Multiple cytotoxic factors involved in IL-21 enhanced antitumor function of CIK cells signaled through STAT-3 and STAT5b pathways.

BACKGROUND/OBJECTIVES: 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.

METHOD: 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.

CONCLUSION: 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.