A serum-free protocol for the ex vivo expansion of Cytokine-Induced Killer cells using gas-permeable static culture flasks.

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-γ, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.

DC-CIK as a widely applicable cancer immunotherapy.

Introduction: Immunotherapy is now a standard treatment for many malignancies. Although immune checkpoint inhibition has demonstrated substantial efficacy by enhancing T cell activation and function in the tumor microenvironment, adoptive transfer of T and NK cell products promises to provide activated cells capable of immediate and direct tumor destruction. A widely applicable, non-MHC dependent, cellular therapy, consisting of in vitro generated dendritic cells (DC) combined with cytokine-induced killer cells (CIK), is highly efficient to produce from individual patients and has demonstrated safety and efficacy alone or with chemotherapy.Areas covered: We summarize the clinical data from studies of DC-CIK and discuss future research directions.Expert opinion: Patients with a wide variety of tumor types who have received DC-CIK therapy may experience clinical responses. This versatile therapy synergizes with other anti-cancer therapies including chemotherapy and immunotherapy.

A novel simplified method of generating cytomegalovirus-specific cytokine-induced killer cells of high specificity and superior potency with GMP compliance.

We describe a method of rendering polyclonal cytokine-induced killer cells (CIK) specific against cytomegalovirus (CMV), focusing on GMP compliance. Peripheral blood mononuclear cells (PBMNC) are stimulated with pooled CMV peptides pp65 and IE-1 for 16-24 h and the reactive T cell subset which up-regulate CD137 is further co-stimulated with anti-CD137, followed by expansion in G-Rex flasks under standard CIK culture condition. This method generates a large number CMV-specific CIK with superior potency compared to published method currently in clinical trials. The cytotoxicity as measured by chromium release assay correlates with the upregulation of CD107a upon peptide re-challenge as measured by flow cytometry. CMV-CIK at maturity consist of mainly late effector memory CD8 T cells and have a skewed TCR repertoire with preferential expansion of a few families. Such CMV-CIK retain their function after freezing and thawing. CMV-CIK thus generated is ready for clinical trial against drug-resistant CMV disease.

Epithelial ovarian cancer stem­like cells are resistant to the cellular lysis of cytokine­induced killer cells via HIF1A­mediated downregulation of ICAM­1.

Epithelial ovarian cancer (EOC) is the most lethal of all gynecologic tumors. Cancer spheroid culture is a widely used model to study cancer stem cells. Previous studies have demonstrated the effectiveness of cytokine­induced killer (CIK) cell­based therapies against cancer and cancer stem cells. However, it is not clear how EOC spheroid cells respond to CIK­mediated cellular lysis, and the mechanisms involved have never been reported before. A flow cytometry­based method was used to evaluate the anti­cancer effects of CIK cells against adherent A2780 cells and A2780 spheroids. To demonstrate the association between hypoxia inducible factor­1α (HIF1A) and intercellular adhesion molecule­1 (ICAM­1), two HIF1A short hairpin RNA (shRNA) stable transfected cell lines were established. Furthermore, the protein expression levels of hypoxia/HIF1A­associated signaling pathways were evaluated, including transforming growth factor­ß1 (TGF­ß1)/mothers against decapentaplegic homologs (SMADs) and nuclear factor­κB (NF­κB) signaling pathways, comparing A2780 adherent cells and cancer spheroids. Flow cytometry revealed that A2780 spheroid cells were more resistant to CIK­mediated cellular lysis, which was partially reversed by an anti­ICAM­1 antibody. HIF1A was significantly upregulated in A2780 spheroids compared with adherent cells. Using HIF1A shRNA stable transfected cell lines and cobalt chloride, it was revealed that hypoxia/HIF1A contributed to downregulation of ICAM­1 in A2780 spheroid cells and adherent cells. Furthermore, hypoxia/HIF1A­associated signaling pathways, TGF­ß1/SMADs and NF­κB, were activated in A2780 spheroid cells by using western blotting. The findings indicate that EOC stem­like cells resist the CIK­mediated cellular lysis via HIF1A­mediated downregulation of ICAM­1, which may be instructive for optimizing and enhancing CIK­based therapies.

