[Effects of humanized interleukin 21 on anti-leukemic activity of cytokine induced killer cells and the mechanism].

OBJECTIVE: To explore the effects of humanized interleukin 21 (IL-21) on anti-leukemic activity of cytokine induced killer(CIK) cells derived from peripheral blood(PB) and the mechanism.

METHODS: Mononuclear cells were separated from peripheral blood and cultured with cytokines to induce CIK cells. Proliferation of CIK cells with or without IL-21 stimulation and their cytotoxic activity against K562 cells was measured by MTT method. IL-21 receptor (IL-21R) and immunophenotypes of CIK cells were measured by flow cytometry. The expression of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), tumor necrosis factor-β (TNF-β), perforin, granzyme A, granzyme B, FasL and NKG2D mRNA were measured by semi-quantitative RT-PCR. FasL on the surface of CIK cells and intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry. The concentration of IFN-γ and TNF-α in the cultured supernatant were measured by enzyme immunoassay. JAK-STAT signalling pathway of CIK cells were measured by Western-blot.

RESULTS: After IL-21 stimulation, the proportion of CIK cells increased from (17.5 ± 4.7)% to (26.5 ± 2.1)%. Cytotoxic activity against K562 cells by CIK cells increased from (22.8 ± 2.8)% to(44.6 ± 8.3)%. The expression of IL-21R increased about 2 folds. The mRNA expression of IFN-γ increased almost 2 folds from (0.3760 ± 0.2358) to (0.7786 ± 0.2493), TNF-α increased almost 2 folds from (0.6557 ± 0.1598) to (1.3145 ± 0.2136), perforin increased almost 1.5 folds from (0.6361 ± 0.1457) to (0.9831 ± 0.1265), granzyme B increased almost 2 folds from (0.4084 ± 0.1589) to (0.7319 ± 0.1639), FasL increased almost 2 folds from (0.4015 ± 0.2842) to (0.7381 ± 0.2568), the expression of granzyme A, TNF-β and NKG2D were similar with control. Flow cytometry analysis showed that the expression of FasL of CIK cells was higher than that of control (0.19% vs 0.04%), the expression of perforin increased from 35.28% to 53.16%, and the expression of granzyme B increased from 43.16% to 78.82%. The concentration of IFN-γ in the culture supernatant increased almost 2 folds from (25.8 ± 6.1) ng/L to (56.0 ± 2.3) ng/L, and TNF-α increased almost 3 folds from (5.64 ± 0.61) µg/L to (15.14 ± 0.93) µg/L. Western blot showed that the expression of STAT1 and STAT5a had no significant differences, but the expression of STAT3 and STAT5b were higher than that of control.

CONCLUSION: Humanized IL-21 could enhance the anti-leukemic activity of CIK cells via increasing IL-21R, perforin, granzyme B, FasL, IFN-γ and TNF-α, as well as activating JAK-STAT signaling pathway. These data indicate that IL-21 has a potential clinical value in the enhancement of anti-leukemic immunotherapy.