The Effect Of IL-15and IL-21on Human Cytokine-induced Killer Cells In Vitro

ObjectiveTo investigate the effect of IL-15and IL-21on human cytokine-induced killer(CIK) cells in vitro.MethodsCIK cells were induced from healthy volunteers peripheral blood mononuclearcells (PBMCs) by Human Lymphocyte Separation Medium, and then were induceddifferentiating into CIK within IL-15, IL-21and the combination, as experimentalgroup (IL-15group, IL-21group and IL-15combined IL-21group). The controlgroup only added to conventional medium. The cells were cultured in incubator with5%CO2、37℃for10days.1) We analyzed the proliferation of CIK cells by cellcounting trypan blue exclusion test.2) The CIK cell phenotypes ofCD3+/CD3+CD4+/CD3+CD8+/CD3+CD56+were determined by Flow cytometer(FCM).3) The cytokines production of CIK cell, such as IFN-γ, TNF-α, IL-12, wasanalyzed by Enzyme-linked immunosorbent assay (ELISA). CIK cells co-culturedwith esophageal cancer cell line EC9706.4) Cytotoxicity of esophageal carcinomacell line EC9706cells with CIK cells was analyzed by Cell Counting Kit-8(CCK-8).5. Apoptosis cells of esophageal carcinoma cell line EC9706cells with CIKcells were determined by FCM using Annexin V-FITC/PI staining. ResultsThe results of CIK cells induced by IL-15, IL-21are shown:1. The number of cells in control group was (7.83±1.18)×105/ml,(26±9.86)×105/ml in IL-15group,(14.58±1.81)×105/ml in IL-21group, and (11.53±1.70)×105/ml in IL-15+IL-21group. The number of CIK cells in each experimental groupwere higher than control group (P <0.05).2. The CD3+of CIK cells phenotype in the IL-15group and IL-21group wasmore than control group,and the difference was statistically significant (P<0.05).The CD3+CD4+of CIK cell phenotype in the IL-15group and IL-21group washigher than the control (P<0.05). The CD3+CD8+of CIK cells phenotype in theexperimental groups percentile were higher than the control, and the difference wasnot statistically significant (P<0.05). The CD3+CD56+of CIK cell phenotype in theIL-15+IL-21group was higher than control group, and the difference also wasstatistically significant (P <0.05).3. The cytokines production IFN-γ of CIK cell in the control group was lowerthan the IL-15group, and the difference was statistically significant (P<0.05). IL-21group was and IL-15combined IL-21group were more than the control group, butthe difference was not statistically significant. The content of TNF-α in IL-15groupand IL-21group were higher than the control group,and the difference wasstatistically significant (P<0.05). The content of TNF-α in IL-15combined IL-21wasless than the control group, and the difference was statistically significant (P<0.05).However, IL-12was not detected in the supernatant of CIK cells.4. After the CIK cells co-cultured with esophageal cancer EC9706, the cellinhibition rate was (26.56±11.2)%in IL-15group,(8.56±3.12)%in IL-21group,(18.41±8.72)%in the combined group. The differences was statistically significantbetween the two groups (P <0.05).5. Apoptosis cells of esophageal carcinoma cell line EC9706cells with CIK cellswas detected: the apoptosis rate of control group was (13.25±1.17)%. Comparing tocontrol group, the apoptosis rate of IL-15group was (15.71±1.31)%,(15.33±0.91)%in IL-21group,(18.25±4.37)%in IL-15and IL-21combined group. Eachexperimental group apoptosis rate was higher than control group, and the differencewas statistically significant (P <0.05). ConclusionsThe study suggests that:1.It can promote CIK cell proliferation induced withIL-15, IL-21alone or combination.2. The CD3+and CD3+CD8+of CIK cell phenotype with IL-15alone can beincreased expression. The CD3+, CD3+CD8+and CD3+CD56+expression of CIKcell phenotype with IL-21alone can be increased. The CD3+, CD3+CD8+andCD3+CD56+expression of CIK cell phenotype with IL-21and IL-15can beincreased, too.3. The cytokines production IFN-γ and TNF-α of the CIK cells induced withIL-15alone can be increased. The cytokines production the TNF-α of the CIK cellsinduced with IL-21alone. It was no effect in IL-15and IL-21. IL-12was not detectedin the supernatant.4. It can enhance the cytotoxicity of CIK cells induced with IL-15and IL-21forEC9706esophageal cancer cell lines. And the IL-15group and the combined group isbetter than the cytotoxic effect of IL-21group.