The combination of dendritic cells-cytotoxic T lymphocytes/cytokine-induced killer (DC-CTL/CIK) therapy exerts immune and clinical responses in patients with malignant tumors
- Ying Wang†1,
- Zenghui Xu†1,
- Fuping Zhou†2,
- Yan Sun1,
- Jingbo Chen1,
- Linfang Li1,
- Huajun Jin1 and
- Qijun Qian1, 2Email author
© Wang et al. 2015
Received: 3 July 2015
Accepted: 30 October 2015
Published: 10 November 2015
The clinical trials using immunotherapy have been performed for the treatment of variety of malignant tumors. However, large-scale meta-analysis of combined DC-CTL/CIK therapy on immune and clinical response in patients has not been well studied yet. The purpose of this study is to investigate the role of DC-CTL/CIK therapy and evaluate the changes of immune indicators and tumor serological markers both at an individual level and at a system level, which is an important basis for immunotherapy as well as prognosis estimation.
Three cohorts were designed to estimate therapeutic effects on patients with malignant tumors. Tumor serological markers were detected pre- and post-treatment by immunoradiometric methods using commercially available diagnostic kits. Lymphocyte subsets were identified by flow cytometry. The quality of life was assessed by EORTC QLQ-C30 questionnaire.
In this study, we found out that Tregs was significantly reduced after transfusion of DC-CTL/CIK cells companied by decreasing serological tumor markers including AFP, CA199 and CA242 in primary liver cancer and CA724 in gastric cancer. A system-level analysis showed that lower percentages of Tregs were detected in patients with long-lasting courses of immunotherapy. Strikingly, a tumor progression indicator, myeloid-derived suppressor cells (MDSC), was dramatically decreased in patients after DC-CTL/CIK treatment. These results suggested that DC-CTL/CIK therapy improves immune functions and the quality of life post-treatment versus pre-therapy, indicating that DC-CTL/CIK therapy might block the deterioration of invasive cancers in these patients.
This study demonstrated that DC-CTL/CIK therapy could reduce Tregs, MDSCs, and several crucial serological tumor markers in particular tumors, and improve the function of T cells immune systems and the quality of life in patients with malignant tumor.
KeywordsDC-CTL/CIK immunotherapy Serological tumor markers Immune indicators Quality of life Tregs
Malignant tumors are refractory diseases that cause death and high economic burden in the world. Traditional therapeutic regimens such as surgery, radiation, or chemotherapy are usually accompanied by adverse side-effects. Nowadays, immunotherapy has emerged as an effective treatment for patients with malignant tumors. It has been broadly applied in clinical trials for the treatment of various malignant tumors. Lots of trials have demonstrated that the therapeutic use of DC vaccines, CTL cells and CIK cells in the improvement of cancer therapy and showed promising outcomes of DC-CTL/CIK therapy alone or combined with conventional therapies [1, 2]. In previous reports, several studies have focused on improving curative effect and evaluating the safety and efficacy of immunotherapy [3–8], founding that DC vaccines combined with CIK cells might have a potential to prevent tumor recurrence, increase progression-free survival rates, and improve the quality of life for cancer patients. Moreover, the side effects and toxicity of immunotherapy were mild and easily controllable.
