Cytokine-induced killer (CIK) cells are a cluster of heterogeneous cells uniting a T cell and natural killer cell‐like phenotype in their terminally differentiated CD3+CD56+ subset, and exert anti-tumor activity in a non-MHC restricted manner. CIK cells are expanded ex vivo with the sequential addition of multiple cytokines, including interferon‐γ, monoclonal antibodies against CD3 and interleukin‐2.
CIK cells represent a heterogeneous cell population comprising CD3+CD56- cells, CD3-CD56+ cells and CD3+CD56+ cells. Schmidt-Wolf, et al. first published the protocol for CIK cells generation in 1991 and their remarkable antitumor activity and minimal toxicity were also reported simultaneously . CIK cells can be easily amplified ex vivo from peripheral blood mononuclear cells (PBMCs) with sequential addition of interferon-γ (IFN-γ) 1000 IU/ml on day 0, and 50 ng/ml monoclonal antibody against CD3 (anti-CD3 mAb), 100 IU/ml interleukin-1β (IL-1β) and 600 IU/ml interleukin-2 (IL-2) on the next day . IL-2 and fresh culture medium need to be supplemented regularly until after 2-3 weeks. IFN-γ priming 24h before the mitogenic stimulation anti-CD3 mAb and IL-2 is crucial for the cytotoxic activity of CIK cells, as IFN-γ increases the activation of IL-2-responding cells and activates monocytes to regulate the immunomodulatory factor IL-12. Studies also demonstrated that CIK cells stimulated by or transfected with IL‐6, IL‐7, IL‐12, IL‐15, IL-21 or thymoglobulin manifested phenotype alteration, proliferation improvement and cytotoxicity enhancement .
It is reported that CD3+ cells accounted for >90% of CIK cells and the amount of the main effector cells CD3+CD56+ cells ranged between 7.6% and 65% with a median of 35.3% . It is interesting to find that the proportion of CD3+CD8+ subset increased faster than its CD3+CD56+ counterpart after IL-2 and anti-CD3 mAb stimulation as the latter kept low proliferation until after day 7. The CD3+CD56+ subset exerts its anticancer activity in a major histocompatibility complex (MHC)‐unrestricted manner with higher portion of CD8+ cells and granzyme content. This double positive population shows a more terminally differentiated effector phenotype CD27+CD28- or CD27-CD28- than its CD3+CD56- precursors .
Besides polyclonal T-cell receptor (TCR) repertoire, CIK cells also express NK-like structures, including natural killer group 2 member D (NKG2D), DNAX accessory molecule-1 (DNAM-1) and low densities of NKp30. But lack expression of NK-specific activating receptors (NKp44, NKp46) and inhibitory receptors (KIR2DL1, KIR2DL2, KIR3DL1, NKG2A, CD94) . A transcriptomic study found that CD8 and Lck kinase, a member of the Src kinase family, contributed largely to the cytotoxicity of CIK cells . When comparing the mature CIK cells with the ones in the early stage, the expression of tumor cytotoxic molecules, including perforin, granzyme, Fas ligand (FasL) and CD40 ligand (CD40L) expectedly increased, while immune checkpoints PD-1, CD28, CD137, and VSIR were down-regulated with the upregulation of LAG3, CTLA4, and TIM3 .
Researchers are still exploring the decisive molecules and pathways involved in CIK cell cytotoxicity. One key mechanism is the engagement of NKG2D. NKG2D is a member of the c-type lectin-activating receptor family that originally identified in NK cells but also expressed by activated CD8+ T cells, γδT cells and NK1.1+ T cells . The interaction between NKG2D and its ligand MHC class I-related molecules A and B (MIC A/B) and members of the UL16-binding protein family (ULBP1-4) , whose expression are relatively restricted to malignancies, accounts for the majority of the MHC-independent cytotoxicity of CIK cells . Besides NKG2D, CIK cells can also exert their antitumor activity in a TCR-mediated lytic manner, which is the so called “dual-functional capability” . Both pathways above could be blocked concurrently by the antibodies against lymphocyte function-associated antigen (LFA-1) and DNAM-1. Moreover, NKp30 which usually expressed on NK cells but also on CIK cells with a low density was indicated to take part in CIK cell-mediated tumor eradication. Some groups also reported that the CD3+CD56+CD16+ subset demonstrated dramatic antitumor response after therapeutic monoclonal antibodies, such as trastuzumab or cetuximab administration via the antibody-dependent cell-mediated cytotoxicity (ADCC) with the expression of FcyRIII receptor (CD16) in a donor-dependent manner .
Novel combinational immunotherapies based on CIK cells are emerging so as to achieve higher efficacy and specific recognition against malignancies. CIK cells in combination with dendritic cells have been shown to improve the antitumor effectiveness for cancer preclinically and clinically as DCs possess high ability of antigen recognition and presentation . For the purpose of reversing the inhibition of T lymphocytes signaling in tumor microenvironment, blockades of immune checkpoints including PD-1, PD-L1, KIR, LAG-3 or TIM-3 were adopted and suggested to increase the antitumor potency of CIK cells against myeloid leukemia, gastrointestinal cancer and advanced non-small cell lung cancer (NSCLC) . Apart from monoclonal antibodies against CD20 (rituximab or GA101) and CD30 (Brentuximab Vedotin), some studies revealed that bispecific antibodies (BsAbs) in combination with CIK cells exerted augmented antitumor efficacy . With the ability of recognizing two distinctive epitopes, BsAbs redirect CIK cells to tumor cells and strengthen their cytotoxicity in B cell lymphoma by CD19/CD5 BsAb, gastric cancer by EGFR/CD3 BsAb or CD133high cancer cells by CD3/CD133 BsAb . With the development of gene editing technology and clinical success achieved by second-generation chimeric antigen receptor (CAR) T cells targeting CD19, genetically engineered CIK cells to express CAR targeting tumor associated antigens (TAAs) have become one of the favorable immunotherapeutic strategies . CIK cells with engineered CAR targeting TAAs including carcinoembryonic antigen (CEA), CD123 and CD33 have demonstrated exciting results with significantly elevated killing activity of the modified CIK cells .
Since the very first report describing the generation protocol and promising antitumor efficacy on B cell lymphoma by CIK cells in 1991, rapidly updated technologies and expanding clinical practice have been increasing our understanding about how to optimize the therapeutic regimen based on CIK cells to gain durable responses and ideal treatment effectiveness in patients . In order to collect exhaustive information of clinical trials worldwide and standardize the CIK cell-based treatment system, the IRCC was established in 2010 and three reports have been published to provide an overview of the CIK cell therapy state . New trials based on CIK cells can be registered on the homepage: http://www.ciobonn.de/cik/.