Selenium alleviates lead-induced CIK cells pyroptosis and inflammation through IRAK1/TAK1/IKK pathway.

The toxic heavy metal lead is widely found in rivers and soils as an environmental pollutant, posing a threat to the health of aquatic organisms. Selenium is an essential trace element and a powerful antioxidant that has been shown to have anti-inflammatory and antioxidant properties as well as alleviating heavy metal poisoning. Many studies have shown that lead poisoning produces inflammatory responses and damage to the kidneys of a wide range of animals, but the effects on cellular pyroptosis and immune function and selenium antagonism in CIK cells are not clear. In this study, 500 µM Pb and 20 nM Se were applied to grass carp kidney cells, and the results showed that Pb exposure to CIK cells resulted in oxidative stress, activation of the IRAK1/TAK1/IKK pathway, up-regulation of the expression of cellular pyroptosis markers GSDMD and NLRP3, and cellular pyroptosis of CIK cells, as well as up-regulation of IL-1ß and IL-18, and the generation of cellular inflammatory response. In contrast, Se treatment significantly reduced the ROS level, the expression of cellular pyroptosis markers GSDMD, NLRP3 and inflammatory element IL-1ß and IL-18. Taken together, Se alleviated cellular pyroptosis and immune dysfunction caused by Pb exposure through oxidative stress and activation of the IRAK1/TAK1/IKK pathway. This study complements the harmful effects of the heavy metal Pb on fish and the real-life application of selenium in the healthy culture of fish as a reference will be provided.

Cytokine-Induced Killer Cells in Combination with Heat Shock Protein 90 Inhibitors Functioning via the Fas/FasL Axis Provides Rationale for a Potential Clinical Benefit in Burkitt's lymphoma.

Constant efforts are being made to develop methods for improving cancer immunotherapy, including cytokine-induced killer (CIK) cell therapy. Numerous heat shock protein (HSP) 90 inhibitors have been assessed for antitumor efficacy in preclinical and clinical trials, highlighting their individual prospects for targeted cancer therapy. Therefore, we tested the compatibility of CIK cells with HSP90 inhibitors using Burkitt's lymphoma (BL) cells. Our analysis revealed that CIK cytotoxicity in BL cells was augmented in combination with independent HSP90 inhibitors 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin) and ganetespib. Interestingly, CIK cell cytotoxicity did not diminish after blocking with NKG2D (natural killer group 2, member D), which is a prerequisite for their activation. Subsequent analyses revealed that the increased expression of Fas on the surface of BL cells, which induces caspase 3/7-dependent apoptosis, may account for this effect. Thus, we provide evidence that CIK cells, either alone or in combination with HSP90 inhibitors, target BL cells via the Fas-FasL axis rather than the NKG2D pathway. In the context of clinical relevance, we also found that high expression of HSP90 family genes (HSP90AA1, HSP90AB1, and HSP90B1) was significantly associated with the reduced overall survival of BL patients. In addition to HSP90, genes belonging to the Hsp40, Hsp70, and Hsp110 families have also been found to be clinically significant for BL survival. Taken together, the combinatorial therapy of CIK cells with HSP90 inhibitors has the potential to provide clinical benefits to patients with BL.

Paraquat induces apoptosis, programmed necrosis, and immune dysfunction in CIK cells via the PTEN/PI3K/AKT axis.

Paraquat (PQ) is a highly water-soluble, non-selective herbicide. Due to water pollution and lack of specific medicines, it is extremely harmful to humans and aquatic animals. Oxidative stress and apoptosis can affect the immune function of the body. However, the effects and mechanisms of PQ on the immune function, apoptosis and programmed necrosis on CIK cells are still unclear. Therefore, we constructed low (L, 50 µmol/L), medium (M, 100 µmol/L), and high (H, 150 µmol/L) dose models of PQ exposure on CIK cells. The expression of oxidative stress-related indexes (MDA, CAT, GSH-Px and SOD) and interrelated genes were examined by flow cytometry, qRT-PCR, and western blotting methods. Our data demonstrated that PQ treatment caused an increase in MDA content and the decreases in the activities of antioxidase and antioxidants (SOD, GSH-Px and CAT) on CIK cells (p < 0.05). We also discovered the PTEN/PI3K/AKT pathway was significantly activated in a dose dependent manner (p < 0.05). Furthermore, the proportion of programmed necrosis cells increased dramatically at PQ doses from 0 µmol/L to 150 µmol/L. Apoptosis and necrosis-related genes also showed dose-dependent changes (p < 0.05). Briefly, PQ exposure leads to apoptosis and programmed necrosis via the oxidative stress and PTEN/PI3K/AKT pathway, thereby causing immune dysfunction of CIK cells. This study enriches the toxic influences of PQ on the cells of aquatic organisms and provides a reference for comparative medicine.

