title: [Effects and mechanism on anti-leukemic activity of cytokine-induced killer cells with an endogenous expression of interleukin-21].
abstract: OBJECTIVE
  To explore the effects and mechanism on anti-leukemic activity of cytokine inducing killer (CIK) cells with an endogenous expression of interleukin-21 (IL-21).
  
  
  METHODS
  Mononuclear cells were isolated from peripheral blood and cultured with cytokines to generate CIK cells. IL-21 lentiviral vector was constructed and used to transfect 293T cells. Then the culture supernatant with virus infected CIK cells was identified. Proliferation of CIK cells and their cytotoxic activity against K562 cells were measured by methyl thiazolyl tetrazolium (MTT). The expressions 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 reverse transcription-polymerase chain reaction (RT-PCR). Immunophenotypes of CIK cells, IL-21 receptor (IL-21R) and FasL on the surface of CIK cells, intra-cellular perforin and granzyme B of CIK cells were measured by flow cytometry. And the concentrations of IFN-γ and TNF-α in cultured supernatant were measured by enzyme immunoassay.
  
  
  RESULTS
  By restriction enzyme digestion and sequencing, IL-21 lentiviral vector was identified, after transfecting virus supernatant into CIK cells, the expression of IL-21 was detected in CIK cells. Compared to control, (1) the total number of cells remained unchanged, but the proportion of cells expressing CD3(+)/CD56(+) phenotype increased from 16.95% ± 4.70% to 24.60% ± 2.10%. (2) Cytotoxic activity against K562 cells by CIK cells increased from 23.3% ± 2.8% to 58.4% ± 8.3% and stayed at 61.2% ± 6.2% after 5 days. It was stronger and longer compared to the exogenous effect of IL-21 (from 22.8% ± 2.8% to 44.6% ± 8.3%). (3) The expression of IL-21R increased around 2 folds. (4) The mRNA expressions of IFN-γ and TNF-α increased almost 1.5 folds, perforin, granzyme B, FasL rose almost 2 folds, the expressions of granzyme A, TNF-β and NKG2D were similar with those of controls. (5) Detected by flow cytometry, the expression of FasL of CIK cells was higher than that of control (0.56% ± 0.37% vs 0.06% ± 0.02%), the expression of perforin increased from 12.23% ± 2.35% to 25.86% ± 6.13%, the expression of granzyme B rose from 14.56% ± 1.36% to 37.58% ± 2.30%, the concentration of IFN-γ in culture supernatant spiked from (23.2 ± 5.6) to (55.3 ± 3.5) ng/L and TNF-α jumped from (5.6 ± 0.6) to (15.6 ± 0.6) µg/L.
  
  
  CONCLUSIONS
  CIK cells with an endogenous expression of IL-21 have stronger anti-leukemic activity through an up-regulation of IL-21R, perforin, granzyme B, FasL, IFN-γ and TNF-α. Thus IL-21 may potentially enhance the anti-leukemic immunotherapy.
authors.affiliation: First Center Clinical College of Tianjin Medical University, Tianjin 300192, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhao
authors.forename: Ming-feng
authors.initials: MF
authors.lastname: Zhao
authors.forename: Sajin
authors.initials: S
authors.lastname: Rajbhandary
authors.forename: Wen-yi
authors.initials: WY
authors.lastname: Lu
authors.forename: Hai-bo
authors.initials: HB
authors.lastname: Zhu
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Quan
authors.initials: Q
authors.lastname: You
authors.forename: Xia
authors.initials: X
authors.lastname: Xiao
authors.forename: Qi
authors.initials: Q
authors.lastname: Deng
authors.forename: Yu-ming
authors.initials: YM
authors.lastname: Li
mesh_terms: D002478:Cells, Cultured; D055612:Cytokine-Induced Killer Cells / Q000378:metabolism*; D053222:Fas Ligand Protein / Q000378:metabolism; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism*; D020014:K562 Cells; D008233:Lymphotoxin-alpha / Q000378:metabolism; D054353:Perforin / Q000378:metabolism; D053655:Receptors, Interleukin-21 / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000098546:Interleukin-21
chemical_list: C505661:FASLG protein, human; D053222:Fas Ligand Protein; D007378:Interleukins; D008233:Lymphotoxin-alpha; D053655:Receptors, Interleukin-21; D014409:Tumor Necrosis Factor-alpha; D054353:Perforin; D007371:Interferon-gamma; D053804:Granzymes; D000098546:Interleukin-21