DC-CTL/CIK treatment have displayed encouraging results in tumor therapy, however, several critical issues remain to be solved. First of all, the study of immunotherapy in solid tumors is still rare although several clinical trials had been carried out in the treatment of metastatic renal cell carcinoma, colorectal cancer and other malignancies [7, 9]. The sample sizes for most of trials are small. Secondly, there is still no reliable biomarker for evaluating clinical responses and monitoring prognosis of DC-CTL/CIK therapy. The indicators such as CD3, CD4, and CD8 have been selected as common indicators to assess the efficacy of immunotherapy. The changes of those indexes were statistically significant in patients post-treatment versus pre-therapy, however, whether this or other biomarkers correlate with clinical outcome remain less explored [10–12]. Thirdly, there is still no standard cell preparation system of DC-CTL/CIK therapy. A lot of robust cell culture regimens have been established, however, different laboratories performed variously in cells culture regimens such as numbers of infused cells, types of additional cytokines, concentration and incubation time [13–16], which could have an impact on the efficacy of DC-CTL/CIK therapy. In the previous studies, Qu et al.  reported the number of CIK cells cultured in vitro increased to 14.3 ± 2.6 times at 12 days culture period, and this number could reach to 20.5 ± 3.2 times when CIK cells was co-cultured with DC cells. Pan et al.  and Tao et al.  depicted that the main functional properties of CIK cells might be limited by Tregs. Theoretically, improvement of DC-CTL/CIK culture strategy and depletion of Tregs could bring benefit to tumor therapy. Thus, a more standardized and efficient cell production process is needed to be well-studied.
Therefore, in the present study, we designed three cohorts, totally including 83 patients, to investigate therapeutic effect of DC-CTL/CIK therapy in patients with malignant solid tumors. To identify the essential features overall and investigate the therapeutic role of DC-CTL/CIK therapy, we attempted to assess several common serological tumor markers and immune indicators across a wide range of tumor types at an individual level. And then, at a systematic level we performed to analyze the correlation between changes of Treg cells and prognosis and to assess the long-term quality of life. Additionally, a novel tumor progression indicator, myeloid-derived suppressor cells (MDSC), was also being approached preliminarily in this study.
We consecutively analyzed cohorts of 83 patients in three subgroups. The first subgroup (cohort 1) contained 60 patients with detailed the clinicopathologic information about sex, age, Tumor Classification, tumor-node-metastasis (TNM) stage, adjuvant therapeutic strategies, serological tumor markers detection, immune indicators detection and follow-up of prognosis, who were from Biotherapy Department of Eastern Hepatobiliary Surgery Hospital, received DC-CTL/CIK therapy from February 2012 to March 2014 (25 patients with primary liver cancer, 10 patients with cholangiocarcinoma, 11 patients with lung cancer, 7 patients with gastric cancer and 7 patients with colon cancer). In Cohort 2, 14 patients with negative for all serological tumor indexes were reviewed. Cohort 3 included nine patients for MDSC detection. The peripheral blood samples were accessed in Department of Biotherapy, Eastern Hepatobiliary Surgery Hospital, the Second Military Medical University (China). All patients signed informed consents.
DC-CTL/CIK cells culture regimen
Peripheral blood mononuclear cells (PBMCs) were harvested by blood cell separator (FreseniusKabi, Germany). About 1 × 109–3 × 109 PBMC cells/40–60 ml white cells were collected from each patient, and were cultured in AIM-V cell culture solution (Gibco, America) for 2 h. The adherent cells were initiated into culture for DC maturation by 100 ng/ml GM-CSF (PeproTech, America) and 100 ng/ml IL-4 (PeproTech, America) in AIM-V cell culture solution. Part of the non-adherent cells were stored at −80 °C and thawed for co-cultivation, and the rest were applied for inducing CIKs.
On Day 6, the recombinant non-proliferative adenovirus (serotype 35) carrying the expression cassettes of tumor-associated antigens (TAAs) were used to infect the DC cells with a multiplicity of infection (MOI) of 5. After 24 h cultivation, IL-1β (25 ng/ml; Novoprotein, China) and TNF-α (100 ng/ml; Novoprotein, China) were added into the culture for DC-maturation for another 24 h.
On day 8, about 2 × 106 DC cells were harvested and co-cultured with T cells (about 4 × 107 cells) at a DC/T cell ratio of 1:20 for another 4 days to induce antigen-specific CTL cells which were stimulated with CD3 monoclonal antibody (mAb) (50 ng/ml; Novoprotein, China) pre-coated onto plastic plates and amplified by IL-2 (500 IU/ml; Novoprotein, China). The rest cells were applied as DC vaccine. The applied TAAs included p53, survivin, hTERT and additional AFP/CEA for AFP/CEA positive patients.