A Phase IB Trial of Autologous Cytokine-Induced Killer Cells in Combination with Sintilimab, Monoclonal Antibody Against Programmed Cell Death-1, plus Chemotherapy in Patients with Advanced Non-Small-Cell Lung Cancer.

INTRODUCTION: Can the Cytokine-induced killer (CIK) cells in combination with immune checkpoint inhibitor further improve the efficacy of chemotherapy in non-small cell lung cancer (NSCLC) patients? What are the adverse reactions of this combination therapy? But these problems are not clear. Therefore, we conducted a phase 1b trial to evaluate the safety and efficacy of autologous CIK cells therapy combined with Sintilimab, antibody against programmed cell death-1, plus chemotherapy in untreated, advanced NSCLC patients. PATIENTS AND METHODS: Patients with stage IIIB/IIIC/IV NSCLC received Sintilimab, platinum-based doublet chemotherapy, and CIK cells every 3 weeks for 4 cycles, then maintenance treatment with Sintilimab in squamous and with Sintilimab plus pemetrexed in non-squamous NSCLC until disease progression or unacceptable toxicity or 2 years. The primary endpoints were safety and objective response rate (ORR). RESULTS: Thirty-four patients received the treatment. 94.1% of patients experienced treatment-related adverse events (TRAEs). Grade 3 or greater TRAEs occurred in 64.7% of patients. One (2.9%) patient died of grade 5 immune-related pneumonia. The ORR and DCR were 82.4% (95% CI, 65.5%-93.2%) and 100.0% (95% CI, 89.7%-100.0%), respectively. Objective responses were evaluated in 14 of 15 non-squamous patients (93.3%; 95% CI, 68.1%-99.8%) and in 14 of 19 squamous patients (73.7%; 95% CI, 48.8%-90.9%). Median PFS was 19.3 months (95% CI, 8.3 months to not available). CONCLUSION: Autologous CIK cells immunotherapy in combination with Sintilimab plus chemotherapy was well tolerable and showed encouraging efficacy in patients with previously untreated, advanced NSCLC (ClinicalTrials.gov number, NCT03987867).

Xanthan gum enhances peripheral blood CIK cells cytotoxicity in serum-free medium.

As a water-soluble macromolecule polysaccharide, xanthan gum (XG) has several biological activities, such as antitumor, antiviral, and immunomodulatory function. However, the effect of XG on the proliferation and cytotoxicity of cytokines induced killer (CIK) cells is rarely studied. In this study, the effect of XG on CIK cells derived from peripheral blood was investigated by analyzing the expansion fold of total cells, phenotype, cytotoxicity, degranulation, and apoptosis in serum-free medium. The results showed that the expansion fold of total cells with 100 µg/ml XG which molecule weight is 2.95 × 106 Da reached 4534.0 folds, significantly higher than that without XG (1299.0 folds, p < 0.05). The percentage of main effector cells-CD3+ CD56+ cells increased to 25.5% and the cytotoxic activity of CIK cells increased to 45.3%. The cell proportions of expression granzyme B and perforin that related to cytotoxicity in CIK cells reached 53.6% and 48.3%, respectively, significantly higher than 27.5% and 37.5% in the group without XG (p < 0.05). Collectively, XG could stimulate the ex vivo expansion of CIK cells and enhance the cytotoxicity of expanded CIK cells. The above results provide technical support for optimizing the expansion process of CIK cells ex vivo.

CCL17 and CCL22 induce CCR4 receptor expression and promote cytokine-induced killer cells migration.

