Wilfoside C3N Promotes Tumor Cell Death by Activating Gammadelta T Cells-Mediated Anti-tumor Immunity

HOU Jin-yu,WANG Jing

(1.Zhangjiakou NO.1 High School,Zhangjiakou,Hebei Province 075000,China; 2.Life Science Research Center,Hebei North University,Zhangjiukou,Hebei Province,China)

Wilfoside C3N Promotes Tumor Cell Death by Activating Gammadelta T Cells-Mediated Anti-tumor Immunity

HOU Jin-yu1,WANG Jing2

(1.Zhangjiakou NO.1 High School,Zhangjiakou,Hebei Province 075000,China; 2.Life Science Research Center,Hebei North University,Zhangjiukou,Hebei Province,China)

Vδ1+γδ T lymphocytes are known to play important roles in anti-tumor immunity.We recently reported an anti-tumor activity of wilfoside C3N,an active component extracted from Chinese medicinal herbs.In the current study,we evaluated the role of Vδ1+γδ T cells in C3N anti-tumor activity using aninvitrocell co-culture model.We found that C3N induced the ECA109 tumor cells to undergo apoptosis in the presence of Vδ1+γδ T cells.The level of ECA109 apoptosis maximized when both C3N and Vδ1+γδ T cells were present,which correlated with the increased expression of Fas on ECA109 and Fas ligand on Vδ1+γδ T cells induced by C3N.In addition,C3N also enhanced secretion of cytokines,perforin and granzymes by Vδ1+γδ T cells.These observations suggest that activation of Vδ1+γδ T cells may play a critical role in C3N-mediated anti-tumor activity.

wilfoside C3N;Vδ1+gammadelta T cells;anti-tumor;ECA109 cells

The principle of Chinese medicine is to strengthen the resistance,and eliminate the invading pathogenic factors.Strengthening the body resistance can be able to improve the immunity of body which is due to the fact that it can afford to activate T cell immunity.Eliminating the pathogenic factors mainly dreams of inhibition of cancer cell proliferation and induction of cancer cell apoptosis.C21steroidalglycoside(C21),which is a component of the traditional Chinese medical herbBaishouwu,may reach the unity of them.TheC21,isolated from the roots ofCynanchumauriculatum,was found to inhibit the growth of several human tumor cell lines.The constituents ofC21 in this herb are complex and most of them show good pharmacological activities.Wilfoside C3N(Caudatin 3-O-β-D-cymarophranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside)and wilfoside C1N(Caudatin 3-O-α-L-cymarophranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside)are the two most abundant and active C21 in it.Wilfoside C3N showed a much better absorption than wilfoside C1N[1-3].

Because of its antitumor properties,C21 holds promise as a treatment for many kinds of tumor[4-5].However,other than our recent reports thatC21 induced apoptosis in hepatocellular cancer cells(Heps)by down-regulating Bcl-2 expression and induced apoptosis[6],virtually no work has been done to elucidate the mechanisms of the action.Moreover,rare of antitumor effect of C3N has been researched.

Whereas the vast majority of T cells express a T-cell receptor(TCR)composed of αβ heterodimers,a smaller population expresses a γδ TCR.In contrast to αβ T cells,γδ T cells show less TCR diversity,are particularly enriched at epithelial surfaces and appear to respond to self-molecules that signal potential danger or cellular stress.In addition,various subsets of γδ T cells have shown antitumor and immunoregulatory activities[7].The increasing evidence that γδ T cells have potent antitumor activity suggests their value in immunotherapy,particularly in areas of unmet need such as metastatic carcinoma[8].More recently,the relative contributions of αβ and γδ T cells to tumor immunosurveillance have been systematically studied.

Tumors form more readily in γδ-deficient mice after injection of squamous-cell carcinoma or melanoma tumor cell lines[9].In addition to infiltrating γδ T cells have also been shown to produce IFN-γ early in tumor development.γδ T cells not only inhibit the early stages of tumor development but also limit progression to carcinoma[10].

γδ T cells appear to perform Perforin and FasL-mediated cytotoxicity utilizing a common secretory mechanism based on cytolytic granule exocytosis.In the presence of calcium,perforin lyses a variety of target cells non-specifically.Cell surface-associated FasL was expressed by very few freshly isolated γδ T cells.There were obvious differences in the frequency of FasL+ T cells after stimulated with pathogene,antigen and some medicine during in vitro cultivation.FasL+ T cells can be representing a three to fivefold increase,targeting to Fas and effecting to Fas-mediated cytotoxicity and apoptosis[11].Moreover,most T cells in epithelial tissues express the V1 gene segment and respond to stress-associated factors,including heat shock proteins and the MHC-related molecules MHC-I-related protein A(MICA)and MHC-I-related protein B(MICB),which are induced on tumor and virus-infected cells[12].

