Gemcitabine treatment enhanced the anti-tumor effect of cytokine induced killer cells by depletion of CD4+CD25bri regulatory T cells

Immunology Letters 181 (2017) 36–44

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Gemcitabine treatment enhanced the anti-tumor effect of cytokine induced killer cells by depletion of CD4+ CD25bri regulatory T cells Peng Zhao a,1 , Danni Zhu a,1 , Zhen Zhang a , Bin Han b , Daiqing Gao a , Xiaofang Wei a , Xihe Xie a , Changyou Li a , Weihong Sun a , Qingqing Wang c , Qingming Guo a,∗ a

Biotherapy Center, Qingdao Central Hospital, The Second Affiliated Hospital of Qingdao University Medical College, Qingdao 266042, PR China Institute of Transfusion Medicine, Qingdao Blood Center, Qingdao 266071, PR China c Institute of Immunology, Zhejiang University School of Medicine, Hangzhou 310058, PR China b

a r t i c l e

i n f o

Article history: Received 22 August 2016 Received in revised form 19 October 2016 Accepted 16 November 2016 Available online 17 November 2016 Keywords: Gemcitabine cytokine induced killer cells CD4+ CD25bri regulatory T cells cytotoxicity

a b s t r a c t Cytokine induced killer (CIK) cells have a powerful tumor cells killing activity both in vitro and in vivo and transfusion of these cells have become an adjuvant treatment for tumors. CIK cells are induced and amplified from peripheral blood mononuclear cells (PBMCs) with multiple cytokines. As CD4+ CD25bri regulatory T cells can be also induced by high dose of interleukin 2 (IL-2) which is used for CIK cells amplification in the CIK cell culture system, the anti-tumor activity of CIK cells was suppressed to some extent. In order to overcome this unwanted suppressive factor, we found that low dose of gemcitabine could reduce the proportion of CD4+ CD25bri regulatory T cells in the CIK cell culture system and significantly enhance the anti-tumor activity of CIK cells in vitro. The levels of interleukin-10 (IL-10) and transforming growth factor-␤ (TGF-␤) were also reduced significantly following the depletion of CD4+ CD25bri regulatory T cells in gemcitabine treated CIK cell culture system. In vivo experiment showed that low dose of gemcitabine treated CIK cells significantly suppressed tumor growth and prolonged their lifespan in tumor-bearing nude mice, with the proportion of CD4+ CD25bri regulatory T cells reduced. Meanwhile, we detected lower levels of IL-10, TGF-␤ and a higher level of interferon-␥ (IFN-␥) in tumor-bearing nude mice that received gemcitabine treated CIK cells transfusion than those in other groups. The possible mechanism involved in the enhanced anti-tumor activity in vivo was that gemcitabine treated CIK cells created a strengthened anti-tumor immune microenvironment with the changed levels of cytokines such as IL-10, TGF-␤ and IFN-␥. These results suggested a strategy to improve the adoptive immune therapy in recent use by removing the suppressive factors and a more effective tumor treatment combining chemotherapy and immunotherapy. © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Recently, the adoptive cell therapy (ACT) has been attempted as an alternative treatment option for tumor patients to eliminate tumor cells. It refers the transfusion of autologous or allogeneic T cells, NK cells, dendritic cells (DC) or any other immune cells into tumor-bearing hosts or patients considering that these cells can help to control tumor growth [1]. It has made a rapid progress and the immunotherapy has been recognized as the fourth anti-tumor modality after operation, chemotherapy, and radiotherapy [2].

∗ Corresponding authors at: Biotherapy Center, Qingdao Central Hospital, 127 Siliu, South Road, Qingdao 266042, Shandong, PR China. E-mail address: qingming [email protected] (Q. Guo). 1 Contributed equally to this work.

Among the various types of immune cells studied as potential candidates for transfusion, cytokine induced killer cells (CIK) have a powerful anti-tumor effect both in vitro and in vivo and have been used as a palliate or adjuvant treatment for many types of solid tumors. CIK cells are a population of heterogeneous cells generated in vitro by the amplification of peripheral blood mononuclear cells (PBMCs) with multiple cytokines including IFN-␥, IL-2 and anti-CD3 monoclonal antibody [3]. Among CIK cells, the lymphocytes with co-expression of the T cell marker CD3 and NK cell marker CD56 have the strongest tumor killing activity. They can kill a broad range of tumor cells via non-major histocompatibility complex (MHC) restriction [4,5]. Also, CIK cells have the capacity to migrate toward tumor sites and display anti-tumor activity in vivo [6,7]. Currently, CIK cells are widely used as an adjuvant therapy for tumors such as the lung, liver and kidney cancer after surgery, radiotherapy

http://dx.doi.org/10.1016/j.imlet.2016.11.009 0165-2478/© 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