Recently, cytokine-induced killer (CIK) cells have been shown to possess effective cytotoxic activity against some tumor cells both in vitro and in clinical research. Furthermore, dendritic cell-activated CIK (DC-CIK) cells display significantly increased antitumor activity compared to unstimulated CIK cells. Study findings indicate DC cells can secrete chemokine C-C motif ligand 17 (CCL17) and chemokine C-C motif ligand 22 (CCL22) with a common receptor molecule, C-C chemokine receptor type-4(CCR4). CCL17 and CCL22 levels were measured by ELISA from CIK cell culture supernatants and the expression of CCR4 on CIK and DC-CIK cells was analyzed by flow cytometry. Through Migration and Killing assays, further analyzed the effects of the altered expression levels of CCR4 on the chemotactic ability and the tumor-killing efficiency of CIK cells. We found markedly increased CCL17 and CCL22 in supernatants of DC-CIK co-cultures. Similarly, the expression of CCR4 was also increased on CIK cells in these co-cultures. Further, the stimulation of CCL17 and CCL22 increased expression of the CCR4 and enhanced the migratory capacity and antitumor efficacy of CIK cells. Simultaneously, similar effects had achieved by transfecting the CCR4 gene into CIK cells. DC cells may promote the expression of CCR4 on CIK cells by secreting CCL17 and CCL22, thereby promoting infiltration of DC-CIK cells into the tumor microenvironment, and exerting stronger antitumor activity than CIK cells.

30 years of CIK cell therapy: recapitulating the key breakthroughs and future perspective.

Emerging evidence from the numerous clinical trials involving cytokine-induced killer (CIK) cell therapy suggests that its optimization in combination with other contemporary cancer therapies in a complementary manner (rather than as competition) will be a key to combat cancer.

NKG2D Engagement Alone Is Sufficient to Activate Cytokine-Induced Killer Cells While 2B4 Only Provides Limited Coactivation.

Cytokine-induced killer (CIK) cells are an ex vivo expanded heterogeneous cell population with an enriched NK-T phenotype (CD3+CD56+). Due to the convenient and relatively inexpensive expansion capability, together with low incidence of graft versus host disease (GVHD) in allogeneic cancer patients, CIK cells are a promising candidate for immunotherapy. It is well known that natural killer group 2D (NKG2D) plays an important role in CIK cell-mediated antitumor activity; however, it remains unclear whether its engagement alone is sufficient or if it requires additional co-stimulatory signals to activate the CIK cells. Likewise, the role of 2B4 has not yet been identified in CIK cells. Herein, we investigated the individual and cumulative contribution of NKG2D and 2B4 in the activation of CIK cells. Our analysis suggests that (a) NKG2D (not 2B4) is implicated in CIK cell (especially CD3+CD56+ subset)-mediated cytotoxicity, IFN-γ secretion, E/T conjugate formation, and degranulation; (b) NKG2D alone is adequate enough to induce degranulation, IFN-γ secretion, and LFA-1 activation in CIK cells, while 2B4 only provides limited synergy with NKG2D (e.g., in LFA-1 activation); and (c) NKG2D was unable to costimulate CD3. Collectively, we conclude that NKG2D engagement alone suffices to activate CIK cells, thereby strengthening the idea that targeting the NKG2D axis is a promising approach to improve CIK cell therapy for cancer patients. Furthermore, CIK cells exhibit similarities to classical invariant natural killer (iNKT) cells with deficiencies in 2B4 stimulation and in the costimulation of CD3 with NKG2D. In addition, based on the current data, the divergence in receptor function between CIK cells and NK (or T) cells can be assumed, pointing to the possibility that molecular modifications (e.g., using chimeric antigen receptor technology) on CIK cells may need to be customized and optimized to maximize their functional potential.

Innovative therapeutic strategy for B-cell malignancies that combines obinutuzumab and cytokine-induced killer cells.

