Preliminary Study On The Induction Of ECCE-CIK And Its Anti-tumor And Antiviral Effects

Cytokine induced killer cells(CIK)can effectively inhibit the replication of hepatitis b virus(HBV)in patients with chronic hepatitis b(CHB).It has been considered to be an effective adoptive immunotherapy treatment strategy for the treatment of CHB patients.However,due to the existence of individual differences and the immunosuppression status in some CHB patients,there is defection in the quantity and quality of CIK cultured by traditional method.Engineered cells for costimulatory enhancement(ECCE)is a 4-1BBL-expressing K562 cell line,which can combine with 4-1BB on the surface of T cells to promote the proliferation of T cells.We studied the expansion of CIK cultured with ECCE and preliminarily established the culture system of ECCE-CIK by detecting the phenotypes,cytotoxicity to the tumor cell lines and secretion of cytokines.Then,the ECCE-CIK culture system was implemented in CHB patients.And we also explored the inhibitory effect of ECCE-CIK on HBV in human cell line HepG2.2.15,which could provide experimental basis for the usage of ECCE-CIK cells in the treatment of CHB.[Objective]1.To construct ECCE-CIK and detect its proliferation,phenotypes,cytotoxicity to the tumor cell lines and secretion of cytokines for providing a new method to improve the cultivation of CIK.2.To explore the inhibitory effect of ECCE-CIK from the patients with CHB on the HBV replication in HepG2.2.15 cell lines preliminarily for providing experimental basis for usage of ECCE-CIK in the treatment of CHB.[Methods]1.Peripheral blood mononuclear cells(PBMC)were collected and isolated from 10 healthy people.PBMC were induced with IFN-?,CD3 monoclonal antibodies,and IL-2 to produce conventional CIK.The irradiated ECCE cells were co-cultured with PBMC at proportions of 1:1,10:1 or 1:10,then induced by adding IFN-?,CD3 monoclonal antibodies and IL-2 to produce the ECCE-CIK.Expansion of conventional CIK and ECCE-CIK were assessed at regular intervals.The phenotypes and cytotoxicity of conventional CIK and ECCE-CIK were evaluated by CFSE/PI,CCK-8 and cytokines were measured by ELISA.2.Peripheral blood were collected from 10 CHB patients and ECCE-CIK were cultured.We established direct and indirect co-culture system in vitro.In direct co-culture system,ECCE-CIK and HepG2.2.15 cells contacted directly.In the indirect co-culture system,ECCE-CIK and HepG2.2.15 cells were separated by a semipermeable membrane 0.4?m,which allowed the passage of soluble factors produced by ECCE-CIK.ECCE-CIK were co-cultured with HepG2.2.15 at different effector to target ratios for 3h,24h,or 48h separately.At the end of the experiments,the corresponding culture supernatants were harvested for measurement of the cytotoxicity by lactate dehydrogenase(LDH)release assay,HBV DNA by real-time PCR,HBsAg by Chemiluminescence microparticle immunoassay,IFN-?and TNF-?by ELISA.[Results]1.On the 21st day,the proliferation fold of traditional CIK was 73.79±7.28(PBMC(10^5)),the proliferation fold of ECCE-CIK were 398.05±36.37(ECCE:PBMC=1:1)and 567.23±108.30(ECCE:PBMC=10:1)respectively,when the 1×10^5/ml PBMC from healthy people were used as the initial concentration.And the proliferation fold of traditional CIK was 523.51±62.81(PBMC(10^6)),the proliferation folds of ECCE-CIK were 2856.21±94.17(ECCE:PBMC=1:10)and4123.25±158.54(ECCE:PBMC=1:1)respectively,when the 1×10^6/ml PBMC from healthy people were used as the initial concentration.Compared with traditional CIK,the expansion fold of ECCE-CIK in healthy people were significantly higher,and the difference was statistically significant.The expansion fold of ECCE-CIK(1×10^6/ml PBMC,ECCE:PBMC=1:1)was the highest.2.The proportion of