We have found the C21 could improve the specific and non-specific cellular immunity in promoting antitumor cytokines(such as IL-2 and TNF-α)secretion in vivo.As well as after treatment of the C21,inhibitory effect on the transplanted Heps was observed in the tuner-bearing mice.

The purpose of this paper is to investigate and confirm the reinforcing immunity effect of C3N by observing the changes of phenotypes,proliferation activity,cytotoxicity and FasL expression of Vδ1+γδ T lymphocytes isolated from normal human peripheral blood and esophageal carcinoma tissue after treatment.

1 Materials and Methods

1.1 Reagents

Wilfoside C3N was synthesized from the tuber ofCynanchumauriculatumRoyle,planted in Jiangsu,and was extracted and identified as C3N with special purified-grade(98% C3N content)by Chinese Medicine Preparing Center of Zhejiang University of Traditional Chinese Medicine.All other chemicals were of reagent grade.

1.2 Cell line

Esophageal squamous carcinoma(ESCC)cell line ECA109 was obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences(Shanghai Institute of Cell Biology,Chinese Academy of Sciences),cultured in RPMI 1640 medium(Invitrogen)supplemented with 10% fetal bovine serum(FBS)(Invitrogen),50 ug/ml gentamycin and incubated at 37 ℃ in a humidified atmosphere containing 5% CO2.

1.3 Peripheral blood mononuclear cells preparation

Heparinized,peripheral blood samples were obtained from a total of 20 healthy donors，HD,female 10,male 10,age(30±10)years.Peripheral blood samples from each HD were obtained upon informed consent.Peripheral blood mononuclear cells(PBMCs)were isolated using Lymphoprep Ficoll-Hypaque(Tianjin MD Pacific Technology Co.,Ltd.,Tianjin,China)density gradient centrifugation.This study was approved by Ethical Approval Committee of Hebei North University.

1.4 γδ-T-cell purification and expansion

PBMCs were cultured at 1×106cells/ml,1ml/well in 24-well culture plates in complete medium RPMI 1640(Invitrogen),supplemented with 10%(v/v)fetal bovine serum(FBS)(Invitrogen),2 mmol·L-1L-glutamine(Gibco)and 50 ug/ml gentamycin at 37 ℃,5% CO2,95% humidity,in the presence of 10 U·mL-1rhIL-2(Changchun Changsheng Gene Pharmaceutical Co.Ltd.,Changchun,China)for 14 days,with added rhIL-2(10 U/mL)every 3-4 days.γδ T lymphocytes were isolated by positive selection with a magnetic affinity cell sorter(MACS)TCRγ/δ MicroBead kit(Miltenyi Biotec,USA).The purity of γδ T cells was measured by FACSAria(Becton Dickinson)flow cytometer with mAbs of anti-CD3-FITC and anti-TCR γδ-PE(BD Biosciences)(Purity>99%).Positively selected γδ T lymphocytes were cultured with 10% heat-inactivated autologous serum,L-glutamine(2 mmol·L-1),phytohaemagglutinin(PHA)(1μg·mL-1;Sigma))and rhIL-2(10 U·mL-1)for 7 days.Pamidronate(100 μM;Hangzhou Longhill Bio-Medication Technology Co.Ltd.,Hangzhou,China)was added to 50% of the cell pools to facilitate preferential outgrowth of Vδ1+γδ T lymphocytes.Vδ1+γδ T lymphocytes were enriched and followed by depletion of Vδ2+γδ T lymphocytes in these cultures[13].Highly purified Vδ1+γδ T cells were used at various test groups during extended culture as effector T cells in cytotoxicity or cytokine release assays.The purity of the respective Vδ1+γδ T cells was determined by FACSAria(Becton Dickinson)flow cytometer before each experiment.The γδ T cells were stained with mAbs to Vδ2 and Vδ1,followed by FITC-conjugated rabbit anti-mouse IgG(All of these mAbs were products of BD Biosciences).γδ T cells exhibited a stable Vδ1+γδ T TCR phenotype purity(>98%).