BACKGROUND: Patients affected by aggressive B-cell malignancies who are resistant to primary or salvage chemoimmunotherapy have an extremely poor prognosis and limited therapeutic options. Promising therapeutic success has been achieved with the infusion of CD19 chimeric antigen receptor-T cells, but several limits still restrain the administration to a limited proportion of patients. This unmet clinical need might be fulfilled by an adoptive immunotherapy approach that combines cytokine-induced killer (CIK) cells and monoclonal antibodies (mAb) to the CD20 antigen. Indeed, CIK cells are an effector population endowed with antitumor activity, which can be further improved and antigen-specifically redirected by clinical-grade mAb triggering antibody-dependent cell-mediated cytotoxicity. METHODS: CIK cells were generated from peripheral blood of patients affected by different B-cell malignancies using a blinatumomab-based cell culture protocol. Effector cells were combined with the anti-CD20 mAb obinutuzumab and their therapeutic activity was assessed both in vitro and in vivo. RESULTS: CIK cells were successfully expanded in clinically relevant numbers, starting from small volumes of peripheral blood with extremely low CD3+ counts and high tumor burden. This relied on the addition of blinatumumab in culture, which leads to the simultaneous expansion of effector cells and the complete elimination of the neoplastic component. Moreover, CIK cells were highly cytotoxic in vitro against both B-cell tumor cell lines and autologous neoplastic targets, and had a significant therapeutic efficacy against a B-cell malignancy patient-derived xenograft on in vivo transfer. CONCLUSIONS: The combination of an easily expandable CIK cell effector population with a mAb already in clinical use establishes a tumor antigen-specific redirection strategy that can be rapidly translated into clinical practice, providing an effective therapeutic alternative for B-cell malignancies without any need for genetic modifications. Additionally, the approach can be potentially applied to an extremely vast array of different tumors by simply substituting the targeting mAb.

Effect of RFA and TACE combined with postoperative cytokine-induced killer cell immunotherapy in primary hepatocellular carcinoma.

PURPOSE: To evaluate the efficacy and safety of radiofrequency ablation (RFA) and transcatheter arterial chemoembolization (TACE) combined with postoperative cytokine-induced killer (CIK) cell immunotherapy in the treatment of primary hepatocellular carcinoma (HCC). METHODS: The clinical data of 116 patients with primary HCC treated in our hospital from March 2016 to January 2018 were collected. 58 patients were treated with RFA+TACE (RFA+TACE group), and the other 58 patients underwent RFA+TACE+CIK cell immunotherapy (RFA+TACE+CIK group). Before and after treatment, the proportions of cluster of differentiation 3+ (CD3+), CD3+CD4+, and CD3+CD8+ T cells, regulatory T cells (Tregs) and natural killer (NK) cells and the CD4+/CD8+ ratio were detected via flow cytometry, and the levels of serum interferon-γ (IFN-γ), interleukin-2 (IL-2) and IL-6 were detected via enzyme-linked immunosorbent assay (ELISA). The incidence of adverse reactions and the quality of life score of patients after treatment were compared between the two groups, and the patient's survival status was recorded through follow-up. RESULTS: After treatment, the levels of CD3+ T cells, CD3+CD4+ T cells, CD4+/CD8+ ratio, Tregs and NK cells were significantly higher, while the level of CD3+CD8+ T cells was significantly lower in RFA+TACE+CIK group than those in RFA+TACE group. After treatment, the level of alpha fetoprotein (AFP) obviously declined in both groups compared with that before treatment, and it was significantly lower in RFA+TACE+CIK group than that in RFA+TACE group. After treatment, the scores of the QLQ-C30 questionnaire were all significantly higher in RFA+TACE+CIK group than those in RFA+TACE group. After treatment, the general functioning score rose from (58.55±11.82) and (59.39±10.97) points to (74.74±15.58) and (68.42±14.85) points, respectively, in RFA+TACE+CIK group and RFA+TACE group, and it was significantly higher in RFA+TACE+CIK group than that in RFA+TACE group. According to the follow-up results, the mean overall survival (OS) of patients was (42.1±5.6) months and (37.8±4.8) months, and the 5-year OS rate was 29.3% (17/58) and 13.8% (8/58), respectively, in RFA+TACE+CIK group and RFA+TACE group. The results of log-rank test showed that the OS in RFA+TACE+CIK group was significantly superior to that in RFA+TACE group. CONCLUSIONS: RFA and TACE combined with postoperative autologous CIK cell reinfusion have significant efficacy in the treatment of primary HCC, which can enhance the immune function, improve the postoperative quality of life and raise the survival rate of patients, with tolerable adverse reactions.

Inhibition of glioma by adenovirus KGHV500 encoding anti-p21Ras scFv and carried by cytokine-induced killer cells.