P. Zhao et al. / Immunology Letters 181 (2017) 36–44

or chemotherapy and a palliative treatment for advanced tumors [8–11]. However, some inhibitory factors limited the anti-tumor activity of CIK cells [12]. One of the most important factors is CD4+ CD25+ regulatory T cells (Tregs) which are induced and expanded greatly when high dose of IL-2 was used to amplify CIK cells. CD4+ CD25+ regulatory T cells are potent immunosuppressive cells that can promote tumor growth and invasion by inducing immune escape and suppressing anti-tumor immune response [4,13]. Many tumors develop and progress with an accumulation of CD4+ CD25+ regulatory T cells in tumor sites and peripheral blood. The fork-head/winged-helix transcription factor 3 (foxp3 or scurfin), a member of the forkhead family, is required for the development of CD4+ CD25+ regulatory T cells [14]. Foxp3 coordinates a transcriptional program that results in the expression of genes important for the regulatory function of Tregs [15]. Tregs can suppress many immune cells such as CD4+ T cells, CD8+ T cells, monocytes, dendritic cells and B cells through cell-to-cell interactions and/or secretion of cytokines such as IL-10 and TGF-␤ [16–18]. Some studies reported that Tregs significantly decreased the anti-tumor activity of CIK cells and played an important role in immune suppression via secretion of IL-10 and TGF-␤ [19,20]. So, it is believed that the depletion or down-regulation of Tregs and the released classical inhibitory cytokines in the CIK cell culture system might enhance the anti-tumor activity of CIK cells [19–21]. This impedes us to design an effective method to deplete the unwanted induced Tregs in the process of induction and expansion of CIK cells. Recently, low-dose chemotherapy has emerged as a promising approach for selective Tregs depletion since most of the chemotherapeutic drugs mainly target the proliferative cells. For instance, administration of low dose of cyclophosphamide (CTX) or gemcitabine (Gem) selectively decreased the proportion of Tregs in the blood of tumor patients and tumor-bearing mice [22,23]. However, without biotransformation in vivo, CTX has no anti-tumor effect in vitro. So we focus on the function of gemcitabine in regulating Tregs. Gemcitabine is a pyrimidine nucleoside analogue that has been used clinically to treat various cancers. Here we investigated whether administration of gemcitabine to the CIK cell culture system could inhibit Tregs and improve the anti-tumor effects of CIK cells. In this study, we determined a low concentration of gemcitabine for targeting the unwanted induced CD4+ CD25bri regulatory T cells in the CIK cell induction and culture system. Without CD4+ CD25bri regulatory T cells or with less CD4+ CD25bri regulatory T cells, gemcitabine treated CIK cells exhibited an enhanced tumor cells killing activity when co-cultured with tumor cells in vitro. Moreover, the tumor-bearing mice that received transfusion of gemcitabine treated CIK cells had a relatively slower tumor growth rate and longer survival periods than those received untreated CIK cells.

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complete RPMI1640 supplemented with 10% heat-inactivated FCS, and incubated at 37 ◦ C in humidified air with 5% CO2 . Cells were cultured to cover 70–80% of the flasks. Trypsin (Corning, USA) was used for digestion, counting and passage. 2.2. CIK cells induction and expansion Blood and leukopheresis were taken from cancer patients who intended to receive adoptive immunotherapy. CIK cells were generated from peripheral blood mononuclear cells with Ficoll (GE Healthcare, Uppsala, Sweden) separation. Briefly, PBMCs were cultured overnight in RPMI 1640 at a cell density of 1.5 × 106 /ml supplemented with 10% heat-inactivated autologous plasma and recombinant human IFN-␥ (1000 U/ml; Shanghai Kaimao, China). After 18 to 24 h in culture at 37◦ C in a 5% CO2 atmosphere, antiCD3 antibody (50 ng/ml; OKT3, eBioscience, USA) and recombinant human IL-2 (300U/ml; Quangang, Jinan, China) were added. Thereafter, fresh medium with IL-2 was added every 2 or 3 days without opening the culture system. 2.3. Gemcitabine treatment A series of low doses of gemcitabine (LILLY, France) were added to the CIK cell culture system mentioned above on day 5 and the final concentrations increased from 10 ng/ml to 150 ng/ml. After receiving gemcitabine treatment for about 6 h, cells were washed twice and recovered in the CIK cell culture system for at least 2 days. 2.4. Flow cytometry analysis and isolation of CD4+ T cells

2. Materials and methods

PBMCs were cultured in the CIK cell culture system for 9 days and then harvested. After washing twice with phosphatebuffered saline (PBS), the cells were double or triple stained with peridinin-chlorophyll-protein complex (PerCP)-anti-CD3, fluorescein isothio-cyanate (FITC)-anti-human CD4, FITC-anti-human CD8, APC-anti-human FoxP3, phycoerythrin (PE)-anti-human CD25, PE-anti-human CD56 (BD Biosciences, USA). The cells (1 × 106 ) were incubated with various conjugated mAbs for 20 min at room temperature. After incubation, cells were washed twice and then analyzed by using FACS Calibur (BD Biosciences, USA) and data on 10000 cells were acquired and analyzed by Cellquest software. To determine the percentage of Tregs in the stained and detected CIK cells, lymphocytes were gated by plotting forward vs side scatter followed by gating on CD3+ CD4+ T cells. Gated cells were then analyzed for CD25 expression. In order to harvest CD4+ CD25bri T, CD4+ CD25dim T and CD4+ CD25neg T cells, FACS sorting was used to isolate each type of the T cells from CIK cells. For intracellular detection of foxp3, the isolated CD4+ CD25bri T, CD4+ CD25dim T and CD4+ CD25neg T cells were fixed/permeabilized and washed twice with stain buffer. Then the cells were stained with APC-anti human FoxP3 according to the BD protocol.