Enhanced metabolic activities for ATP production and elevated metabolic flux via pentose phosphate pathway contribute for better CIK cells expansion.

OBJECTIVE: Ex vivo expansion is an effective way to produce cytokine-induced killer (CIK) cells needed for clinical trials. Here, ex vivo expansion and metabolism characters of CIK cells in static and dynamic cultures and the relationship between cell expansion and metabolism were investigated. MATERIALS AND METHODS: Oxygen transfer efficiency was assessed by computational fluid dynamics technique. Cell phenotype, apoptosis and of transporter expression were determined by flow cytometry and Western blotting. Metabolites and enzyme activities were assessed by biochemical methods. RESULTS: Dynamic cultures favoured better CIK cell expansion without impairing their phenotype and cytotoxicity, enhanced oxygen transfer efficiency. The glucose metabolism flux of cells in dynamic cultures was enhanced by upregulating surface glucose transporter 1 expression and phosphofructokinase activity. Moreover, pentose phosphate pathway (PPP) metabolic flux was enhanced through upregulating glucose-6-phosphate dehydrogenase activity. Glutaminolysis was also accelerated via boosting glutamine transporters expression, glutaminase (GLS) and glutamate dehydrogenase activities. Together with higher oxygen consumption rate and extracellular acidification rate, it was suggested that cells in dynamic cultures were in a more vigorous metabolic state for ATP production. CONCLUSION: Dynamic cultures accelerated glucose and glutamine metabolic flux to promote ATP production, elevated glucose metabolic flux through PPP to promote biosynthesis for better cell expansion. These findings may provide the basis for ex vivo CIK cell expansion process optimization.

A dynamic transcriptomic atlas of cytokine-induced killer cells.

Several clinical immunotherapy trials with cytokine-induced killer (CIK) cells have been reported. However, molecular evidence of cell expansion, acquisition of tumor cytotoxicity, and safety of CIK cells is required before putting them to clinical use. Here, we performed dynamic transcriptomic analyses of CIKs generated from primary peripheral blood mononuclear cells exposed to interferon-γ, OKT3, and interleukin-2. CIK mRNAs were extracted and sequenced at days 0, 1, 7, and 14 and subjected to bioinformatics analyses. Using weighted correlation network analysis (WGCNA), we identified two major gene modules that mediate immune cell activation and mitosis. We found that activation and cytotoxicity of CIK cells likely rely on cluster of differentiation 8 (CD8) and its protein partner LCK proto-oncogene, Src family tyrosine kinase (LCK). A time-course series analysis revealed that CIK cells have relatively low immunogenicity because of decreased expression of some self-antigens. Importantly, we identified several crucial activating receptors and auxiliary adhesion receptors expressed on CIK cells that may function as tumor sensors. Interestingly, cytotoxicity-associated genes, including those encoding PRF1, GZMB, FASL, and several cytokines, were up-regulated in mature CIK cells. Most immune-checkpoint molecules and inflammatory tumor-promoting factors were down-regulated in the CIK cells, suggesting efficacy and safety in future clinical trials. Notably, insulin-like growth factor 1 (IGF-1) was highly expressed in CIK cells and may promote cytotoxicity, although it also could facilitate tumorigenesis. The transcriptomic atlas of CIK cells presented here may inform efforts to improve CIK-associated tumor cytotoxicity and safety in clinical trials.

Dynamic suspension culture improves ex vivo expansion of cytokine-induced killer cells by upregulating cell activation and glucose consumption rate.

