title: Multiple cytotoxic factors involved in IL-21 enhanced antitumor function of CIK cells signaled through STAT-3 and STAT5b pathways.
abstract: OBJECTIVE
  Maintenance of cellular function in culture is vital for transfer and development following adoptive immunotherapy. Dual properties of IL-21 in activating T cells and reducing activation induced cell death led us to explore the mechanism of action of IL-21 enhanced proliferation and cytotoxic potential of CIK cells.
  
  
  METHODS
  CIK cells cultured from PBMCs of healthy subjects were stimulated with IL-21 and cellular viability and cytotoxicity to K562 cells were measured. To elucidate the mechanism of action of IL-21, mRNA expression of cytotoxic factors was assessed by RT-PCR and protein expression of significantly important cytotoxic factors and cytokine secretion were determined through flow cytometry and ELISA. Western blotting was performed to check the involvement of the JAK/STAT pathway following stimulation.
  
  
  RESULTS
  We found that IL-21 did not enhance in vitro proliferation of CIK cells, but did increase the number of cells expressing the CD3+/ CD56+ phenotype. Cytotoxic potential was increased with corresponding increase in perforin (0.9831±0.1265 to 0.7592±0.1457), granzyme B (0.4084±0.1589 to 0.7319±0.1639) and FasL (0.4015±0.2842 to 0.7381±0.2568). Interferon gamma and TNF-alpha were noted to increase (25.8±6.1 ng/L to 56.0±2.3 ng/L; and 5.64±0.61 μg/L to 15.14±0.93 μg/L, respectively) while no significant differences were observed in the expression of granzyme A, TNF-alpha and NKG2D, and NKG2D. We further affirmed that IL-21 signals through the STAT-3 and STAT- 5b signaling pathway in the CIK cell pool.
  
  
  CONCLUSIONS
  IL-21 enhances cytotoxic potential of CIK cells through increasing expression of perforin, granzyme B, IFN-gamma and TNF-alpha. The effect is brought about by the activation of STAT-3 and STAT-5b proteins.
authors.affiliation: Department of Hematology, Tianjin First Central Hospital, Tianjin, China E-mail : zmfzmf@hotmail.com.
authors.forename: S
authors.initials: S
authors.lastname: Rajbhandary
authors.forename: Ming-Feng
authors.initials: MF
authors.lastname: Zhao
authors.forename: Nan
authors.initials: N
authors.lastname: Zhao
authors.forename: Wen-Yi
authors.initials: WY
authors.lastname: Lu
authors.forename: Hai-Bo
authors.initials: HB
authors.lastname: Zhu
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: D000970:Antineoplastic Agents / Q000378:metabolism*; D017252:CD3 Complex / Q000378:metabolism; D019002:CD56 Antigen / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000502:physiology; D055612:Cytokine-Induced Killer Cells / Q000378:metabolism*; D053804:Granzymes / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D007378:Interleukins / Q000378:metabolism*; D020014:K562 Cells; D055655:NK Cell Lectin-Like Receptor Subfamily K / Q000378:metabolism; D054353:Perforin / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D050799:STAT5 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000098546:Interleukin-21
chemical_list: D000970:Antineoplastic Agents; D017252:CD3 Complex; D019002:CD56 Antigen; D007378:Interleukins; C527085:KLRK1 protein, human; D055655:NK Cell Lectin-Like Receptor Subfamily K; D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D050799:STAT5 Transcription Factor; C494095:STAT5B protein, human; D014409:Tumor Necrosis Factor-alpha; D054353:Perforin; D007371:Interferon-gamma; D053804:Granzymes; D000098546:Interleukin-21