1.5 Transwell coculture

To evaluate the interaction between ECA109 cells and Vδ1+γδ T cells and the effects of C3N environment on the cells,cells were harvested and washed with PBS three times after incubation. Vδ1+γδ T cells at 1×106cells/ml(3 mL/well)were placed into the upper wells and ECA109 cells at 1×105cells·mL-1were seeded in the lower chamber of 6-well Transwell plates with polycarbonated membrane(Costar,pore diameter 0.4 μm)between upper and lower wells with C3N(4 μg·mL-1)in the microenvironment.Cells in Transwell were cultured with L-glutamine(2 mmol·L-1),PHA(1μg·mL-1)and rhIL-2(10 U·mL-1)for 48 h as described above.ECA109 cells and Vδ1+γδ T cells were paralleled cultured alone as control 1 and control 2,respectively.The same Transwell coculture of ECA109 cells and Vδ1+γδ T cells without C3N were used as control 3.ECA109 cells treated with C3N(4 μg·mL-1)for 48h were performed as control 4.CD95(Fas)-induced apoptosis of human Jurkat cell line by monoclonal antibody EOS9.1 was served as a positive control.

1.6 Analysis of apoptosis using FACS

Cells were harvested and washed twice with cold PBS.The cells were centrifuged at 500 g for 5 min at 4 ℃,then the supernatant was discarded and the pellet was resuspended in 500 μL of 1×annexin binding buffer(BD Biosciences)with 5 μL of annexin V- fluorescein isothiocyanate(BD Biosciences)and 0.5 μL of propidium iodide(BD Biosciences)for 30 min at room temperature in the dark.The percentage of cells with annexin V+/propidium iodide(PI)-was measured using flow cytometry(FACSAria,BD Biosciences)within 1 h.

1.7 Detection of Fas/FasL and perforin expression by flow cytometry

Cells were resuspended and stained for cell surface markers with FITC labeled mouse anti-human Fas mAb(BD Biosciences)and PE labeled mouse anti-human FasL mAb(Santa Cruz)(1 μg/106cells).Cells were incubated for 40 minutes in dark,washed with PBS three times and measured with the FACS Aria(Becton Dickinson).Perforin staining was performed by incubating Vδ1+γδ T cells.Cells were fixed with 4% paraformaldehyde(10 minutes at 4 ℃)and incubated with permeabilization buffer(0.5% saponin,5% FCS,and 0.1% sodium acid)for 10 minutes.PE labeled mouse anti-human perforin mAb(BD Biosciences)was added for 40 minutes in dark.Cells were immediately analyzed with 3μl isotype control antibody by flow cytometry as above.

1.8 RNA extraction and real-time fluorescent quantitative RT-PCR

Total Vδ1+γδ T cells cellular RNA was immediately extracted from harvested cells using Trizol reagent(TaKaRa Biotechnology Co,Ltd.,Dalian)according to the protocol of the supplier.RNA purity was estimated by the absorbance ratio A260/A280.The calculated ratios were in the range of 1.7-2.0.The SYBR Green I dye method was adopted and amplification was performed with a 7300 Real-Time PCR System(Applied Biosystemswity).Thirty-six cycles of pre-degeneration at 95 ℃ for 2 min,degeneration at 95 ℃ for 10 s,annealing at 50 ℃ for 10 s,and extension at 72 ℃ for 45 s were used for all experiments.Table 1 shows the synthetic oligonucleotide primer sequences used for the reaction.GAPDH served as a control.Critical threshold(Ct)cycle numbers were obtained from amplification of each group,and Δ Ct values were calculated by subtracting the Ct value of GAPDH from the Ct value of Fas or FasL. The ΔΔCt value was determined by subtracting the Δ Ct value for the controls from the Δ Ct value for the samples and calculating the Relative Quantity of Expression(RQE)of Fas or Fas by the equation:RQE=2-ΔΔCt.

Table 1 The synthetic primers used for RT-PCR

1.9 Measurement of other cytokines

After coculture of Vδ1+γδ T cells and ECA109 cells,supernatants were harvested and tested for release of IFN-γ,TNF-α,MIP-1α,MIP-1β and RANTES by ELISA using standard kits(BD Biosciences),according to the manufacturer’s recommendations.The absorbance per well was measured at 450 nm with a stat fax-2100(Awareness Technology Inc.)ELISA reader.Sample concentrations were calculated using the appropriate standard calibration.The concentration of cytokines in the supernatant was determined by regression analysis.

1.10 Statistical analysis

Statistical analyses were performed using independent-samples t-test procedure by the SPSS 10.0 software package for Windows.Differences were considered to be statistically significant atP<0.05.

