as well as the optimization of cell processing parameters and combination treatment regimens.

We describe a method to utilize tumor-infiltrating lymphocytes (TILs) from mice through flow cytometry for adoptive cell transfer. This protocol aims to verify the specific cytotoxicity of TILs against tumors in a syngeneic pancreatic cancer mouse model, providing insights into the development of adoptive cell therapies for pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a dismal prognosis and limited therapeutic options. Adoptive cell therapy, which involves isolating and ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Purification and Expansion of Mouse Invariant Natural Killer T Cells for in vitro and in vivo Studies

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or microbial lipid antigens presented by the non-polymorphic MHC class I-related molecule CD1d. Preclinical and clinical studies support a role for iNKT cells in cancer, autoimmunity and infectious diseases. iNKT cells are very conserved throughout species and their investigation has been facilitated by mouse models, including CD1d-deficient or iNKT-deficient mice, and the possibility to unequivocally detect them in mice and men with CD1d tetramers or mAbs specific for the semi-invariant TCR. However, iNKT cells are rare and they need to be expanded to reach manageable numbers for any study. Because the generation of primary mouse iNKT cell line in vitro has proven difficult, we have set up a robust protocol to purify and expand splenic iNKT cells from the iV&#945;14-J&#945;18 transgenic mice (iV&#945;14Tg), in which iNKT cells are 30 times more frequent. We show here that primary splenic iV&#945;14Tg iNKT cells can be enriched through an immunomagnetic separation process, yielding about 95-98% pure iNKT cells. The purified iNKT cells are stimulated by anti-CD3/CD28 beads plus IL-2 and IL-7, resulting in 30-fold expansion by day +14 of the culture with 85-99% purity. The expanded iNKT cells can be easily genetically manipulated, providing an invaluable tool to dissect mechanisms of activation and function in vitro and, more importantly, also upon adoptive transfer in vivo.

We describe a rapid and robust protocol to enrich invariant natural killer T (iNKT) cells from mouse spleen and expand them in vitro to suitable numbers for in vitro and in vivo studies.

Invariant Natural Killer T (iNKT) cells are innate-like T Lymphocytes expressing a conserved semi-invariant T cell receptor (TCR) specific for self or ...

CD Marker Recognition of Human Cytokine-Induced Killer Cells by Flow Cytometry

Source: Hsiao, C. H., et al. <a href="https://app.jove.com/v/60420">Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment.</a> J. Vis. Exp. (2020)
In this video, we demonstrate a flow cytometry-based technique to determine the proportion of cytokine-induced killer T-cells, CIK cells, expanded from a peripheral blood mononuclear cell culture.

Source: Hsiao, C. H., et al. Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment. J. Vis. Exp. (2020)
In this video, ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Assays and Functional Profiling

An In Vitro Assay to Study the Cytotoxic Capability of Cytokine-Induced Killer Cells

Source: Hsiao, C. H., et al. <a href="https://app.jove.com/v/60420">Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment.</a> J. Vis. Exp. (2020).
This video demonstrates an in vitro technique to measure the cytotoxic ability of cytokine-induced killer (CIK) cells against cancer cells. Upon co-culturing target cancer cells with CIK cells, the dead cancer cells are identified using a fluorescent viability dye and are quantified using flow cytometry, estimating the cytotoxic effect of CIK cells.

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

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

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