Additionally, CIKs were cultured in 4 × 40 Ml serum-free medium supplemented with 1000 U/mL IL-2, 5 μg/mL CD3 monoclonal antibodies, 12.5 μg/ml RetroNectin (Novoprotein, China) and 1000 U/mL IFN-γ (Novoprotein, China).
Cell collection and centrifugation
Cells were harvested on day 8, transferred from medium bags into centrifuge bottles, and centrifuged at 1500 rpm for 5 min, removed the supernatant by aspiration, washed twice by adding normal saline, then removed the supernatant and re-suspended the cell pellet in 300 ml normal saline and 500 μl IL-2. We transferred cells into transfer bag, heated seal the bag and prepared for clinical infusions. The protocol of cell collection and centrifugation on day 10 and day 12 is the same as day 8.
Preparation for infusions
The infused cells were cultured for detecting levels of bacteria, fungus and endotoxin by using BacT/ALERT® 3D instrument (Biomerieux, America) and and PREVI® Color Gram (Biomerieux, America).
In total, the final volume is 1200 ml (300 ml/per bag × 4 bag = 1200 ml). Promethazine is used to treat allergy symptoms 10 min before starting the treatment. The usual dose is 12.5 mg by intramuscular injection. After the infusion, Sodium bicarbonate is used to make the urine more alkaline to prevent kidney failure. During the procedure of infusion, the drop speed in the first 15 min is 30 drops/min. If patients have no adverse reaction after 15 min, we adjust the drop speed at 80 drops/min.
For each treatment, patients were treated with 3 intravenous infusions. About 1 × 107 DC vaccine, 1 × 109 CIKs and both DC vaccine and CTLs (about 1 × 109 cells) were infused intravenously on day 8, day 10 and day 12 respectively.
Detection of immune indicators by flow cytometry
The immune phenotypes and cytokine production were identified by FC500 flow cytometer (Beckman Coulter, USA) and Guava easyCyte™ flow cytometers (Merck Millipore, Germany). The monoclone antibodies were CD3+ to evaluate total T lymphocytes, CD3+/CD4+ to evaluate Helper T cells, CD3+/CD8+ to evaluate cytotoxic T cells (CTLs), CD3+/CD56+ to evaluate cytokine-induced killer (CIKs), CD4+/CD25+/CD127low/− to evaluate regulatory T cells (Tregs), CD80/CD86/CD83/CD11c to evaluate Dendritic cells (DCs), IFN-r to evaluate production of Interferon gamma and CD11b+/CD33+/HLA−DR− to evaluate Myeloid-derived suppressor cells (MDSCs) . The proportion of Treg cells was calculated by the ratio of CD25hiCD127lo/− cells to CD4+ cells. The proportion of MDSC cells was calculated by the ratio of CD11b+ CD33+ cells to HLADR− cells. The flow cytometry data were analyzed by CXP software and guavaSoft™ 3.1.1.
Detection of serological tumor markers by immunoradiometric methods
Serum AFP (Autobio, China), CEA (Roche, Switzerland), CA19-9 (Bayer, America), CA125 (Roche, Switzerland), CA242 (DPC, America) and CA724 (DPC, America) were detected by immunoradiometric methods with commercially available diagnostic kits, respectively. The recommended cutoff-values for diagnostic purpose were 20 μg/L for AFP, 5 μg/L for CEA, 37 U/ml for CA19-9, 35 U/ml for CA125, 12 U/ml for CA242 and 6 U/ml for CA742. Values above the cutoff concentrations were considered positive in this study.
Evaluation and statistical analysis
Statistical analyses were completed using statistical SPSS 22 software (SPSS, Chicago, IL, USA). Analysis of biomarkers was measured using paired t test with P < 0.05 considered significant. The EORTC QLQ-C30 questionnaire [18, 19], developed by European Organization for Research and Treatment of Cancer (EORTC), is used to assess the quality of life of cancer patients. The hazard ratio was calculated by Cox’s proportional hazards regression method.