2.1. Mice and tumor cell lines

2.5. Apoptosis assay

Male nude BALB/c mice (5 weeks of age) were purchased from Shanghai Slac Animal Inc. (Shanghai, China). All mice were bred in a specific pathogen-free barrier facility in Experimental Animal Center of Qingdao University (Qingdao, China). This study was approved by the ethical review board of Qingdao University and all animal usage was conducted according to protocols approved by the Qingdao University Institutional Animal Care and Use Committee. Human pancreatic cancer cell line PANC-1 and human colorectal cancer cell line HT-29 maintained in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) while human chronic myelogenous leukemia cell line K562 maintained in

The isolated CD4+ CD25bri T cells mentioned above and the residual CIK cells were cultured separately in the same condition as the CIK cell culture system. A series of low doses of gemcitabine were added to each group to obtain a final concentration of 30 ng/ml, 60 ng/ml and 100 ng/ml respectively. After gemcitabine treatment for about 6 h, cells were washed twice and then the apoptosis was quantified by annexin V-FITC and PI-double staining by using a staining kit (Mbchem, China). Briefly, cells were washed twice with PBS and then 400 ␮l binding buffer containing 5 ␮l annexin V-FITC was added. Following gentle vortex, the mixture was incubated for 15 min at 4 ◦ C in the dark and then 10 ␮l PI was added. Following

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gentle vortex, the samples were analyzed by using FACS Calibur flow cytometer within 1 h. 2.6. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for the detection of proliferation of CIK cells Gemcitabine treated CIK cells and untreated CIK cells were cultured in a 96-well flat-bottomed plate at 2 × 104 cells/150 ml of cell suspension respectively. After recovering for 48 h, 20 ␮l of 5 mg/ml MTT tetrazolium substrate (Sigma) dissolved in PBS was added to each well and incubated at 37 ◦ C for 4 h. Then after centrifuging at 400 g/min for 5 min, the supernatant was removed carefully and 150 ␮l DMSO was added to each well. After full oscillation for 10 min, the OD value was analyzed on a Microplate Reader to determine the spectrophotometric absorbance of the samples at 570 nm. 2.7. CIK cells induction from the depleted PBMCs

CD4+ CD25+

regulatory T cells

CD4+ CD25+ T cells were purified with the human CD4+ CD25+ regulatory T cell Isolation Kit (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. During the two-step procedure, all the non-CD4+ cells and the CD4+ CD25− cells were collected and considered as Treg-depleted PBMCs. Then we induced CIK cells from the Treg-depleted PBMCs according to the method mention in 2.2. These CIK cells generated from the Treg-depleted PBMCs were termed as  CIK. 2.8. Co-culture of CIK cells and cancer cells  CIK,

gemcitabine treated CIK cells (Gem-CIK) or untreated CIK cells (CIK) were co-cultured with PANC-1, HT-29 or K562 cells at effector: target ratios of 20:1, 10:1 and 5:1 in 3 ml of medium with IL-2 at a concentration of 100U/ml. There were also 3 control groups containing only labeled target cells (e.g. K562, PANC-1 or HT-29 cells). After 3 h of culture, we collected the supernatant of each culture system for later cytokines determination. 2.9. Cytotoxic assay Gemcitabine treated CIK cells (Gem-CIK),  CIK or untreated CIK cells (CIK) were used as effector cells wile K562, PANC-1 or HT-29 cells were used as target cells. Treatment with CFSE was performed as follows. Fifty microliters of CFSE were added to 1 ml of each target cells suspension (4 × 105 cells/ml) in PBS to obtain a final concentration of 2.5 ␮M CFSE. The cells were incubated for 10 min at 37 ◦ C and gently mixed every 5 min. At the end of incubation, 1 ml of FBS was added to the cell suspension to stop the staining reaction, and the cells were centrifuged at 600 × g for 5 min at room temperature, washed twice with cold PBS, and resuspended in serum-free medium. Gem-CIK,  CIK or CIK cells were co-cultured with CFSE labeled K562, PANC-1 and HT-29 cells with effector: target ratios of 20:1, 10:1 and 5:1 for 3 h in 3 ml of medium with IL-2 at a concentration of 200U/ml. The control target cells with only CFSE labeled were cultured in the same culture system. Plates were gently mixed and incubated at 37 ◦ C in 5% CO2 for 3 h. At the end of the incubation period, we collected all cells in each well into tubes respectively and all tubes were placed on ice. 20 ␮l of propidiumiodide (PI) (1 ␮g/ml) were added to each tube for 10–15 min on ice. Finally, 100 ␮l of complete medium were added before acquisition on a FACS Calibur cytometer (BD Biosciences). The calculation of cytotoxicity was based on the degree of reduction of viable target cells with the ability to retain CFSE and exclude PI (CFSE high PI− ).

2.10. Blocking study Gemcitabine treated CIK cells (Gem-CIK) or untreated CIK cells (CIK) were co-cultured with PANC-1 cells at effector: target ratios of 20:1, 10:1 and 5:1. The culture condition and cytotoxic assay are in accordance with what mentioned in the section of 2.08 or 2.09 respectively. In the neutralizing group, blocking anti-human IL-10 mAb (eBioscience) with a final concentration of 5 ␮g/ml and blocking anti-human TGF-␤ mAb (eBioscience) with a final concentration of 10 ␮g/ml were added at the beginning of co-culture of untreated CIK cells and PANC-1 cells.