Ras gene mutation or overexpression can lead to tumorigenesis in multiple kinds of cancer, including glioma. However, no drugs targeting Ras or its expression products have been approved for clinical application thus far. Adenoviral gene therapy is a promising method for the treatment of glioma. In this study, the human glioma cell line U251 was co-cultured with recombinant adenovirus KGHV500, and the anti-tumor effects of KGHV500 were determined by MTT, scratch test, Transwell invasion, and apoptosis assays. Then, KGHV500 was delivered via the intravenous injection of CIK cells into glioma xenografts. Tumor volume, ki67 proliferation index, apoptosis levels, and anti-p21Ras scFv expression were tested to evaluate targeting ability, anti-tumor efficacy, and safety. We found that the KGHV500 exhibited anti-tumor activity in U251 cells and increased the intracellular expression of anti-p21Ras scFv compared with that in the control groups. CIK cells delivered KGHV500 to U251 glioma cell xenografts and enhanced anti-tumor activity against glioma xenografts compared to that produced by the control treatment. In conclusion, targeting Ras is a useful therapeutic strategy for gliomas and other Ras-driven cancers, and the delivery of anti-p21Ras scFv by recombinant adenovirus and CIK cells may play an essential role in the therapy of Ras-driven cancers.

Autologous cytokine-induced killer (CIK) cells enhance the clinical response to PD-1 blocking antibodies in patients with advanced non-small cell lung cancer: A preliminary study.

BACKGROUND: Programmed death-1 (PD-1) blocking antibodies have been shown to improve progression-free survival (PFS) and overall survival in a subset of patients with non-small cell lung cancer (NSCLC). However, the objective response rate with these agents remains low, and the vast majority of NSCLC patients require alternative combination treatment regimens to prolong their survival. The purpose of this study was to evaluate the clinical efficacy of autologous cytokine-induced killer (CIK) cell infusions combined with PD-1 blocking antibodies in patients with NSCLC. METHODS: In this preliminary study, we investigated the safety and immune function effectiveness of PD-1 blockade antibodies pembrolizumab or nivolumab administered in combination with or without autologous CIK cell infusions in 18 patients with advanced NSCLC. The peripheral blood mononuclear cells were isolated from these patients and the expression level of some cell surface molecules like PD-1 were detected using flow cytometry to reflect the effectiveness of this combination regimen. RESULTS: No treatment-related deaths occurred in either cohort. In comparison with the pretreatment level, CD3+ CD56+ CD16+ T cells were significantly increased with the combination therapy, while myeloid-derived suppressor cells were significantly increased with PD-1 blocking antibody therapy alone but not with combination therapy. Although the serum interleukin-4 level was downregulated following treatment with the combination regimen, interferon-γ levels were unchanged. CONCLUSIONS: The purpose of this clinical study was to report the clinical efficacy and lack of exacerbated autoimmune adverse events with a combination of PD-1 blockade and CIK cell infusions in patients with advanced NSCLC, further supporting assessments of this combination in future clinical trials.

MiR-374b increases the CIK expression and mediates biological function changes in cervical cancer cells by targeting the PD-1/PD-L1 signaling pathway.

OBJECTIVE: To investigate the role of miR-374b in medicating biological function changes in cervical cancer cells by increasing the cytokine-induced killer (CIK) expression. METHODS: Venous blood of 62 cervical cancer patients and 58 healthy individuals including Human cervical cancer cell line (HeLa) and normal human uterine smooth muscle cells (HUSMC) were tested for expression of miR-374b, PD-1, and PD-L1. sh-PD-1, si-PD-1, NC, miR-374b-inhibitor, and miR-374b-mimics were transfected into HeLa cells, and expression of miR-374b, PD-1, and PD-L1 was determined by a qRT-PCR assay, and the proliferation and apoptosis of the cells were detected using a MTT assay and flow cytometry, respectively. RESULTS: PD-1 was highly expressed in cervical cancer, while miR-374b is lowly expressed in it, and the area-under-the-curve (AUC) of both PD-1 and miR-374b was larger than 0.8. The dual luciferase reporter assay confirmed relationship between PD-1 and miR-374b. Expression of PD-1 in HeLa cells was significantly down-regulated after transfection of miR-374b-mimics. Compared with the CIK + NC group, the CIK + miR-374b-mimics group and the CIK + siRNA-PD-1 group showed a significant decrease in the relative mRNA expression of PD-1, compared with other group showed significantly lowered activity of HeLa cells, and the two groups showed significantly reduced tumor volume. CONCLUSION: MiR-374b increases the CIK expression and mediates biological function changes in cervical cancer cells by targeting the PD-1/PD-L1 signaling pathway, so it is expected to be a potential therapeutic target for cervical cancer.