Ex vivo expansion is an effective strategy to acquire cytokine-induced killer (CIK) cells needed for clinical trials. In this work, the effects of dynamic suspension culture, which was carried out by shake flasks on a shaker, on CIK cells were investigated by the analysis of expansion characteristics and physiological functions, with the objective to optimize the culture conditions for ex vivo expansion of CIK cells. The results showed that the expansion folds of total cells in dynamic cultures reached 69.36 ± 30.36 folds on day 14, which were significantly higher than those in static cultures (9.24 ± 1.12 folds, P < 0.05), however, the proportions of CD3+ cells and CD3+CD56+ cells in both cultures were similar, leading to much higher expansion of CD3+ cells and CD3+CD56+ cells in dynamic cultures. Additionally, expanded CIK cells in two cultures possessed comparable physiological functions. Notably, significantly higher percentages of CD25+ cells and CD69+ cells were found in dynamic cultures (P < 0.05). Besides, much higher glucose consumption rate of cells (P < 0.05) but similar YLac/gluc were observed in dynamic cultures. Further, cells in dynamic cultures had better glucose utilization efficiency. Together, these results suggested that dynamic cultures improved cell activation, then accelerated glucose consumption rate, which enhanced cell expansion and promoted glucose utilization efficiency of cells.

Co-culture of dendritic cells and cytokine-induced killer cells effectively suppresses liver cancer stem cell growth by inhibiting pathways in the immune system.

BACKGROUND: Application of dendritic cells (DC) for cancer immunotherapy involves tumor-associated immunogenic antigens for effective therapeutic strategies. The present study investigated whether DC co-cultured with autologous cytokine-induced killer cells (CIK) could induce a more specific immune response against liver cancer stem cells (LCSC) generated from human hepatocellular carcinoma (HCC) cells in vitro and in vivo. METHODS: Human DC and CIK were generated from peripheral blood mononuclear cells (PBMCs) taken from consenting liver cancer patients. Flow cytometry was used to determine the phenotypes of DC and CIK, and cell proliferation. The tumor growth and anti-tumor activity of these cells were further evaluated using a nude mouse tumor model. RESULTS: We demonstrated that DC and CIK significantly enhanced the apoptosis ratio, depending on DC-CIK cell numbers, by increasing caspase-3 protein expression and reducing proliferating cell nuclear antigen (PCNA) protein expression against LCSC. The in vivo data indicated that DC-CIK exhibited significant LCSC cell-induced tumor growth inhibition in nude mice, which was most significant with LCSC antigen loaded DCs. CONCLUSIONS: The results showed, that DC-CIK cells could inhibit HCC and LCSC growths in vitro and in vivo and the most successful DC triggering of cell cytotoxic activity could be achieved by their LCSC antigen loading.

Cord blood-derived cytokine-induced killer cells combined with blinatumomab as a therapeutic strategy for CD19+ tumors.

BACKGROUND: Cytokine-induced killer cells (CIKs) are an advanced therapeutic medicinal product (ATMP) that has shown therapeutic activity in clinical trials but needs optimization. We developed a novel strategy using CIKs from banked cryopreserved cord blood units (CBUs) combined with bispecific antibody (BsAb) blinatumomab to treat CD19+ malignancies. METHODS: CB-CIKs were expanded in vitro and fully characterized in comparison with peripheral blood (PB)-derived CIKs. RESULTS: CB-CIKs, like PB-CIKs, were mostly CD3+ T cells with mean 45% CD3+CD56+ and expressing mostly TCR(T cell receptor)αß with a TH1 phenotype. CB-CIK cultures had, however, a larger proportion of CD4+ cells, mostly CD56-, as well as a greater proportion of naïve CCR7+CD45RA+ and a lower percentage of effector memory cells, compared with PB-CIKs. CB-CIKs were very similar to PB-CIKs in their expression of a large panel of co-stimulatory and inhibitory/exhaustion markers, except for higher CD28 expression among CD8+ cells. Like PB-CIKs, CB-CIKs were highly cytotoxic in vitro against natural killer (NK) cell targets and efficiently lysed CD19+ tumor cells in the presence of blinatumomab, with 30-60% lysis of target cells at very low effector:target ratios. Finally, both CB-CIKs and PB-CIKs, combined with blinatumomab, showed significant therapeutic activity in an aggressive PDX Ph+ CD19+ acute lymphoblastic leukemia model in NOD-SCID mice, without sign of toxicity or graft-versus-host disease. The improved expansion protocol was finally validated in good manufacturing practice conditions, showing reproducible expansion of CIKs from cryopreserved cord blood units with a median of 28.8 × 106 CIK/kg. DISCUSSION: We conclude that CB-CIKs, combined with bispecific T-cell-engaging antibodies, offer a novel, effective treatment strategy for leukemia.

