Enhanced Effects Of Interleukin21on Anti-leukemic Activity Of Cytokine Induced Killer Cells

Objective:To explore the effects of humanized interleukin21(IL-21) and the mechanism on anti-leukemic activity of cytokine inducing killer(CIK) cells derived from peripheral blood(PB).Methods:1. Mononuclear cells were separated from peripheral blood, and cultured with cytokines to induce CIK cells.2. IL-21lentiviral vector was constructed and used to transfect293T cells.The the culture supernatant with virus infected CIK cells was identified. At conditions with or without stimulation by exogenous and endogenesis IL-21:3. Proliferation of CIK cells and their cytotoxic activity against K562cells was measured by MTT method.4. IL-21receptor(IL-21R) and FasL on the surface of CIK cells, intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry.5. The expression of IFN-γ、TNF-α、TNF-β、perforin、 granzyme A、 granzyme B、FasL and NKG2D mRNA were measured by semi-quantitative RT-PCR.6. The concentration of IFN-γ and TNF-α in the cultured supernatant were measured by enzyme immunoassay.7. JAK-STAT signalling pathway of CIK cells were measured by western-blot.Results:1. At conditions with or without stimulation by exogenous IL-21:CIK cells showed the following changes:(1) the total number of cells were unchanged, but the proportion of CIK cells were increased from17.5±4.7%to26.5±2.1%(p<0.05).(2) Cytotoxic activity against K562cells by CIK cells was increased from22.8±2.8%to44.6±8.3%(p＜0.05).(3) The expression of IL-21R was increased about2folds.(4) The mRNA expression of IFN-γ increased from0.3760±0.2358to0.7786±0.2493(p＜0.05), TNF-α increased from0.6557±0.1598to1.3145±0.2136(p<0.05), perforin increased from0.6361±0.1457to0.9831±0.1265(p<0.05), granzyme B increased from0.4084±0.1589to0.7319±0.1639(p＜0.05), FasL increased from0.4015±0.2842to0.7381±0.2568(p＜0.05), the expression of granzyme A、TNF-β and NKG2D were similar with control.(5) Detected by flow cytometry, the expression of FasL of CIK cells was higher than that of control(0.19%vs.0.04%), the expression of perforin increased from35.28%to53.16%, and the expression of granzyme B increased from43.16%to78.82%, the concentration of IFN-γ in the culture supernatant increased almost2folds, from25.8±6.1ng/L to56.0±2.3ng/L (p<0.05), and TNF-α increased almost3folds, from5.64±0.61ug/L to15.14±0.93μg/L (p<0.05).(6) The result of western blot showed expression of STAT1and STAT5a had no significant differences, but the expression of STAT3and STAT5b were higher than that of control.2. At conditions with or without stimulation by endogenesis IL-21:By restriction enzyme digestion and sequencing, IL-21lentiviral vector was identified, after transfecting virus surpematant into CIK cells, the expression of IL-21was detected in CIK cells. Compared to control,(1) the total number of cells were unchanged, but the proportion of CIK cells were increased from16.95±4.7%to24.60±2.1%(p<0.05).(2) Cytotoxic activity against K562cells by CIK cells was increased from23.28±2.8%to58.42±8.3%(p＜0.05), and stayed at61.16±6.2%after5days, it was stronger and longer compared to the exogenous effect of IL-21from22.80±2.8%to44.60±8.3%(p<0.05).(3) The expression of IL-21R increased about2folds.(4) The mRNA expression of IFN-yand TNF-a increased almost1.5folds, perforin,granzyme B, FasL increased almost2folds, the expression of granzyme A, TNF-β and NKG2D were similar with control.(5) Detected by flow cytometry, the expression of FasL of CIK cells was higher than that of control(0.56%vs.0.06%), the expression of perforin increased from12.23%to25.86%, and the expression of granzyme B increased from14.56%to37.58%, the concentration of IFN-y in the culture supernatant increased from23.2±5.6ng/L to55.3±3.5ng/L (p<0.05), and TNF-α increased from5.65±0.58μg/L to15.62±0.62μg/L (p<0.05)Conclusion:Humanized IL-21could enhance the anti-leukemic activity of CIK cells and the CIK cells that express IL-21endogenously had more stronger anti-leukemic activity via increasing IL-21R, perforink, granzyme B, FasL, IFN-y and TNF-a, as well as activating JAK-STAT signaling pathway, and IL-21acts in synergism with IL-2. These data indicate IL-21has a potential clinic value in the enhancement of anti-lekemic immunotherapy.