The criteria used to assess side effects included temperature, blood pressure, allergic reaction, appetite, fatigue and skin eruption.
Characteristics of patients
Characteristics of patients
Cohort 1 N (%)
Cohort 2 N (%)
Cohort 3 N (%)
No. of patients, N
42 (70 %)
8 (57.1 %)
6 (66.7 %)
18 (30 %)
6 (42.9 %)
3 (33.3 %)
Age, median years (range)
10 (16.7 %)
2 (22.2 %)
7 (11.7 %)
1 (7.1 %)
7 (11.7 %)
11 (18.3 %)
4 (28.6 %)
2 (22.2 %)
Primary liver cancer
25 (41.7 %)
1 (7.1 %)
2 (22.2 %)
8 (57.1 %)
3 (33.3 %)
3 (5 %)
1 (7.1 %)
2 (22.2 %)
4 (6.7 %)
1 (11.1 %)
12 (20 %)
2 (14.3 %)
1 (11.1 %)
2 (3.3 %)
9 (15 %)
4 (28.6 %)
1 (11.1 %)
27 (45 %)
6 (42.9 %)
3 (33.3 %)
3 (5 %)
1 (7.1 %)
1 (11.1 %)
Adjuvant therapeutic strategies
24 (40 %)
5 (35.7 %)
7 (77.8 %)
36 (60 %)
9 (64.3 %)
2 (22.2 %)
18 (30 %)
6 (42.9 %)
1 (11.1 %)
8 (13.3 %)
1 (7.1 %)
1 (11.1 %)
2 (3.3 %)
1 (11.1 %)
9 (15 %)
2 (14.3 %)
Molecular targeted therapy
2 (3.3 %)
1 (7.1 %)
Univariate analysis in patients
OR (95 % Cl)
Sex (male vs. female)
Age (≤54 vs. >54)
TNM stage (I, II vs. III, IV)
Chemotherapy (no vs. yes)
Radiation therapy (no vs. yes)
Surgery (no vs. yes)
Molecular targeted therapy (no vs. yes)
Physiotherapy (no vs. yes)
Interventional treatment (no vs. yes)
Quality control and phenotypes of infused cells
Individual analysis of the changes of immune indicators and serological tumor markers pre- and post-treatment
Systematic analysis of correlation between proportions of Tregs and prognosis
Additionally, a heat map was performed to describe by each six indicators of cancer to compare the difference between different types of cancer pre-therapy. As shown in Fig. 4c, in fact, it is difficult to find a single marker that has high sensitivity and specificity to achieve clinical utility. An elevated level of a tumor marker is different in many kinds of tumors. In our study, cases in Cohort 1 were positive for one of six tumor markers, the percentage of Treg cells significantly decreased post-treatment in patients with all types of tumors. Thus, we further observed other 14 cases in Cohort 2 that were negative for all six tumor markers pre-therapy. Interestingly, the changes of immune indicators (Fig. 4d) were basically consistent with those of tumor marker positive cases, showing that the percentage of CD3+ CD8+ T cells increased and Tregs reduced significantly post-treatment. Thus, we demonstrated that the percentage of Tregs was significantly correlated with the survival time of tumor patients and a combination of serological tumor markers and Treg to investigate the therapeutic roles of DC-CTL/CIK therapy could be more efficiently in the patients with malignant tumors.
Assessment of quality of life (QoL)
We measured quality of life of patients undergoing DC-CIK/CTL treatment. The QLQ-C30 has been suggested to have multi-item subscales measuring function and symptom. A quality-of-life feature, QoL score, was designed to evaluate the balance between side effects and quality of life. It is inspiring that the QoL score of 25 (41.7 %) patients improved obviously post treatment, and 12 (20 %) patients did not take a turn for the worse. Thus, it indicated that DC-CIK/CTL therapy could at least partially block the deterioration of cancers in the patients with malignant tumors with elevated quality of life.