2 Results

2.1 Effects of C3N on the ECA109 cells morphology in the presence of Vδ1+γδ T cells

Treatment with C3N at a final concentration of 4 μg·mL-1induced significant morphological changes of the ECA109 tumor cells and the effect was most pronounced in the presence of Vδ1+γδ T cells.The untreated control ECA109 cells were short spindle-shaped and triangle-shaped and exhibited characteristics resembling those of epithelial cells(Figure 1A).After co-cultivation with Vδ1+γδ T cells for 48h,the ECA109 cells grew slowly with long spindle connections between adjacent cells(Figure 1B).Upon treatment with 4 μg·mL-1concentration of C3N for 48h,morphological changes were observed.ECA109 cells retracted and gradually became round-shaped(see Figure 1C).Whereas,when treated with C3N and cocultured with Vδ1+γδ T cells together,ECA109 cells not only became round-shaped but also increased shedding(see Figure 1D).

Fig 1 Effects of C3N and Vδ1+γδ T cells co-cultivation on the ECA109 cells morphology A.ECA109 cells in the control group;B.ECA109 cells cocultured with Vδ1+γδ T cells for 48h; C.ECA109 cells treated with 4 μg·mL-1concentration of C3N for 48h; D.ECA109 cells treated with 4 μg·mL-1concentration of C3N and cocultured with Vδ1+γδ T cells for 48h.

2.2 Induction of tumor apoptosis apoptosis by C3N in the presence of Vδ1+γδ T cells

The tumor cells treated with C3N and co-cultured with Vδ1+γδ T cells were detected for apoptosis using Annexin V and PI double staining followed by FCM.The percentage of early apoptotic CA109 cells cocultured with Vδ1+γδ T cells or treated with C3N alone or in combination was(5.9±0.7)%,(9.3±1.5)%,and(27.9±5.6)%respectively.However,the untreated control tumor cells maintained a minimal level of apoptosis(1.7±0.3)%.The apoptosis rate in tumor cell cultures containing both C3N and T cells was statistically significantly higher than those in the control or singly treated cultures(see Figure 2).

2.3 Induction of Fas/FasL expression by C3N

Upon C3N treatment,strong expression of FasL in Vδ1+γδ T cells was induced at both protein and mRNA levels while Fas was significantly induced in ECA109 cells(see Figure 3).

Fig 2 Apoptosis rate of ECA109 cells detected by flow cytometry after various treatments.

Fig 3 Expressions of FasL and Fas on Vδ1+γδ T cells and ECA109 cells after different treatments.

A.Expressions on Vδ1+γδ T cells;B.Expression on ECA109 cells;C.Relative expression of mRNA on Vδ1+γδ T cells;D.Relative expression of mRNA on ECA109 cells.T Cells:Vδ1+γδ T cells in the control group;ECA109 Cells:ECA109 cells in the control group;T Cells+ECA109 Cells:ECA109 cells cocultured with Vδ1+γδ T cells for 48 h;ECA109+C3N:ECA109 cells treated with 4 μg·mL-1concentration of C3N for 48 h;T Cells+ECA109+C3N:ECA109 cells treated with 4 μg·mL-1concentration of C3N and cocultured with Vδ1+γδ T cells for 48 h.*P<0.05;**P<0.01.

2.4 Induction of perforin expression by C3N

As shown in Figure 4,the percentage of perforin positive Vδ1+γδ T cells was significantly higher in cultures containing both ECA109 cells and C3N than that of cultures containing either alone.

Fig 4 Expressions of perforin on Vδ1+γδ T cells after different treatments.T Cells:

Vδ1+γδ T cells in the control group;T Cells+ECA109:ECA109 cells cocultured with Vδ1+γδ T cells for 48 h;T Cells+ECA109+C3N:ECA109 cells treated with 4 μg/mL concentration of C3N and cocultured with Vδ1+γδ T cells for 48 h.

2.5 Induction of cytokine expression by C3N

We further monitored the cytokine secretion by Vδ1+γδ T cells upon C3N treatment and we found that IFN-γ,TNF-α,MIP-1α,MIP-1β and RANTES reached the highest levels when the T cell cultures were treated C3N in the presence of ECA109 cells.Vδ1+γδ T cells cultured alone did not secrete any significant levels of these cytokines.Only a low level expression was detected in the supernatant of the ECA109-co-cultured Vδ1+γδ T cells in the absence of C3N.Significant cytokine secretion was only detected in Vδ1+γδ T cells treated with both ECA109 and C3N(see Table 2).