Increase in Efficacy of Checkpoint Inhibition by Cytokine-Induced-Killer Cells as a Combination Immunotherapy for Renal Cancer.

Cytokine-induced killer (CIK) cells are heterogeneous, major histocompatibility complex (MHC)-unrestricted T lymphocytes that have acquired the expression of several natural killer (NK) cell surface markers following the addition of interferon gamma (IFN-γ), OKT3 and interleukin-2 (IL-2). Treatment with CIK cells demonstrates a practical approach in cancer immunotherapy with limited, if any, graft versus host disease (GvHD) toxicity. CIK cells have been proposed and tested in many clinical trials in cancer patients by autologous, allogeneic or haploidentical administration. The possibility of combining them with specific monoclonal antibodies nivolumab and ipilimumab will further expand the possibility of their clinical utilization. Initially, phenotypic analysis was performed to explore CD3, CD4, CD56, PD-1 and CTLA-4 expression on CIK cells and PD-L1/PD-L2 expression on tumor cells. We further treated CIK cells with nivolumab and ipilimumab and measured the cytotoxicity of CIK cells cocultured to renal carcinoma cell lines, A-498 and Caki-2. We observed a significant decrease in viability of renal cell lines after treating with CIK cells (p < 0.0001) in comparison to untreated renal cell lines and anti-PD-1 or anti-CTLA-4 treatment had no remarkable effect on the viability of tumor cells. Using CCK-8, Precision Count Beads™ and Cell Trace™ violet proliferation assays, we proved significant increased proliferation of CIK cells in the presence of a combination of anti-PD-1 and anti-CTLA-4 antibodies compared to untreated CIK cells. The IFN-γ secretion increased significantly in the presence of A-498 and combinatorial blockade of PD-1 and CTLA-4 compared to nivolumab or ipilimumab monotreatment (p < 0.001). In conclusion, a combination of immune checkpoint inhibition with CIK cells augments cytotoxicity of CIK cells against renal cancer cells.

High Expression of Cannabinoid Receptor 2 on Cytokine-Induced Killer Cells and Multiple Myeloma Cells.

Multiple myeloma (MM) is characterized by aberrant bone marrow plasma cell (PC) proliferation and is one of the most common hematological malignancies. The potential effect of cannabinoids on the immune system and hematological malignancies has been poorly characterized. Cannabidiol (CBD) may be used to treat various diseases. CBD is known to exert immunomodulatory effects through the activation of cannabinoid receptor 2 (CB2), which is expressed in high levels in the hematopoietic system. Cytokine-induced killer (CIK) cells are a heterogeneous population of polyclonal T lymphocytes obtained via ex vivo sequential incubation of peripheral blood mononuclear cells (PBMCs) with interferon-γ (IFN-γ), anti CD3 monoclonal antibody, and IL-2. They are characterized by the expression of CD3+ and CD56+, which are surface markers common to T lymphocytes and natural killer (NK) cells. CIK cells are mainly used in hematological patients who suffer relapse after allogeneic transplantation. Here, we investigated their antitumor effect in combination with pure cannabidiol in KMS-12 MM cells by lactate dehydrogenase LDH cytotoxicity assay, CCK-8 assay, and flow cytometry analysis. The surface and intracellular CB2 expressions on CIK cells and on KMS-12 and U-266 MM cell lines were also detected by flow cytometry. Our findings confirm that the CB2 receptor is highly expressed on CIK cells as well as on MM cells. CBD was able to decrease the viability of tumor cells and can have a protective role for CIK cells. It also inhibits the cytotoxic activity of CIKs against MM at high concentrations, so in view of a clinical perspective, it has to be considered that the lower concentration of 1 µM can be used in combination with CIK cells. Further studies will be required to address the mechanism of CBD modulation of CIK cells in more detail.

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 & T)-based flow cytometry system.

FOXP3 Inhibitory Peptide P60 Increases Efficacy of Cytokine-induced Killer Cells Against Renal and Pancreatic Cancer Cells.