Comparison of MDSC levels pre- and post-treatment of DC-CTL/CIK therapy
The side-effects were also recorded in all cases. There were no severe or unusual side-effects recorded that are common in chemotherapy and radiotherapy during or after the transfusion, except for temporary fever. In total, 40 % patients developed a fever during the immunotherapy, and the body temperature was 37.5–38. These patients recovered without any treatment. Six out of 60 (10 %) cases had temperatures >38 °C treated with drugs to lower body temperature.
In this study, we collected three cohorts of patients to investigate therapeutic effect and immune modulation of DC-CTL/CIK cells in malignant tumors both at an individual level (changes of immune indicators and serological tumor markers pre- and post-treatment) and at a system level (correlation between proportions of Tregs and prognosis). We detected several serological tumor markers and immune indicators pre-therapy. Levels of 6 common tumor markers in 60 cases were highly expressed in serum. The abnormal level of T lymphocyte subpopulations indicated severe damage of immune systems in these patients, and the accumulation of Tregs within the tumor microenvironment represents a major obstacle for the development of effective antitumor immune-therapies [16, 20]. Excitingly, we found out that our DC-CTL/CIK therapy significantly reduced several serological tumor markers such as AFP, CA199 and CA242 in primary liver cancer and CA724 in gastric cancer (p < 0.05), elevated the level of CD3+ CD8+ T cells in primary liver cancer and lung cancer (p < 0.05), and decreased the level of CD3+ CD4+ T cells in colon cancer, primary liver cancer and lung cancer (p < 0.05) and Treg cells in all types of tumors (p < 0.05), which indicated the promotion of immune functions in these patients. The quality of life (QoL score) of patients was improved after treatment which could be indicate that it exerts immune and clinical responses in patients with malignant tumors DC/CIK treatment.
A systematic analysis was performed to discuss the correlation between proportions of Tregs and prognosis, and whether a combination of immune indicators especially Treg indicator with serum tumor markers could be a better way to evaluate therapy responses. Tregs within tumors represented a major obstacle for cancer immunotherapy [21, 22]. In previous studies, it has been reported that Tregs decreased the cytotoxicity of CIK cells, and the down-regulation of Tregs could strengthen the killing activity of CIK cells to tumor cells [14, 16, 23]. Tao  and Lin  indicated that the addition of IL-6, IL-7, IL-15 during CIK cell culture in vitro inhibited the production of Tregs. Pan  suggested that DC cells decreased concomitant expanded Tregs and Tregs related IL-35 in CIK cells, to against leukemia cell lines K562 and NB4.
Here, compared with the prior methods, we also provided a novel cell culture program in the yield of DC cells, a cocktail of antigens and incubation time, to significantly reduce the proportion of Tregs. As in our cell culture protocol, it has greatly reduced the percentage of Tregs (less than 7 %). Analysis at a system level showed that long-lasting course of DC/CIK treated patients (>3 cycles) had lower percentages of Tregs than patients who only received short-term therapy, and the percentage of Treg was lower for these patients with immunotherapy alone or combined with other adjuvant therapeutic strategies.