2.11. In vivo xenograft experiments All animal studies adhered to the protocols approved by the Institutional Animal Care and Use Committee of Qingdao University, Qingdao, China. PANC-1 cells were harvested for inoculation of mice with 0.05% trypsin–EDTA, washed twice and re-suspended in PBS. The backs of the 4-week-old male BALB/c nude mice were completely shaved and then a SC injection containing 1 × 107 PANC-1 cells/mouse was administered to the back. After 8 days, we selected 24 mice with similar tumor volumes and were randomly divided into 4 groups: Gem-CIK (n = 6), Gem-CIK & Treg (n = 6), CIK (n = 6) and PBS control (n = 6) groups. On the day 10 and day 14 after tumor cell inoculation, 1 × 107 Gem-CIK cells, 1 × 107 Gem-CIK cells mixed with 1.8 × 106 isolated CD4+ CD25bri T cells or 1 × 107 CIK cells in 0.5 ml PBS were intravenously injected into the tail of the mice in the appropriate group respectively. The mice were monitored daily, and the appearance of a hard texture at the inoculation site was identified as a tumor nodule. The tumor volumes were measured every other day using digital calipers and were calculated using the formula (A x B2 ) x 0.5, in which A was the largest and B was the shortest dimension.

2.12. Cytokine determination The supernatant of the gemcitabine treated or untreated CIK cell culture system and co-culture system was collected and centrifuged for 10 min at 800 × g. We also collected blood of tumorbearing mice in each group from posterior veins of eyeball 5 days after the second CIK cells or PBS injection. The tumor mice blood was collected in tubes and centrifuged for 10 min at 1000 × g within 30 min of collection. The separated supernatant and plasma were stored at −80 ◦ C for later cytokine determination. After centrifugation of the supernatant and plasma collection mentioned above, IL-10, TGF-␤ and IFN-␥ levels in the supernatant of culture system or serum of tumor-bearing mice were determined by ELISA using the human IL-10, TGF-␤ and IFN-␥ ELISA (Cloud–Clone Corp, Houston, USA) according to the manufacturer’s instructions.

2.13. Statistical analysis Data are shown as mean ± standard deviation (SD) for one representative experiment. Statistical significance was determined by Student’s t test or ANOVA, where suitable and a value of P < 0.05 considered to be statistically significant. All the experiments described were performed at least three times in order to warrant the reproducibility of the results.

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Fig. 1. Gemcitabine treatment decreased the proportion of CD4+ CD25bri regulatory T cells in the CIK cell culture system in vitro. The CIK cells were cultured for additional 2 days after 6 h of gemcitabine treatment and then CD makers (CD3, CD8, and CD56) of CIK cells (A) and the proportion of CD4+ CD25bri T cells in the gemcitabine treated CIK cells were detected by flow cytometer (B). (C) The ratio of CD4+ CD25bri T cells/CD4+ T cells in gemcitabine treated CIK cells was calculated and shown. Data are the mean ± SD (n = 3) of one representative experiment out of three performed. (*,p < 0.05; **,p < 0.01). (D) The expression of foxp3 in the isolated CD4+ TCD25bri T, CD4+ CD25dim T and CD4+ CD25neg T cells from the CIK cell culture system was detected by flow cytometer. The filled histograms represent isotype controls staining and the open ones are specific staining. Shown is one representative experiment out of three.

3. Results 3.1. Gemcitabine decreased the proportion of CD4+ CD25bri regulatory T cells in the CIK cell culture system in a dose-dependent manner As a pyrimidine nucleoside analogue that has been used clinically to treat various tumors, it was reported that gemcitabine could deplete regulatory T cells and myeloid derived suppressor cells (MDSCs) in cancer patients [23,24]. According to the concentration of gemcitabine (1250 mg/m2 ) for clinical use, we added a series of low doses of gemcitabine (10, 30, 60, 100, 150 ng/ml) to the CIK cell culture system at day 5. After 6 h of incubation, the cells were washed for 3 times and recovered for additional

2 days. Then we detected the expression of cell surface molecules on CIK cells treated by gemcitabine. It is believed that CIK cells are heterogeneous in vitro expanded T lymphocytes which usually contained three subsets: CD3+ CD56+ T cells, CD3+ CD8+ T cells and CD3+ CD8+ CD56+ T cells. The anti-tumor activity of CIK cells is mostly owing to the high proliferative and cytolytic potential of CD3+ CD56+ T cells whose proportion usually ranged from about 30% to 50% in most cases. As shown in Fig. 1A, gemcitabine treatment has no significant effect on the proportion of CD3+ CD56+ T cells in CIK cells since CD3+ CD56+ T cells are the representative cells of CIK cells and its proportion was between the usual proportion (30%-50%). Then we detected the proportion of CD4+ CD25bri T cells in the gemcitabine treated CIK cells and untreated CIK cells. Fig. 1B showed that the proportion of CD4+ CD25bri T cells and