BACKGROUND/AIM: Cytokine-induced killer (CIK) cells are ex vivo expanded major histocompatibility complex (MHC)-unrestricted cytotoxic cells with promising effects against a variety of cancer types. Regulatory T-cells (T-reg) have been shown to reduce the effectiveness of CIK cells against tumor cells. Peptide P60 has been shown to inhibit the immunosuppressive functions of T-regs. This study aimed at examining the effect of p60 on CIK cells efficacy against renal and pancreatic cancer cells. MATERIALS AND METHODS: The effect of P60 on CIK cytotoxicity was examined using flow cytometry, WST-8-based cell viability assay and interferon γ (IFNγ) ELISA. RESULTS: P60 treatment resulted in a significant decrease in the viability of renal and pancreatic cancer cell lines co-cultured with CIK cells. No increase in IFNγ secretion from CIK cells was detected following treatment with P60. P60 caused no changes in the distribution of major effector cell populations in CIK cell cultures. CONCLUSION: P60 may potentiate CIK cell cytotoxicity against tumor cells.

Clinical Trials with Combination of Cytokine-Induced Killer Cells and Dendritic Cells for Cancer Therapy.

Adoptive cellular immunotherapy (ACI) is a promising treatment for a number of cancers. Cytokine-induced killer cells (CIKs) are considered to be major cytotoxic immunologic effector cells. Usually cancer cells are able to suppress antitumor responses by secreting immunosuppressive factors. CIKs have significant antitumor activity and are capable of eradicating tumors with few side effects. They are a very encouraging cell population used against hematological and solid tumors, with an inexpensive expansion protocol which could yield to superior clinical outcome in clinical trials employing adoptive cellular therapy combination. In the last decade, clinical protocols have been modified by enriching lymphocytes with CIK cells. They are a subpopulation of lymphocytes characterized by the expression of CD3+ and CD56+ wich are surface markers common to T lymphocytes and natural killer NK cells. CIK cells are mainly used in two diseases: in hematological patients who suffer relapse after allogeneic transplantation and in patients with hepatic carcinoma after surgical ablation to eliminate residual tumor cells. Dendritic cells DCs could play a pivotal role in enhancing the antitumor efficacy of CIKs.

In vitro cytotoxic effect of Trastuzumab in combination with Pertuzumab in breast cancer cells is improved by interleukin-2 activated NK cells.

Targeting erb-b2 receptor tyrosine kinase 2 (ERBB2) using the combination of Trastuzumab and Pertuzumab has demonstrated promising results in breast cancer therapy. It has further been revealed that interleukin-2 (IL-2) can activate Natural Killer cells (NK cells) and elevate their cytotoxic potency against tumor cells. In this study, we explored the cytotoxic effect of recombinant human IL-2 in combination with Trastuzumab and Pertuzumab on the ERBB2 positive (SK-BR-3) and negative (MDA-MB-231) breast cancer cell lines. The cytotoxicity level of IL-2 activated NK cells (approximately 75%) were significantly higher than untreated cells (approximately 55%) in the presence of Trastuzumab and Pertuzumab against SK-BR-3 cells, while no difference was observed in the case of MDA-MB-231 cells (about 15%).

Cord blood CD4+/CD25+ T cells down-regulates NKG2D on cytokine-induced killer cells.

Studies have shown that CD4+ CD25+ regulatory T cells (Tregs) could inhibit cytokine-induced killer (CIK) cells against tumor cells, but minimal data have been reported on the underlying mechanisms. The purpose of this study was to investigate the potential suppressive mechanisms of cord blood Tregs on CIK cells in vivo and in vitro. The in vitro study demonstrated that Tregs were normally proliferated and had potent suppressive characteristics. CD4+ CD25+ LAP+ cells were highly expressed as part of activated Tregs, which limited CIK cell-mediated cytotoxicity and reduced the expression of the NKG2D receptor. Interestingly, the inhibitory ability of Tregs could be mimicked by soluble TGF-ß1 and neutralizing TGF-ß1 antibody could abrogate the inhibitory function of Tregs on CIK cells. In vivo results showed that adoptively transferred CIK cells could delay the tumor growth in nude mice. Moreover, depletion of CD4+ CD25+ Tregs in preculture or blockade of TGF-beta 1 strikingly enhanced CIK cells cytotoxicity. These data indicate that Tregs inhibit CIK cells cytotoxicity mainly by down regulating the expression of NKG2D receptor in a TGF-ß dependent manner.