DC-CIK cells derived from ovarian cancer patient menstrual blood activate the TNFR1-ASK1-AIP1 pathway to kill autologous ovarian cancer stem cells.

Ovarian cancer stem cells (OCSCs) are highly carcinogenic and have very strong resistance to traditional chemotherapeutic drugs; therefore, they are an important factor in ovarian cancer metastasis and recurrence. It has been reported that dendritic cell (DC)-cytokine-induced killer (CIK) cells have significant killing effects on all cancer cells across many systems including the blood, digestive, respiratory, urinary and reproductive systems. However, whether DC-CIK cells can selectively kill OCSCs is currently unclear. In this study, we collected ovarian cancer patient menstrual blood (OCPMB) samples to acquire mononuclear cells and isolated DC-CIK cells in vitro. In addition, autologous CD44+/CD133+ OCSCs were isolated and used as target cells. The experimental results showed that when DC-CIK cells and OCSCs were mixed and cultured in vitro at ratios of 5:1, 10:1 and 50:1, the DC-CIK cells killed significant amounts of OCSCs, inhibited their invasion in vitro and promoted their apoptosis. The qPCR and Western blot results showed that DC-CIK cells stimulated high expression levels and phosphorylation of TNFR1, ASK1, AIP1 and JNK in OCSCs through the release of TNF-α. After the endogenous TNFR1 gene was knocked out in OCSCs using the CRISPR/Cas9 technology, the killing function of DC-CIK cells on target OCSCs was significantly attenuated. The results of the analyses of clinical samples suggested that the TNFR1 expression level was negatively correlated with ovarian cancer stage and prognosis. Therefore, we innovatively confirmed that DC-CIK cells derived from OCPMB could secret TNF-α to activate the expression of the TNFR1-ASK1-AIP1-JNK pathway in OCSCs and kill autologous OCSCs.

Innovative Clinical Perspectives for CIK Cells in Cancer Patients.

Cytokine-induced killer (CIK) cells are T lymphocytes that have acquired, in vitro, following extensive manipulation by Interferon gamma (IFN-γ), OKT3 and Interleukin 2 (IL-2) addition, the expression of several Natural Killer (NK) cell-surface markers. CIK cells have a dual "nature", due to the presence of functional TCR as well as NK molecules, even if the antitumoral activity can be traced back only to the NK-like structures (DNAM-1, NKG2D, NKp30 and CD56). In addition to antineoplastic activity in vitro and in several in-vivo models, CIK cells show very limited, if any, GvHD toxicity as well as a strong intratumoral homing. For all such reasons, CIK cells have been proposed and tested in many clinical trials in cancer patients both in autologous and allogeneic combinations, up to haploidentical mismatching. Indeed, genetic modification of CIK cells as well as the possibility of combining them with specific monoclonal antibodies will further expand the possibility of their clinical utilization.

Cytokine-induced killer cells: A novel treatment for allergic airway inflammation.