Moreover, to date, the detection of tumor markers has been widely used in cancer screening, early diagnosis, monitoring efficacy of treatment, and prognostic evaluation [24–27]. In fact, it is difficult to find a single marker that has high sensitivity and specificity to indicate a cancer. For instance, alpha-fetoprotein (AFP), a widely accepted biomarker of hepatocellular carcinomas (HCC), in some patients with advanced HCC may still be negative. AFP in our dataset was elevated in 94 % of liver cancer patients, but 10 cases were AFP-negative. As such, it is likely that the effect of DC-CTL/CIK immunotherapy would not be suitably assessed by methods based on response evaluation criteria in solid tumors (RECST). Therefore, it is urgently necessary to identify and validate reliable biomarkers to evaluate clinical responses and outcomes of immunotherapy including DC-CTL/CIK therapy. A methodology named “Immunoscore”, which numerates lymphocytes both in tumor center (CT) and invasive margin (IM), has been introduced to add to the significance of the American Joint Committee on Cancer (AJCC)-Union for International Cancer Control (UICC)-Tumor Node Metastasis (TNM)-classification, and to establish prognosis of clinical outcome in patients . In our study, we proposed that altered immune phenotype in peripheral blood cells of patients may have clinical value in the evaluation of therapeutic effects and clinical outcome, especially Treg cell as a potent immunosuppressive cell which promote tumor growth and invasion. MDSC also has been proposed to have contribution to immune-suppression, and have association with grades, stages, tumor burden and clinical outcomes in patients with different types of cancer . MDSCs can influence the proliferation and activation of T cells, NK cells, dendritic cells and macrophages, especially in inhibiting CD8+ T cell responses [30–32]. Diaz-Montero  demonstrated that MDSC can be referred to as an independent prognostic indicator by multivariate Cox proportional hazards analysis. Here, we also perform a preliminary investigation of MDSCs in peripheral blood pre and post-therapy, and more verification experiments are needed to undertake this improvement in the future.
In addition, as many papers have published that neo-antigens in cancer immunotherapy and personalized antigen for the treatment , optimization of this program will be further performed to standardize and facilitate the cell culture process. Besides DC-CTL/CIK immunotherapy in this study, other immunotherapeutic agents also have made great progress in clinic, such as immune checkpoint inhibitor antibodies of PD-1/PD-L1 or CTLA-4, and chimeric antigen receptor-T cell (CAR-T) targeting specific tumor antigen. CTLA-4 antibody ipilimumab, PD-1 antibody pembrolizumab and nivolumab have been approved by FDA . The most successfully application of CAR-T is on chronic lymphocytic leukemia patients, which specifically binds to CD19 marker. However, immunosuppressive microenvironment in tumor (surrounding with Treg, MDSC, etc.) may greatly impaired the therapeutic effects of immune checkpoint inhibitors and CAR-T. Moreover, there are rare target antigens for T cell engineering [10, 36]. Our DC-CTL/CIK immunotherapeutic agents can greatly improve the system of immune function and reduce Tregs and MDSCs in peripheral blood, and also with widely antitumor spectrum of solid tumors. Furthermore, combination of DC-CTL/CIK with immune checkpoint inhibitors or CAR-T may bring out better clinical result, and the clinical trials are being undertaken in our hospital which is believed to greatly improve the therapeutic effect of immunotherapy in the future.
In summary, our results demonstrated that DC-CTL/CIK therapy as an immunotherapeutic regimen could influence the immune status, and patients with late-stage malignant tumor may benefit from this therapeutics. Our data conclusively exhibited immune improvement by impairing the immune-suppressive Tregs after DC-CTL/CIK therapy. This immune response is continuously modulated with the increasing of treatment courses. We have also showed a combination of DC-CTL/CIK therapy with other adjuvant therapeutic strategies could reduce the percentages of Treg significantly, and a decrease frequency and depleted suppressive activity of Tregs in peripheral blood in patients which have a strong correlation with prognosis. Thus, this work may provide valuable insights into the clinical curative effect evaluation of DC-CTL/CIK therapy and the design of immunotherapeutic strategies for malignant tumors.
cytotoxic T lymphocytes
myeloid-derived suppressor cells
peripheral blood mononuclear cells
European Organization for Research and Treatment of Cancer
quality of life
American Joint Committee on Cancer
Union for International Cancer Control
tumor node metastasis
YW and ZX designed the pipeline of the analysis, and drafted the manuscript. YW detected the immune indicator and performed the statistical analysis. FZ collected clinical data. YS, JC and LL cultured DC, CIK and CTL cells for immunotherapy. HJ revised the manuscript. QQ conceived and coordinated the overall study and revised the manuscript. All authors read and approved the final manuscript.
This study was funded by the National Natural Science Foundation of China (Grant Number 81301307) and the National Science and Technology Major Project (Grant Number 2013ZX10002-010-007).
The authors declare that they have no competing interests.
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