Table 2 Cytokine levels in supernatant of cultivation measured by ELISA.(x±s).

The data are representative of three separate experiments and are expressed as the means of triplicates,Significant differences between the values for Vδ1+γδ T cells control and co-cultivation without or with C3N(*P<0.05;**P<0.01).

3 Discussion

In human peripheral blood two main populations of γδ T cells have been identified based on the TCR composition.Vδ2+γδ T cells constitute the majority of γδ T cells in peripheral blood whereas Vδ1+γδ T cells reside preferentially in skin epithelium and in the intestine.All previous reports of tumor-reactive γδ T lymphocytes support the exclusive expression of the Vδ1+TCR.Preferential expansion and cytotoxic antitumor responses of the Vδ1+γδ T cells subset have been demonstrated in patients with tumors of epithelial origin,including adenocarcinomas of the lung[14-15]and in renal cell cancer[16].Based on the antitumor properties and the abilities of improving the specific and non-specific cellular immunity by promoting antitumor cytokines(such as IL-2 and TNF-α)secretioninvivo,wilfoside C3N,the main active component in theC21,is suggested that can enhance Vδ1+γδ T cell activity,stimulate the production and activity of cytokines.With this data in mind,we addressed the question of whether C3N activate human Vδ1+γδ T cells.Although the mechanisms underlying C3N anti-tumor effect may be complex,our findings demonstrated that this effect was most likely related to immune regulation to Vδ1+γδ T cells.At the optimal condition of C3N(4 μg·mL-1,48h treatment)which had been demonstrated in our previous reports,the apoptotic percentages of ECA109 cells were 5.9% in cocultured with Vδ1+γδ T cells,9.3% in treated with C3N at 4 μg·mL-1,and 27.9% in incubated with them together for 48h by using flow cytometry.The morphologic experiment showed the same results as those obtained in flow cytometry analysis.In other words,ECA109 cells were significantly inhibited by C3N and Vδ1+γδ T cells treated togetherinvitro.To further determine the effects of C3N on Vδ1+γδ T cells activated and Vδ1+γδ T cells-mediated anti-tumor immunity,Fas/FasL protein and mRNA expression,perforin intracellular expression of Vδ1+γδ T cells,and the cytokine(IFN-γ,TNF-α,MIP-1α,MIP-1β and RANTES)production were evaluated. In accordance with previous studies,the combination of C3N and Vδ1+γδ T cells were the most effective in apoptotic relative cytokines expression compared with C3N and Vδ1+γδ T cells alone.

Apoptotic cell death is initiatedviaa variety of extracellular and intracellular death signals[17-18].Enhanced cytokine(IFN-γ,TNF-α,MIP-1α,MIP-1β and RANTES)expression(caused by C3N)on human Vδ1+γδ T cells was also evident in transwell cocultures of purified Vδ1+γδ T cells stimulated with C3N,provided that Vδ1+γδ T cells were activated with C3N.For well-recognized,apoptosis can be induced by three pathways:a)granule-dependent exocytosis pathway,b)Fas-FasL linkage-mediated pathway,and c)cross-linking of TNF and TNFR type I[19].

Granule-dependent exocytosis pathway is established through intracellular signaling after target cell recognition by a cytotoxic lymphocyte(NK or cytotoxic T cell).In exocytosis or degranulation,there is microtubules mobilization that leads the preformed granules or lysosomes of the cytotoxic cell towards the point of contact with the target cell,releasing stored lytic molecules[20-21].The lytic molecules stored in granules that induce apoptosis are perforin, granzymes(Grzs),and granulysin.The evidence[22]indicates that perhaps perforin is not essential for the entrance of proteases into the target cell,but it is required for cytolysis.For functional studies on the role of perforin,dose-response assays are needed,whereas,perforin is an unstable molecule,therefore,the amounts vary according to the cytotoxic cell population[23].In our study,we found that C3N could increase the percentage of perforin positive Vδ1+γδ T cells significantly in co-culturing Vδ1+γδ T cells and ECA109 cells with C3N group.We speculated that the high percentage of perforin positive Vδ1+γδ T cells may lead to significant apoptosis of ECA109 cells.Fas-FasL linkage-mediated pathway is important in the control of constantly stimulated T cells,and in promoting tolerance to self-antigens,aside from being a homeostatic mechanism of the cytotoxic T cell activity.Effector T cells and NK cells express FasL(CD178),whereas target cells express Fas(CD95 or Apo-1),thus these cells are susceptible to apoptosis mediated by this pathway[24].In our previous reports,we suggest that C3N-driven apoptosis of ECA109 cellsinvitrowas dependent on caspase-2/mitochondria/caspase-9 and independent of the Fas-FasL/caspase-8 pathway.However,in the present study,expression of FasL on Vδ1+γδ T cells and Fas on ECA109 cells were markedly increased when coculture them together with C3N.In accordance with the results of the most effective apoptosis,we suspected that C3N-induced apoptosis of ECA109 cells was dependent on Fas-FasL linkage-mediated pathway when combined with Vδ1+γδ T cells which expressed increased FasL after treated with C3N.Fas belongs to the tumor necrosis factor receptor(TNFR)-I type family.FasL is a member of the TNFR-II type family[25].Binding of Fas with FasL causes trimerization and recruitment of Fas-associated death domain(FADD)proteins through homotypic death domain interactions.The main docking protein of TNFR is the TNFR-associated death domain protein(TRADD),which binds to the TNFR via an interaction between the respective death domains[26].TRADD then recruits other death domain-containing proteins,including the Fas-associated protein with death domain(FADD),via its death domain[27].Finally,FADD can recruit caspase 8 to the complex[28],which in turn is cleaved and activated,triggering the apoptotic response.

