Cytokine induced killer cell immunotherapy in cancer treatment: from bench to bedside Cytokine induced killer cell immunotherapy in cancer treatment: from bench to bedside

— Cytokine-induced killer (CIK) cells are T effector cells generated by monocytes cultured and stimulated by cytokines. CIK cells were studied for more than 20 years ago. They can cause lysis of tumor cells that of both autologous and allogeneic origins, so that they were used in cancer treatment. This review aimed to summarize ad-vancements of CIK cells and their current clinical applications in cancer treatment. In general, CIK cells were widely clinically used for recent 5 years. They gave promising results in hepatocellular carcinoma, lung cancer, breast cancer, renal cancer, and treatment. Looking into the future, CIK cell based immunotherapy will become an important tool in cancer treatment.


INTRODUCTION
Adoptive cell therapy of cancer demonstrated in mice more than 50 years ago (Mitchison, 1955). During 50 years, some adoptive cell therapies were developed with many bright results in both pre-clinic and clinic. To date, more than 10.000 patients with cancer were treated by adoptive cell therapies. Most of them used CIK cell therapy and dendritic cell (DC) therapy. Different to DCs that cause tumor antigenspecific immune responses, CIK cell therapy can create nonantigen specific immune responses. CIK cells possess non-MHC-restricted cytolytic activities against to cancer cells. From some particular mechanisms of cancer cell recognition, CIK cells can detect and attack cancer cells. This review summarizes some characteristics of CIK cells and an update of clinical applications of CIK cells in cancer treatment.

CIK CELL PHENOTYPES
CIK cells are a heterogeneous population of T lymphocytes with NK phenotypes and functional properties. This cell population was named "cytokine induced killer" cells because they were generated under effects of cytokines. These CIK cells can cause toxic malignant cells followed the manner of MHC-unrestricted cytotoxicity. CIK cells were pro-duced by incubation of peripheral blood monocytes in medium plus with INF-gamma, mAb anti-CD3 and IL-2 (Schmidt-Wolf et al., 1991). After 2-3 weeks of culture, CIK cells are expanded in vitro from few to more than 1000 fold (Marin et al., 2006;Pals et al., 2007;Thorne et al., 2006).
In vitro, CIK cells easily were produced by culture of peripheral blood mononuclear cells in medium plus with INFgamma on day 0, and anti-CD3 OKT3 (50 ng/ml) and IL-2 (500 IU/mL) on the next day, followed by the addition of IL-2 during the culture. INF-gamma was used to increase the cytotoxicity of CIK cells, while anti-CD3 acted as a mitogenic agent on T cells. The cocktail with INF-gamma, anti-CD3 and IL-2 holds an important role that triggers the T cell expansion and increases the T cell cytotoxicity. So that by the in vitro cell culture, CIK cells were expanded with a large amount and high activity of T cells and NK cells. Some studies showed that CIK cells produced IL-2, IL-7 and IL-12. So that to produce CIK cells, besides IL-2 addition, some authors used IL-7 and IL-12 also could produce high antitumor activity CIK cells (Farag et al., 2002;Huang et al., 2006). To date, almost studies used CIK cells were induced from peripheral blood, but CIK cells also can be produced from bone marrow and cord blood (Alvarnas et al., 2001;Introna et al., 2006).

MECHANISM OF ANTI-TUMOR ACTIVITY
The mechanism of anti-tumor activity of CIK cells has not completely clarified. However, some pathways related to this activity were identified. It is showed that CIK cells significantly inhibited the anti-tumor activity when they were blocked lymphocyte function associated antigen -1 (LFA-1) and cell adhesion molecule -1 (ICAM-1) (Schmidt-Wolf et al., 1996;Schmidt-Wolf et al., 1993). In fact, CIK cells were treated with dibutyryl (db)-cAMP that prevents the conversion of LFA-11 into a high affinity receptor for ICAM-1, were inhibited to release perforin and granzyme (Mehta et al., 1995).
Another mechanism also was detected about tumor recognition of CIK cells that similar in NK cells. CIK cells recognize the tumor antigen via NKG2D. NKG2D (Natural killer group 2 member D) is one member of the c-type lectinactivating receptor family. NKG2D is expressed on all NK cells. Ligands for NKG2D were restrictedly expressed in malignant cells (Diefenbach et al., 2000;Jamieson et al., 2002). When induced with IFN-gamma, IL-2 and anti-CD3, NKG2D was highly expressed in CIK cells. Interaction between NKG2D and tumor antigen that expressed in tumor cells will cause the cytolytic effect by perforin and granzyme release of CIK cells.
Besides the recognition of CIK cells by NKG2D, CIK cells also specifically attack tumor cells by another mechanism. Recent studies showed that co-culture of CIK cells and induced DCs that can present tumor antigen can enhance the CIK cells to attack tumor cells Nagaraj et al., 2004;Ziske et al., 2001).