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CD4+ CD25dim/− T cells in CIK cells without gemcitabine treatment were 15.75% and 22.13% respectively. However, when a series of low doses of gemcitabine were added to the CIK cell culture system, the proportion of CD4+ CD25bri T cells in the CIK cell culture system was decreased. The statistics in Fig. 1C showed that the ratio of CD4+ CD25bri T cells/CD4+ T cells dropped significantly with the increasing concentration of gemcitabine. In addition, we isolated CD4+ CD25bri T, CD4+ CD25dim T and CD4+ CD25neg T cells from the CIK cell culture system and detected the expression of foxp3 in each subset of CD4+ T cells by flow cytometer. As shown in Fig. 1D, CD4+ CD25bri T cells had a high expression level of foxp3 while CD4+ CD25neg T cells or CD4+ CD25dim T cells had nearly no expression or slight expression of foxp3. This indicated CD4+ CD25bri T cells had the phenotype of regulatory T cells since foxp3 was an important molecule expressed in regulatory T cells. Collectively, these results indicated that low dose of gemcitabine significantly reduced the proportion of CD4+ CD25bri regulatory T cells in CIK cells. 3.2. The effect of low dose of gemcitabine on the viability and proliferation of CD4+ CD25bri T cells and CIK cells Because of the cytotoxic effect of gemcitabine, we tried to determine whether it affected the viability and proliferation of CIK cells. The proliferation ability of different concentrations of gemcitabine treated CIK cells was determined by MTT assay. As shown in Fig. 2A, the proliferation ability of CIK cells treated with 30 ng/ml to 60 ng/ml gemcitabine was strengthened although the differences between these CIK cells and untreated CIK cells were not significant. This might due to the removal of the suppressive factors following the depletion of CD4+ CD25bri regulatory T cells. However, when the concentration of gemcitabine exceeded 100 ng/ml, the proliferation rate of CIK cells was decreased significantly. This indicated that the cytotoxicity of gemcitabine began to negatively affect the proliferation ability of CIK cells when the concentration exceeded 100 ng/ml. In order to classify the direct effect of gemcitabine on CD4+ CD25bri T cells and the residual CIK cells, we isolated CD4+ CD25bri T cells from the CIK cell culture system. After a series of low doses of gemcitabine treatment, CD4+ CD25bri T cells and the residual CIK cells were subjected to Annexin V/PI-double staining. Fig. 2B showed that Annexin V and PI double positive CD4+ CD25bri T cells were dramatically increased in a dose-dependent manner while apoptosis rate of the residual CIK cells was not increased significantly until the concentration reached to 100 ng/ml. This indicated that the viability of CD4+ CD25bri T cells was more susceptible to low dose of gemcitabine than CIK cells. Considering the better effect of depletion of CD4+ CD25bri T cells (Fig. 1C) and the slight influence on the viability and proliferation of CIK cells, we selected 60 ng/ml as an optimal concentration and we used this concentration in the subsequent experiments. 3.3. Low dose of gemcitabine treatment enhanced killing activity of CIK cells Cytotoxicity assays were performed to determine whether the killing activity of gemcitabine treated CIK cells was enhanced as compared with untreated CIK cells. Gem-CIK cells and untreated CIK cells were co-cultured with different tumor cell lines (K562, PANC-1 and HT-29) at different ratios for 3 h, then we detected the cytolytic rates by flow cytometer. As shown in Fig. 3, Gem-CIK cells efficiently killed all 3 different allogenic tumor cells with an average killing of 28.21% (K562), 31.37% (PANC-1) and 29.97% (HT-29) respectively at an effector: target ratio of 20:1. These killing rates were significantly higher than that of untreated CIK cells whose average cytolytic rates were 14.51% (K562), 15.37% (PANC-1) and