TNF-α is a cytokine produced by activated cells that induces cell apoptosis,cell activation,and inflammatory processes.Our present studies showed that high level expression of TNF-α was detected in the supernatant of co-culture Vδ1+γδ T cells and ECA109 cells group. TNF family members exert their biological effects through the TNFR superfamily of cell surface receptors[29].The binding of TNF-α to TNFR1 triggers a series of intracellular events initiated by the recruitment of a key adaptor protein TNFR1-associated death domain protein(TRADD)to the receptor complex.In most cells the combination of the above signaling pathways determines the diverse biological activities of TNF-α,including cell apoptosis[30].We do not yet know if TNF-α binds to TNFR in the present studies. However,we have ruled out the possibility that apoptosis of ECA109 cells associated with TNF high level expression in co-culture Vδ1+γδ T cells and ECA109 cells with C3N group.Besides TNF-α,the study confirmed that IFN-γ was up-regulated in such circumstances.We suggested another way that the combination of C3N and the induction of IFN-γ producing Vδ1+γδ T cells eradicated ECA109 cells.

In addition to the generally accepted notion that Fas ligation induces apoptosis of sensitive cells,increasing evidence indicated that it can also elicit inflammation by secreting CC and CXC chemokines.Chemokines with a CXC structure are potent chemoattractants for neutrophils,while chemokines with a CC structure preferentially attract monocytes.Certain CC chemokines,such as MIP-1α,MIP-1β and RANTES,induce the migration of activated T lymphocytes[31].Chemokines can also influence the tumor environment in ways that can promote or retard tumor growth[32].MIP-1α,MIP-1β and RANTES are strongly chemotactic for mononuclear cells and DC[33].We detected that these chemokines were remarkable increased in the supernant of coculture Vδ1+γδ T cells and ECA109 cells with C3N group.We suggested that some components of the secreted molecules were biologically active as tumor-specific stimulation of effector Vδ1+γδ T cells and induced apoptosis of tumor cellsinvitro.However,we had not evidences whether MIP-1α,MIP-1β and RANTES were direct growth inhibitors of ECA109 cells.

Our current and prior findings indicate that C3N-activated Vδ1+γδ T cells mediated anti-tumor immunity to ECA109 cellsinvitroexhibits pleiotropic effects via both granule-dependent exocytosis pathway and Fas-FasL linkage-mediated pathway.Furthermore,tumor-specific effector Vδ1+γδ T cells were able to change the chemokine expression pattern which augments the anti-tumor effects of C3N-activated T cells.Yet,we still have little knowledge about the mechanisms of the effects.Future studies will need to provide a more quantitative and qualitative analysis of C3N treated Vδ1+γδ T cells anti-tumor effects including remaining to be clarified whether C3N induces apoptosisinvivowithout major side effects on other organs.Current efforts are directed at confirming C3N-activated gammadelta T cells mediated anti-tumor immunity.Such characterization may provide additional insights into the mechanisms that are operational during gammadelta T cell-mediated tumor cells apoptosis and C3N act as potential therapeutic agent for cancers.

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[英文編輯：劉彥哲]

R 737.11

A

10.3969/j.issn.1673-1492.2017.02.001

來稿日期：2017-03-08