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 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 & T)-based flow cytometry system.
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, γδ 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−), NK-T cells (CD3+CD56+), and NK cells (CD3–CD56+). Optimization of the CIK induction protocol by use of a fixed schedule for the addition of IFN-γ, 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 x 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-γ, IL-1α, 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.
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
2. PBMC isolation
3. CIK induction and expansion
4. Immunophenotyping for assessment of CIK cells
5. CD marker recognition
6. Culturing and staining of human chronic myeloid leukemia K562 cells and ovarian cancer OC-3 cells
7. Cytotoxic assay
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+ T cells from healthy donors is illustrated in Figure 1B. Figure 1C shows the statistical analysis of the CIK proportion from three individuals.
Figure 2A shows that the CD3+CD56+ cell proportion (0.65% for the original PBMC, left lower panel and 27.4% for the CIK cells harvested on Day 14th, right lower panel) significantly increased after 14 days of expansion. In our culture system, the CIK cells yielded about half a hundred-fold changes compared to the original number of PBMCs (Figure 2B).
Figure 3 shows the cytotoxic effect of CIK against human chronic myeloid leukemia K562 cells and human ovarian cancer OC-3 cells. K562 or OC-3 cells (target, T) were stained with a non-fluorescent dye (CFSE), which was cleaved by intracellular esterases within viable cells and then became a highly fluorescent dye. In the cytotoxic coculture study, CFSE-stained K562 or OC-3 cells were cotreated with CIK cells for 24 h. At the end of incubation, the total cells were harvested and stained with 7-AAD dye, which is a nucleic acid-binding dye that is used as a viability probe for dead cell exclusion. The size and granularity of the CIK and CFSE+ cells are illustrated in Figure 3A. The CFSE-stained K562 cells (target cells, T) were co-treated with CIK cells (effector cells, E) at a ratio of E/T = 0:1, 5:1, and 10:1, respectively. The 7-AAD+ cells of CFSE+ K562 cells were all evaluated. The statistical results were from three independent experiments. Basal lysis means the percentage of cell death in the absence of effector cells (E:T = 0:1). Figure 3B shows the obvious cytotoxicity of CIK against OC-3 cells (E:T = 10:1) following 24 h of incubation.
Figure 1: Flow chart of cytokine-induced killer cells induction and expansion. (A) PBMCs from consented healthy donors were initially exposed to rhIFN-γ (Day 0), followed by rhIL-2, rhIL-1α, and anti-CD3 mAb (Day 1) every 3 days (Day 4). Subsequently, the medium was refreshed with rhIL-2-containing medium every 3 days and the cells were harvested on Day 14. (B) Morphology of CIK cells during 7 days of induction. The activation and expansion of CIK cells were conducted as described in the protocol. Cells were observed under a light microscope on Days 1, 5, and 7, respectively (magnification = 40x, scale bar = 200 μm). (C) Cell counts were performed weekly. Please click here to view a larger version of this figure.
Figure 2: The proportion of CD3+CD56+ T cells from a representative PBMC sample. (A) Lymphocytes were recognized by specific size and granularity. Selected single cell population for analysis by flow cytometry. (B) Statistical analysis of CIK expansion efficacy from three healthy donors was conducted using a t-test (*, p < 0.01). Please click here to view a larger version of this figure.
Figure 3: Cytotoxic effects of CIK cells against human chronic myeloid leukemia K562 and human ovarian cancer OC-3 cells. (A) Following coculture with the CIK cells for 24 h, K562 target cells were recognized and gated based on the staining of CFSE dye. Quadrant illustration of the total cell population under the selected 7-AAD/CFSE parameter and the cumulative cytotoxicity of CIK cells at the indicated E:T ratio. (B) The cytotoxic effect of CIK cells against OC-3 cells at a E:T = 10:1 ratio. Please click here to view a larger version of this figure.
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 & 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 infusion11. However, the efficacy of CIK induction and expansion exhibits individual differences12,13,14. Moreover, safety is the advantage of the infusion of patient-derived CIK cells for cancer cell therapy. It has been reported that CIK cells exert cytolytic effects on epithelial solid cancer cells mostly in a NKG2D-dependent manner. In hematological cancer cells, blocking NKG2D with a specific antibody significantly inhibits CIK-induced cytotoxicity against NKG2D-low K562 cells; however, this treatment does not have any effect on HL-60 cells lacking NKG2D15. Furthermore, CIK cells exhibit less cell-killing activity against K562 cells as compared to CD8+ CIK cells16. In this study, we found that CIK exhibited a greater cytotoxic potential against ovarian cancer OC-3 cells compared to leukemia K562 cells. These data suggest that the exact molecular mechanisms through which CIK effectors kill tumor cells are not yet clear.