CD3~+CD8~+in ECCE-CIK group was 78.2%±4.6%,while the percentage was 65.9%±5.7%in traditional CIK group(P<0.01).The percentage of CD3~+CD56~-was 43.5%±4.2%in ECCE-CIK group and 37.8%±3.6%in traditional CIK group(P<0.01).3.The traditional CIK and ECCE-CIK were used as effector cells to co-culture with different tumor cell lines SMMC7721,MGC803,A375 and A549.The inhibition rate significantly increased with the effector-target ratio for both traditional CIK and ECCE-CIK group,and the cytotoxic effect of ECCE-CIK was stronger than traditional CIK.The tumor cell lines were used as target cells at the ratio of E:T 20:1and the time of co-culture was 3 hours.The cytotoxic effect of ECCE-CIK and traditional CIK were 83.2%±3.5%and 20.5%±4.3%to SMMC7721 cells respectively(P<0.01).The cytotoxic effect of ECCE-CIK and traditional CIK were 48.7%±5.5%and 20.4%±6.3%to MGC803 cells respecively(P<0.01).When A375 were used as target cells,the cytotoxic effect of ECCE-CIK and traditional CIK were 53.6%±4.5%and 22.3%±3.3%respectively(P<0.01).When A549 were used as target cells,the cytotoxic effect of ECCE-CIK and traditional CIK were 53.5%±3.5%and23.6%±4.3%respectively(P<0.01).4.The levels of IFN-?and TNF-?in supernatants of ECCE-CIK,which were co-cultured with SMMC7721,were higher obviously than that of traditional CIK(ECCE-CIK VS traditional CIK,IFN-?,1090.99±127.52 pg/ml VS 396.58±49.1pg/ml,P<0.01;TNF-?,732.71±234.49 pg/ml VS 117.41±30.01 pg/ml,P<0.01).5.The proliferation of CIK from CHB patients was enhanced by ECCE.The proliferation folds of ECCE-CIK were 1563.76±186.55,while the folds of traditional CIK were 228.53±65.82(P<0.01).The percentage of CD3~+CD8~+in ECCE-CIK was higher than that in traditional CIK(90.5%±13.6%VS 80.3%±8.3%,P<0.05).6.The cytotoxic effect of ECCE-CIK from CHB patients when co-cultured with HepG2.2.15 in direct system increased dramatically in the E:T-and time-depended manners.When the timepoint was 24h,the cytotoxic effect was 23.6%±2.6%,63.9%±5.3%and 98.2%±2.0%for the E:T of 1:1,5:1 and 10:1 respectively.When the E:T was 5:1,the cytotoxic effect was 23.5%±2.5%,63.9%±5.3%,95.6%±2.5%for the timepoint of 3h,24h,48h respectively.However,the cytotoxic effect was lower than 2%in indirect co-culture system.7.The HBV DNA and HBsAg from HepG2.2.15 inhibited by ECCE-CIK from patients with CHB gradually increased with the effector-target ratio and the extension of incubation time in both direct and indirect co-culture system.And there was no statistical difference between two co-culture systems.8.When ECCE-CIK from CHB patients were cultured with HepG2.2.15,the level of IFN-?and TNF-?in the supernatants increased with the effector-target ratio in direct system.The level of IFN-?and TNF-?in indirect system were similar with that in direct system.[Conclusions]1.ECCE can significantly enhance the proliferation of CIK generated from healthy donors in vitro and generate ECCE-CIK.Compared with the traditional CIK,the proportion of CD3~+CD8~+subgroup in ECCE-CIK increased,while the cytotoxicity to the tumor cell lines and the secretion of IFN-?and TNF-?were enhanced.2.ECCE can significantly enhance the proliferation of CIK generated from patients with CHB.ECCE-CIK from CHB patients can kill HepG2.2.15 in the manner of direct contact and independent of HBV.The cytotoxic effect increased with the effector-target ratio and the extension of incubation time.3.ECCE-CIK from CHB patients can inhibit the replication of HBV in HepG2.2.15 cells in vitro by non-cell lysis.The effect of suppression increased along with the increase of effector-target ratio and the extension of incubation time.4.IFN-?and TNF-?secreted by ECCE-CIK from CHB may be the main effector molecule for the suppression of HBV generated by HepG2.2.15.