OBJECTIVETo 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.

METHODSMononuclear 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.

RESULTSAfter 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.

CONCLUSIONHumanized 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.