The effectiveness of cytokine-induced killer (CIK) cells for treatment of cancers has long been appreciated. Here, we report for the first time that CIK cells can be applied to treat allergic airway inflammation. Adopting from an established protocol with some modifications, we generated CIK cells ex vivo from mouse T cells, and examined their effectiveness in treatment of allergic airway inflammation using the ovalbumin-induced model of allergic airway inflammation. Based upon evaluation of bronchoalveolar lavage cellularity, T helper type2 cytokine levels and lung histology, all of which are important parameters for determining the severity of allergic airway inflammation, diseased mice treated with CIK cells showed significant reductions in all the parameters without any obvious adverse effects. Interestingly, the observed effects were comparable to those treated with dexamethasone. Thus, our study provides a novel application of CIK cells in treatment of allergic airway inflammation.

Knockdown of PD-L1 in Human Gastric Cancer Cells Inhibits Tumor Progression and Improves the Cytotoxic Sensitivity to CIK Therapy.

Background/Abstract: PD-L1 has been an important target of cancer immunotherapy. We have showed that in human gastric cancer tissues, over-expression of PD-L1 was significantly associated with cancer progression and patients' postoperative prognoses. However, as of now, how PD-L1 regulates the biological function of gastric cancer cells still remains elusive. METHODS: We constructed the stable PD-L1 knockdown expression gastric cancer cell lines by using RNAi method, and further investigated the changes of biological functions including cell viability, migration, invasion, cell cycle, apoptosis, tumorigenicity in vivo, and the cytotoxic sensitivity to CIK therapy, in contrast to the control cells. RESULTS: In the current study, we demonstrated that the knockdown of PD-L1 expression in human gastric cancer cells could significantly suppress the cell proliferation, migration, invasion, apoptosis, cell cycle, tumorigenicity in vivo and the cytotoxic sensitivity to CIK therapy. CONCLUSION: Our results indicate that PD-L1 contributes towards transformation and progression of human gastric cancer cells, and its intervention could prove to be an important therapeutic strategy against gastric cancer.

Dendritic cells infected by Ad-sh-SOCS1 enhance cytokine-induced killer (CIK) cell immunotherapeutic efficacy in cervical cancer models.

BACKGROUND AIMS: Cervical cancer constitutes a major problem in women's health worldwide, but the efficacy of the standard therapy is unsatisfactory. Cytokine-induced killer (CIK) cells exhibit antitumor activity against a variety of malignancies in preclinical models and have proven safe and effective in clinical trials. However, current CIK therapy has limitations and needs to be improved to meet the clinical requirements. The aim of this study was to investigate whether suppressing the expression of cytokine signaling 1 (SOCS1) in dendritic cells (DCs) can shorten in vitro CIK culture time and improve its antitumor efficacy. METHODS: DCs were pre-cultured for 3 days before infected with adenovirus-mediated-SOCS1 short hairpin RNA (Ad-sh-SOCS1) and pulsed with CTL epitope peptides E7. The DCs infected by Ad-sh-SOCS1 (gmDCs) and CIKs were then co-cultured for 5 or 9 days, and CIK proliferation and antitumor activity were evaluated both in vitro and in vivo. RESULTS: Our data show that gmDCs significantly stimulated the expansion of co-cultured CIKs and increased the secretion of interferon-γ and interleukin-12. Moreover, gmDCs-activated CIKs showed higher cytotoxic activity against TC-1 cells expressing HPV16E6 and E7. Our in vivo study showed that the mice infused with gmDCs-activated CIKs on day 10 had an increased survival rate and prolonged survival time compared with the controls. CONCLUSIONS: Taken together, these results indicate that DCs modified by adenovirus-mediated SOCS1 silencing can promote CIKs expansion and enhance the efficacy of antitumor immunotherapy both in vitro and in vivo, which represents an effective therapeutic approach for cervical cancer and other tumors.

Current Status of Leukemia Cytotherapy - Exploitation with Immune Cells.