13.89% (HT-29) respectively. The cytotoxicity of Gem-CIK against these three allogenic tumor targets was also significantly higher than that of untreated CIK cells at effector: target ratios of 10:1 and 5:1. These results indicated that low dose of gemcitabine treatment could enhance the killing activity of CIK cells in vitro. The enhanced cytotoxicity of Gem-CIK cells might due to the depletion of the suppressive CD4+ CD25bri regulatory T cells. Then we detected the killing activity of CIK cells generated from the Treg-depleted PBMCs (termed as  CIK) against the 3 different tumor cell lines. As shown in Fig. 3,  CIK had a significantly higher cytotoxicity against these three allogenic tumor targets than that of untreated CIK cells at the three different effector: target ratios. However, the cytotoxicity of  CIK was approximately the same as Gem-CIK cells. These results further confirmed the conclusion that the enhanced cytotoxicity of Gem-CIK was due to the depletion of the suppressive CD4+ CD25bri regulatory T cells by low dose of gemcitabine. 3.4. Low dose of gemcitabine treatment created a favorable environment for the proliferation and tumor killing activity of CIK cells Following the removal of gemcitabine, CIK cells recovered for another 3 days. We then detected the concentrations of IL-10 and TGF-␤ in the culture supernatant. As shown in Fig. 4, the average concentrations of IL-10 and TGF-␤ in gemcitabine treated group were significantly lower than those in untreated group (Fig. 4A and B respectively). At the same time, we detected the levels of IL-10, TGF-␤ and IFN-␥ in the co-culture system mentioned above. As expected, these results showed that the levels of IL-10 and TGF-␤ in gemcitabine treated group were significantly lower when compared with untreated group (Fig. 4C and D respectively). However, there was no significant difference in the level of IFN-␥ between gemcitabine treated group and untreated group (Fig. 4E). Besides, we neutralized IL-10 and TGF-␤ by using anti-IL-10 mAb and antiTGF-␤ mAb in untreated CIK and PANC-1 co-culture system and then detected the cytotoxicity of the untreated CIK cells. As shown in Fig. 4F, although the killing rates of the CIK cells in IL-10 and TGF-␤ neutralized group were still lower than those of Gem-CIK cells (not significantly), they were significantly enhanced when compared with the killing rates of the CIK cells in non-neutralized group. One possible reason for these results was that CD4+ CD25bri regulatory T cells could suppress the killing ability of CIK cells via cell to cell contact in addition to IL-10 and TGF-␤. Collectively, these results indicated that the depletion of CD4+ CD25bri regulatory T cells by gemcitabine treatment and the accompanying significant reduced levels of IL-10 and TGF-␤ created a favorable environment which facilitated the induction and expansion of CIK cells. 3.5. Low dose of gemcitabine treated CIK cells significantly alleviated tumor progression in tumor bearing nude mice We developed a pancreatic cancer xenograft mouse model to explore the in vivo anti-tumor efficacy of gemcitabine treated CIK cells. 1 × 107 PANC-1 cells suspended in 100 ␮l PBS were injected s.c. into the back of each balb/c nude mouse. After mice of each group received 2 times of CIK cells or PBS injection, we measured tumor volumes every 2 days. According to the tumor index analysis shown in Fig. 5A, there were no significant differences in tumor volumes between tumor mice received CIK cells injections and PBS injections from day 8 to day 14. However, the tumor growth was significantly reduced in Gem-CIK, Gem-CIK & Treg and CIK groups as compared with PBS group from day 16 and day 18 respectively. Furthermore, the tumor volumes in the Gem-CIK group were significantly smaller than those in the Gem-CIK & Treg group or CIK group from day 22. Besides, because the adoptive transferred cells in GemCIK & Treg group and CIK group contained approximately the same

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Fig. 2. The effect of low dose of gemcitabine on the viability and proliferation of CD4+ CD25bri T cells and CIK cells. The proliferation of gemcitabine treated CIK cells was detected by MTT assay (A). Data are the mean ± SD (n = 3) of one representative experiment out of three and results were compared with the 0 ng/ml gemcitabine group. (*,p < 0.05). The isolated CD4+ CD25bri T cells and the residual CIK cells with different concentrations of gemcitabine treatment in the CIK cell culture system were stained with Annexin V-FITC and PI and analyzed by flow cytometry (B). Shown is one representative experiment out of three.

Fig. 3. Low dose of gemcitabine treatment enhanced the killing activity of CIK cells. Gem-CIK cells and  CIK cells had a significantly enhanced killing activity when co-cultured with K562(A), PANC-1(B) or HT-29(C) cell lines at ratios of 20:1, 10:1 and 5:1 in vitro when compared with that of untreated CIK cells. Gem-CIK cells and  CIK cells had similar killing rates. Cytotoxicity was evaluated by flow cytometry after co-culture of Gem-CIK cells,  CIK cells or untreated CIK cells with CFSE–stained tumor targets for 3 h. Data are the mean ± SD (n = 3) of one representative experiment out of three performed. (**,p < 0.01).

quantity of CD4+ CD25bri regulatory T cells, there were no significant differences in tumor volumes between these two groups. At the same time, we collected the venous blood of tumor mice in each group 5 days after receiving the second CIK cells or PBS injection and detected the concentrations of IL-10, TGF-␤ and IFN-␥ in serum. We found that IL-10 and TGF-␤ levels in Gem-CIK group were significantly lower (except for TGF-␤ level compared with PBS group), while IFN-␥ level was significantly higher than those in other groups (Fig. 5B–D). Besides, the median survival periods of the Gem-CIK, Gem-CIK & Treg, CIK and PBS control groups were 57.5, 49.5, 46.5 and 33.0 days respectively. The mice of Gem-CIK group had significantly longer average survival periods than other 3 groups (Fig. 5E). These results indicated that due to the reduced proportion of CD4+ CD25bri regulatory T cells, adoptive transfer of low dose of gemcitabine treated CIK cells could create a strengthened anti-tumor immune microenvironment with low levels of IL-10, TGF-␤ and high level of IFN-␥ which helped to alleviate tumor progression and prolong the lifespan of tumor bearing mice. 4. Discussion The adoptive cell therapy, as a potential new approach, holds great promises in the treatment of tumors resistant to conventional therapies [25–27]. Schmidt Wolf et al. first reported that CIK cells,

which are now considered as primary candidates for ACT, had a strong anti-proliferative capacity and cytotoxicity against tumor cells [3]. It is believed that CIK cells are heterogeneous in vitro expanded T lymphocytes with mixed NK like T cells. The antitumor activity of CIK cells is mostly owing to the high proliferative (about 100 to1000 folds) and cytolytic potential of CD3+ CD56+ NKT cells. These cells express the natural killer-cell receptor NKG2D (NK group2, member D), through which they recognize and kill tumor cells expressing the stress associated ligands MHC class I polypeptide related sequence A and B (MICA and MICB) [28–30]. Therefore, CIK cells can be used to specifically target tumor cells in vivo despite not knowing any tumor specific antigens. Adoptive immunotherapy using CIK cells has shown significant anti-tumor activity in pre-clinical experiments and animal tumor models [31–33]. However, many studies also found that CD4+ CD25bri regulatory T cells were concomitantly expanded in the CIK cell culture system. The reason is that CIK cells are usually generated by the in vitro culture of mononuclear cells with high dose of IL-2 which is a critically lymphocyte stimulator [7]. However, high dose of IL-2 is also required for the generation and function of Tregs [34,35]. Compared with PBMCs, Tregs were always significantly up-regulated within 16 days in the CIK cell culture system and the peak was at about day 6 to day 9. Many studies showed that Tregs could not only compete with effector immune cells for