Tracking target cell viability and evaluating the cytotoxic potential of effector cells using flow cytometry has become a standard and conventional method for clinical examination17. It is has been suggested that a negative effect is observed on the cell viability and the expression of activation markers, such as the CD3+ population in CFSE-stained lymphocytes with flow cytometry18,19. Thus, staining the target cancer cells is a more effective strategy for evaluating the cytotoxic effects of primary CIK cells. IFN-γ, OKT3, and IL-2 are major cytokines or stimulators for CIK differentiation and proliferation. Furthermore, other factors such as thymoglobulin, IL-1α, IL-10, IL-15, are also stimulators. Currently, human serum, human platelet-rich plasma, and even fetal bovine serum are used as medium supplements that can enhance the proliferation of CIK cells. Although serum or plasma are enriched with nutrients and growth factors, the addition of allogeneic animal products presents source, batch, and lot variations that result in experimental variability, and inevitably disconcert studies with therapeutic outcomes for cultured cells. In this study, we used a commercially available serum-free, albumin-free, and xeno-free GMP-grade media supplemented with clinical-grade cytokines to successfully culture the CIK cells. The disadvantage of using xeno-free or allogeneic-free supplements is that they reduce the efficacy of cell proliferation.
The two-color cell tracking methods provided in the protocols independently calculated viable or dead effector target cells in a direct cytotoxicity assay. In our gating strategies, CFSE+ target cells can be obviously distinguished from CIK effector cells (Figure 3). Most importantly, the process of CIK induction and expansion must be qualified and show high viability. For further multiple-dosage infusions, the condition of cryopreservation, and the viability and the cytotoxicity after thawing, are other critical challenges. The actual ratio of specific lysis equals the proportion of 100 x (%Sample lysis – %Basal lysis)/(100 – %Basal lysis). In contrast to other studies18,20, it is recommended that all target cells be investigated to reveal the exact and actual cytotoxicity of CIK cells.
In conclusion, the protocol described in this study is designed to increase the number of PBMC-derived CIK cells from healthy donors and to evaluate their cytotoxic functions against cancer cells with two-color photoactivatable probes for selective tracking of target cells by a flow cytometry with an in vitro diagnostic (IVD) system.
The authors have nothing to disclose.
This study was supported by China Medical University Hospital (DMR-Cell-1809).
7-Amino Actinomycin D | BD | 559925 | ||
APC Mouse Anti-Human CD56 antibody | BD | 555518 | B159 | |
APC Mouse IgG1, κ Isotype Control | BD | 555751 | MOPC-21 | |
BD FACSCanto II Flow Cytometer | BD | 338962 | SN: R33896202856 | |
Carboxyfluorescein diacetate succinimidyl ester (CFSE) | BD | 565082 | Reconstritution of CFSE dye (500 mg) with 90 mL of DMSO | |
D-(+)-Glucose solution | SIGMA | G8644 | For K562 cell culture. Add 12.5 mL to 500 mL of complete medium | |
Dulbecco's Modified Eagle Medium/F12 | HyClone | SH30023.02 | Basal medium for OC-3 cell culture | |
Fetal bovine serum | HyClone | SH30084.03 | For K562 and OC-3 cell culture. Complete medium contains 10% of FBS | |
Ficoll-Paque Plus | GE Healthcare Life Sciences | 71101700-EK | Density gradient solution | |
FITC Mouse Anti-Human CD3 antibody | BD | 555332 | UCHT1 | |
FITC Mouse IgG1, κ Isotype Control | BD | 555748 | MOPC-21 | |
Human anti-CD3 mAb | TaKaRa | T210 | OKT3 | Add 2.5 mL of stock (1 mg/1 mL) to 50 mL of Induction medium. Storage stock at -80 °C |
Penicillin-Streptomycin | Gibco | 15140122 | Add 5 mL of stock (10,000 U/mL) to 500 mL of complete medium. Storage stock at 4 °C. | |
Proleukin | NOVARTIS | Reconstitution of Proleukin Powder (22×106 IU) with 1.2 mL of sterile water and add 2.7 mL to 50 mL of Induction medium. Storage stock at -20 °C | ||
Recombinant Human Interferon-gamma | CellGenix | 1425-050 | Reconstitution of rh IFN-g (5×105 IU/50 µg) with 200 µL of sterile water and add 20 mL to 50 mL of Induction medium. Storage stock at -20 °C | |
Recombinant Human Interleukin-1 alpha | PEPROTECH | 200-01A | Reconstitution rh IL-1α (10 µg) with 1 mL of sterile water and add 5 mL to 50 mL of Induction medium. Storage stock at -20 °C | |
RPMI1640 medium | Gibco | 11875-085 | Basal medium for K562 cell culture. Storage stock at 4 °C | |
Sigma 3-18K Centrifuge | Sigma | 10295 | ||
TrypLE Express Enzyme | Gibco | 12605028 | Cell dissociation enzyme; For deattachment of adheren cells. Storage at room temperature | |
X-VIVO 15 medium | Lonza | 04-418Q | Basal medium for PBMC and CIK cells. Storage at 4 °C |