With the development of chemotherapy and hematopoietic stem cell transplantation (HSCT), the prognosis of leukemia patients has been improved greatly in the past few decades. However, relapsed and refractory leukemia is still the major cause of mortality in leukemia patients. Besides, advancing age, poor performance status and severe co-morbidities limit the applicability of cytotoxic chemotherapy in certain groups of leukemia patients. Novel agents including nucleoside analogs, kinase inhibitors targeting oncoproteins and monoclonal antibodies are under investigation for the management of leukemia. Nevertheless, the outcome remains disappointing. Since immune system plays an important role in eradicating tumor cells, a lot of studies have been conducted in the administration of cytotherapy of immune cells as an alternative method when chemotherapy and transplantation fail to cure the disease, including usage of natural killer (NK) cells, cytokine-induced killer (CIK) cells, donor lymphocyte infusion (DLI), chimeric antigen receptor (CAR)-modified T cells, dendritic cell (DC)-based vaccine and antigen-specific cytotoxic T lymphocytes (CTL). Due to overexpression of several leukemia associated antigens (LAA), leukemic cells are potentially suitable for cellular therapy approach. Here we review the recent literature regarding the different types of cytotherapy against leukemia, and talk about both efficacy and adverse effects related to the strategy.

Cytokine-induced killer cell infusion combined with conventional treatments produced better prognosis for hepatocellular carcinoma patients with barcelona clinic liver cancer B or earlier stage: A systematic review and meta-analysis.

BACKGROUND AIMS: To investigate the clinical benefits of cytokine-induced killer (CIK) cell infusions on hepatocellular carcinoma (HCC) patients, combined with other conventional treatments. METHODS: This was a systematic review and meta-analysis conducted among phase II and III randomized control trials worldwide. Review manager 5.2 version was used to pool the effect size across studies. Sensitivity analyses and risk of bias were estimated among included studies. Egger's test was used to characterize the publication bias. RESULTS: Eight randomized controlled trials and 945 patients with HCC were included in the study. CIK infusion reduced cancer recurrence risk to 0.74 (95% confidence interval [CI] 0.5-0.92), I2 75% (P <0.001), and reduced cancer death risk to 0.76 (95% CI 0.65-0.88), I2 50% (P = 0.09). Among studies blinded for outcome assessment and Barcelona Clinic Liver Cancer stages of 0, A and B, CIK infusion reduced recurrence risk by 18% (relative risk [RR] = 0.82, 95% CI 0.70-0.96) and death risk by 37% (RR = 0.63, 95% CI 0.47-0.85); heterogeneity was 0% and 39%, respectively (P > 0.05). The intercepts of linear regressions for recurrence and death were -2.17 and -2.07, respectively, but the P value was 0.17 and 0.38; no significant publication bias was observed with Egger's test. DISCUSSION: Among hepatocellular carcinoma patients with Barcelona Clinic Liver Cancer score of B or less, CIK cell infusions combined with conventional treatments significantly prolonged recurrence-free and overall survival. This adoptive immunotherapy could be recommended to HCC patients.

Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy.

Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer.

Dendritic Cell Immunotherapy Combined with Cytokine-Induced Killer Cells Effectively Suppresses Established Hepatocellular Carcinomas in Mice.

BACKGROUND: The response of hepatocellular carcinoma (HCC) to immunotherapy is often disappointing and new strategies are clearly needed. The aim of the present study was to investigate whether cytokine-induced killer (CIK) cells combined with a dendritic cell vaccination enhanced cytotoxicity against hepatocarcinoma tumor cells in an in vivo animal model. METHODS: CIKs and DCs were prepared from C3H/HeJ mice by conventional methods, the dendritic cell (DC) pulsed with a MH134 cell lysate, DC or CIK alone were used as controls. Cell phenotypes were analyzed by flow cytometry, cytokine secretion levels were determined by enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was assessed by means of an in vitro lactate dehydrogenase (LDH) release assay. A mouse hepatocarcinoma cell MH134-bearing mice model was established to test the in vivo anti-tumor efficacy of the system. RESULTS: CIK cells combined with DC therapy resulted in significant inhibition of tumor growth compared with the control group, whereas the decrease in tumor growth in mice that had been treated with CIK or DC alone did not reach the level of statistical significance. The combination therapy led to a further increase in the population of cytotoxic T cells (CTLs) in vivo, compared to the CIK or DC alone therapy. In addition, the combination therapy significantly enhanced cytotoxic activity against MH134 cells. CONCLUSION: Taken together, these results show that a DC + CIK vaccination is more effective than DC or CIK alone therapy for the treatment of hepatocarcinoma cancer.