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Fig. 4. Low levels of IL-10, TGF-␤ and high level of IFN-␥ were detected in gemcitabine treated CIK cells group. The supernatant of low dose of gemcitabine treated CIK cells and untreated CIK cells was collected and the levels of IL-10 (A) and TGF-␤ (B) were determined by ELISA. The supernatant of co-culture system was collected and the levels of IL-10 (C),TGF-␤ (D) and IFN-␥ (E) in each group were determined by ELISA. (F) Untreated CIK cells with or without blocking anti-IL-10 mAb & anti-TGF-␤ mAb and Gem-CIK cells were co-cultured with PANC-1 cells at effector: target ratios of 20:1, 10:1 and 5:1 respectively for 3 h and cytotoxicity was evaluated by flow cytometry. Data are the mean ± SD (n = 3) of one representative experiment. Similar results were obtained in three independent experiments. (*, p < 0.05; **, p < 0.01).

Fig. 5. Tumor growth curve, cytokines levels in serum and comparison between survival time of tumor-bearing mice in each group. (A) BALB/c mice were completely shaved at the back and then a SC injection containing 1 × 107 PANC-1 cells/mouse was administered to the back. After 8 days, 24 mice with similar tumor volumes were selected and randomly divided into 4 groups (Gem-CIK, Gem-CIK & Treg, CIK and PBS control goup). On the day 10 and day 14 after tumor cells inoculation, 1 × 107 Gem-CIK cells (or contained 1.8 × 106 isolated CD4+ CD25bri T cells), or CIK cells in 0.5 ml PBS were intravenously injected into the tail of the mice in the appropriate group respectively. The tumor volumes of each group were measured every 2 days. The tumor volumes in Gem-CIK group were significantly smaller than those in Gem-CIK & Treg, CIK and PBS control groups. The plasma of tumor mice in each group was collected 5 days after the second CIK cells or PBS injection and IL-10 (B), TGF-␤ (C) and IFN-␥ (D) levels were determined by ELISA. Data are the mean ± SD (n = 6) of one representative experiment. Similar results were obtained in three independent experiments. (*, p < 0.05; **, p < 0.01). (E) The average survival periods of mice in Gem-CIK group were 57.5 days, while the Gem-CIK & Treg, CIK and PBS control groups exhibited average survival periods of 49.5, 46.5 and 33.0 days respectively. The differences were statistically significant. (P &z.lsquo; 0.05).

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IL-2 via the high affinity IL-2R, but also release a series of suppressive cytokines such as TGF-␤ and IL-10, which result in the suppression of CD4+ T cells, CD8+ T cells and natural killer (NK) cells-mediated anti-tumor immune responses [36,37]. Therefore, Tregs are thought to play a pivotal role in the induction of immune tolerance to tumor antigens and promotion of tumor progression and metastasis. Furthermore, many recent studies reported that the immunosuppressive Tregs not only just concomitantly expanded in CIK cell culture system but also had the strong biological activity of inhibiting the antitumor activity of CIK cells [19,20]. Recently, it is believed that abrogating the effect of Tregs and Tregs related cytokines via immunological or genetic engineering approaches could significantly improve the anti-tumor activity of CIK cells [12]. Many studies reported that depletion of Tregs or attenuation of the suppressive function of Tregs could therefore evoke effective tumor immunity in otherwise nonresponsive hosts. Dannull et al. reported the direct elimination of CD4+ CD25+ Tregs by using the recombinant IL-2-diphtheria toxin followed by vaccination with tumor RNA-transfected DCs significantly improved the stimulation of tumor specific T cell responses in patients with renal cell cancer [38]. Michael A. Morse et al. found that depletion of human regulatory T cells specifically by using denileukin diftitox (ONTAK), a fusion between the active domain of diphtheriatoxin and IL-2 could enhance antigen specific immune responses to cancer vaccines [39]. David Coe et al. conducted experiments in mice and reported that depletion of regulatory T cells by anti-GITR mAb could evoke effective tumor immunity [40]. There are also some clinical trials reported a significantly enhanced tumor immunity by attenuation of the suppressive function of Tregs using anti-CTLA4 antibody [41,42]. However, the monoclonal antibodies such as anti-GITR mAb or anti-CTLA4 mAb can attenuate the immune suppressive function of Tregs but cannot deplete Tregs in the CIK cell culture system. Although anti-CD25 mAb can deplete Tregs in vivo, it cannot deplete these cells in the CIK cell culture system. The reason is that the Tregs depletion function of anti-CD25 mAb requires the involvement of the complement system but there is no complement in the CIK cell culture system. What’s more, most of CIK cells also express CD25 under the stimulation of the high concentration of IL-2 in the CIK culture system, so denileukin diftitox or anti-CD25 mAb can inevitably deplete CIK cells in vitro or in vivo respectively. Therefore, we turned to explore a new strategy to deplete the concomitant expanded Tregs in the process of induction and expansion of CIK cells. Many studies reported that Chemo-drugs with non-cytotoxic concentrations were able to change immune system and facilitate immune activation with little toxicity. Ghiringhelli et al. reported that CTX with low dose could selectively deplete Tregs while with high dose lost its specificity in Tregs depletion in tumor patients [43]. Vincent et al. reported that 5-Fu injection at 50 mg/kg in mice could selectively induce apoptosis of MDSCs in tumor microenvironment and spleen and the lowest point was around day 5 after treatment [44]. The chemotherapeutic drug gemcitabine, given at a dose similar to or less than the equivalent dose used in tumor patients, could attenuate the tumor-suppressive environment mainly by eliminating MDSCs and preserves the number of CD4+ T cells and CD8+ T cells in mice [24,44,45]. It is also reported that low dose of Gem (50 mg/kg) plus CTX (50 mg/kg) at 8-day intervals, could attenuate Tregs and MDSCs mediated immunosuppression concurrently and suppress tumor growth without impairing effector T cells function [46]. In conclusion, these chemotherapy drugs mentioned above are predominant in immunomodulation, and the application is different from conventional clinical chemotherapy. However, CTX cannot deplete Tregs in the CIK culture system ex vivo for its pharmacological function