CLINICAL TRIALS
CIK cells were used in clinical trials for some diseases ( Table  1, 2). Particularly, CIK cell therapy in combination with some conventional therapies or dendritic cells was studied in Phase III of lung cancer, in Phase IV of hepatocellular carcinoma. Moreover, CIK cell therapy also used in clinical trials for other cancers such as ovarian cancer, colorectal cancer, renal cancer, leukemia, breast cancer… In HCC, to increase the efficacy of trans-catheter arterial chemoembolization (TACE) on HCC treatment, Hao et al. (2010) combined TACE and CIK cells. The results showed that the efficacy of TACE was significantly improved. Wang et al. (2013) showed that the disease control rate was 67.7% in patients transfused with CIK cells (Wang et al., 2013). Similarly, in HCC patients, Huang et al. (2013) showed that CIK cell immunotherapy was valuable therapeutic strategy to prevent recurrence and metastasis in HCC patients after TACE and RFA (Huang et al., 2013). Effects of CIK cell transfusion caused a significant increased of CD3+, CD4+, CD4+CD8+ and CD3+CD4+ T cells , improved the immunological status in HCC patients (Shi et al., 2004), reduce the level of serum AFP and anti-HBV and decrease the 1-year recurrence rate of patients with HCC after curative TACE plus RFA (Pan et al., 2010).
CIK cells represent a promising immunotherapy for the treatment of gastrointestinal tumors (Jakel et al., 2014). Adjuvant transfusion of CIK cells prolongs DFS in patients with colorectal cancer from 29.35 ± 6.39 % in control without CIK cells to 59.65 ± 24.80 % with CIK transfusion, and no immediate adverse reactions to the CIK cell transfusions (Zhu et al., 2013). DC/CIK therapy also reduced the risk of postoperative disease progression with an increased OS in gastric and colorectal cancer patients (Gao et al., 2014). DC-CIK cell therapy markedly prolongs survival time enhances immune function and improves the efficacy of the treatment of breast cancer patients. In treated patients, the percentage of T cells (CD3 + , CD4 + and CD4 + CD8 + ), CD16 + monocytes, and CD3 + CD56 + natural killer T cells, the levels of interleukin-2, interleukin-12, tumor necrosis factorα, interferon-γ, and nucleolar organizer region protein in the peripheral blood of cancer patients was significantly increased (Wang et al., 2014b). DC-CIK was feasible and effective in treating advanced renal cancer (Kim et al., 2014;Su et al., 2010). Wang et al. showed an objective response rate (ORR) of 39% and a disease control rate (DCR) of as 75%. No clinically significant side effects were observed (Wang et al., 2014a). In another study, Zhan et al. combined CIK cells with DCs and showed that TL-pulsed DC-CIK cells could prevent recurrence/metastasis and increase the overall survival rate after surgery in localized or locally advanced renal carcino-ma with the overall survival rates significantly higher in the DC-CIK group and IFN-α group than that in the control group (Zhan et al., 2012).
CIK cells also were successfully used in ovarian cancer treatment.  transfused CIK cells in a clinical with 94 patients with IIB-IV grade ovarian cancer. They showed that a median PFS were 37.7 months in the treatment group and 22.2 months in the control group. After 2 courses of CIK cells transfusion, the proportion of CD4CD25CD127 regular T cells in the peripheral blood significantly decreased .
In lung cancer, CIK cells have important effects on antitumor in both early-stage disease (I-IIIA)  and advanced stage disease (IIIB-V) Shi et al., 2012;Zhong et al., 2011). By the research with 87 paired patients, Li et al. (2012) suggested that CIK cell immunotherapy could improve the efficacy of conventional chemotherapy in NSCLC patients, and increased frequency of CIK cell treatment could further enhance the beneficial effects. Yang et al. (2013) also confirmed that CIK cells plus DC could increased the 1-and 2-year overall survival rates up to 57.2 compared to 27.0 % in control (Yang et al., 2013).
Recent studies used CIK cells to target cancer stem cells (CSCs). Gammaitoni et al (2013) demonstrated that CIK tumor killing activity against melanoma CSCs was intense and comparable with results reported against differentiated metastatic melanoma cells. CIK cell transplantation resulted in delayed tumor growth, increased necrotic areas, and lymphocyte infiltration at tumor sites (Gammaitoni et al., 2013).

CONCLUSION
Immune therapies are promising therapies for cancer treatment. CIK cells hold high anti-tumor activity in MHC unrestricted manner. These cells are easily produced with a large amount from peripheral blood by cultured peripheral blood mononuclear cells with INF-gamma, IL-2 and anti-CD3. CIK cells have used to treat several cancers with good results. Especially, CIK cells based therapy clinically studied in phase IV in hepatocellular carcinoma, and phase III in lung cancer. From these results, it hopes that CIK cell therapy rapidly pushes into routine application in lung cancer or hepatocellular carcinoma in the near future.

Competing interests
The authors declare that they have no competing interests.

Open Access
This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0) which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. Mononuclear cells were collected aseptically with blood cell separator composition apheresis 3 days before concurrent chemoradiation, and cultured DC-CIK cells for 10 days. Cells were infused back to the patients in 3 times between the Chemoradiation intermittent period.