Increased Efficacy of Brentuximab Vedotin (SGN-35) in Combination with Cytokine-Induced Killer Cells in Lymphoma.

Brentuximab vedotin (SGN-35) is an antibody-drug conjugate with a high selectivity against CD30⁺ cell lines and more than 300-fold less activity against antigen-negative cells. In the last years, the results of many in vitro and in vivo studies have led to the fast approval of this drug to treat lymphoma patients. Another innovative method to treat tumor cells including lymphoma cells is the use cytokine-induced killer (CIK) cells, which have also been approved and proven to be a safe treatment with only minor adverse events. In this study, a possible additive effect when combining SGN-35 with CIK cells was investigated. The combinational treatment showed that it reduces the viability of CD30⁺ cell lines significantly in vitro. Additionally, the amount of lymphoma cells was significantly reduced when exposed to CIK cells as well as when exposed to SGN-35. A significant negative effect of SGN-35 on the function of CIK cells could be excluded. These results lead to the assumption that SGN-35 and CIK cells in combination might achieve better results in an in vitro setting compared to the single use of SGN-35 and CIK cells. Further investigations in in vivo models must be conducted to obtain a better understanding of the exact mechanisms of both treatments when applied in combination.

[Clinical Analysis of Autologous Cytokine-induced Killer Cells Combined with IL-2 for Treating of Elderly Patients with B-cell Malignant Lymphoma].

OBJECTIVE: To explore the values of autologous cytokine-induced killer cells combined with rhIL-2 for therapy of elderly patients with B-cell malignant lymphoma. METHODS: Eighty-five elderly patients with B-cell malignant lymphoma were treated by cytokine induced killer cells combine with rhIL-2 (CIK+IL-2 group), 85 elderly patients with B-cell malignant lymphoma treated without cytokine induced killer cells combined with rhIL-2 were used as the control group. The patients in CIK+IL-2 group and control group were divided into 4 subgroups accerding to lymphoma types: group A: diffuse large B cell lymphoma (DLBCL), group B: mucosa-associated lymphoid tissue type (MALT), group C: lymphoplas macytic lymphoma (LPL) and group D: hodgkin's lymphma (HL). The clinical effects, T-lymphocyte, ß2 microglobulin level, quality of life and long-term survival were observed. RESULTS: The levels of CD3+, CD3+/CD8+, CD3+/CD56+ after treatment in the 4 subgroups of CIK+IL-2 group were higher than levels before treatment and the control group (P<0.05); the levels of ß2 microglobulin after treatment for the 4 groups were lower than before treatment and the control group (P<0.05); with 1 case death, 16 cases were turned from CRu and PR to CR; the CR rate was not significantly different among the 4 subgroups (P>0.05); the scores of physical performance, role function, cognitive function, emotional functioning, and social function after treatment in the 4 subgroups were higher than the those before treatment (P<0.05); the survival time of patients in the CIK+IL-2 group lasted for 8-76 months, their median survival time was (22.36±5.38) months; the survival of the control group lasted for 7-55 months, their median survival time was (16.15±3.62) months. The survival time of the CIK+IL-2 group was longer than that of the control group (P<0.05). CONCLUSION: The treatment of aged patients with B-cell malignant lymphoma by autologous cytokine-induced killer cells combined with rhIL-2 can effectively improve the T-lymphocyte subsets, ß2 microglobulin level and quality of life, and can prolong survival time of patients.