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needs a series of biotransformation in vivo. Therefore, we selected 5-FU and gemcitabine as our study subjects. In our study, we found that 5-FU did not have the function of depletion of Tregs in vitro at a series of low doses below clinical use (data not shown) while gemcitabine could deplete Tregs at a series of low doses. Our study showed that only when the concentrations of gemcitabine exceeded 60 ng/ml, could the proportion of CD4+ CD25bri T cells reduce significantly. And the higher concentration of gemcitabine, the more favorable to reduce the proportion of CD4+ CD25bri T cells in the CIK cell culture system (Fig. 1B, C). However, when the concentrations exceeded 100 ng/ml, the cytotoxicity of gemcitabine became more and more obvious and the proliferation of CIK cells was inhibited significantly. Therefore, in order to deplete Tregs more efficiently and not affect the proliferation ability of CIK cells, the concentration of gemcitabine should be kept between 60 and 100 ng/ml (Fig. 1C, 2). Furthermore, compared with untreated CIK cells, our data showed that gemcitabine treated CIK cells exhibited a higher efficacy on killing tumor cells as a result of the reduced proportion of CD4+ CD25bri regulatory T cells while preserving CD4+ CD25dim/− T cells. With the depletion of CD4+ CD25bri regulatory T cells, the concentrations of TGF-␤ and IL-10 in the CIK cell culture system also reduced. We all know that CD4+ CD25bri regulatory T cells can suppress the function of other T cells not only by releasing suppressive cytokines such as TGF-␤ and IL-10 but also by cell to cell contact. That is why neutralizing TGF-␤ and IL-10 in the co-culture system can only partially enhance the cytotoxicity of CIK cells. Because of the removal of suppressive factors mentioned above, the proliferation and activation of CIK cells were enhanced and a larger amount of IFN-␥ were released by the activated CIK cells. IFN-␥ is a cytokine with multiple anti-tumor effects such as direct inhibition of tumor growth, blocking angiogenesis, or stimulation of macrophages [1]. It also seems to be important for the elevation of expression of MHC class II molecules which can improve the sensitivity of tumor cells to immunological effector cells [47]. In the co-culture system in vitro, we also detected significantly reduced levels of TGF-␤ and IL-10 and an increasing although not significant level of IFN-␥ in gemcitabine treated group (Fig. 4). Similar results were also obtained in tumor mice serum in vivo experiment (Fig. 5B, C, D). The in vivo study also showed that the mice received adoptive immunotherapy with gemcitabine treated CIK cells had a significantly slower tumor growth rate and longer lifespan when compared with the mice that received untreated CIK cells transfusion (Fig. 5A, E). All these results illustrate that adoptive transfusion of CIK cells with CD4+ CD25bri regulatory T cells depleted and the consequent up regulation of INF␥ and down regulation of TGF-␤ and IL-10 shift tumor-bearing nude mice’ tumor-suppressive environment to a beneficial one which can stimulate a more effective tumor immunity. In conclusion, our study suggested an effective strategy to optimize the induction and expansion of CIK cells which had an enhanced tumor cell killing activity in vitro and a more effective tumor therapy in vivo. We also concluded that the changed cytokines production followed CD4+ CD25bri regulatory T cells depletion may serve as an important mechanism by which gemcitabine treated CIK cells exert their enhanced anti-tumor effects. This strategy of Tregs depletion in vitro by using low dose of gemcitabine can not only be employed in the CIK cells amplification but also be applied to the culture of other immune cells such as NK cells or tumor infiltrating lymphocytes (TILs) whose induction and amplification also need a high dose of IL-2. In the following study, we plan to adoptive transfer the gemcitabine treated autologous CIK cells to tumor volunteers and determine the clinical effects for this strategy represents a safe and effective therapy. If confirmed by larger scale studies, these promising results may have a favorable impact on conventional treatment strategy of tumors.

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