Multiple Cytotoxic Factors and JAK/STAT Pathways Are Involved in the Enhanced Antitumor Function of CIK Cells Induced by IL-21
Authors: Rajbhandary Sajin , Zhao Mingfeng , Zhao Nan , Lu Wenyi , Zhu Haibo , Meng Juanxia , Chai Xiao , Xiao Xia , Deng Qi , Li Yuming , Wang Yuliang 
Published: October 2019

Abstract
Abstract 1059
Adoptive transfer of activated T and NK cells has had significant clinical benefits in certain tumor models. Cytokine induced killer (CIK) cells are a group of cells that possess both T and NK cell like recognition of target cells. These cells are generated after extensive ex vivo manipulation of PBMCs. Maintenance of not only CIK cells but other activated effector T and NK cells in culture is vital for their effective transfer and development following adoptive immunotherapy. IL-21 is the newest member of the common γ chain family which has been shown to increase cytotoxic factors and cytokine secretion in immune cells without over stimulation. Such qualities make IL-21 a suitable agent in immunotherapy of tumors. IL-21 has shown effective antitumor function and is currently going clinical trials for tumors such as renal cell carcinoma, melanoma and lymphoma. Our previous experiment showed that like in T cells and NK cells, IL-21 significantly improves the cytotoxicity of CIK cell on K562 cells and primary leukemic cells from patients. Although proliferation of cells in a CIK cell pool was not observed we found that it helped maintain and grow the CD3+ and CD56+ phenotype. Our present experiment aims to explain the mechanism through which IL-21 promotes CIK cells survival and cell cytotoxicity.
In our experiment, blood from healthy donors was collected and PBMCs were transformed into CIK cells following 14 days of culture using appropriate methods. The cells were then stimulated with IL-21 for a defined period of time and subjected to MTT assays to measure cellular viability and cytotoxicity to K562 cells. To elucidate the mechanism of action of IL-21, CIK cells were checked for the level of mRNA expression of perforin, Granzyme B, FasL, INF-γ, TNF-α,Granzyme A,NKG2D, TNF-β using RT-PCR. Furthermore the expression of significantly important cytotoxic factors and cytokines was measured through flow cytometry and ELISA. Western blot was performed to check the involvement of JAK/STAT pathway following stimulation.
We found that IL-21 doesn't enhance in vitro proliferation of CIK cells, but does increase the number of cells expressing the CD3+/CD56+ phenotype. IL-21 can also significantly increase the cytotoxic potential of CIK cells to K562 cells. It does so with significantly increased production of perforin which increased almost 2 folds from (0.5592±0.1457) to (0.9831±0.1265); Granzyme B also by almost 2 folds from (0.4084±0.1589) to (0.7319±0.1639) and FasL which increased by almost 2 folds from (0.4015±0.2842) to (0.7381±0.2568). Increase in secretion of cytokines such as INF-γ was observed from (25.8±6.1)ng/L to (56.0±2.3)ng/L; and TNF-α from (5.64±0.61)ug/L to (15.14±0.93)ug/L while no significant difference was observed in the expression of Granzyme A,TNF-β and NKG2D. Measurement of IL-21R receptor on CIK cell surface following IL-21 stimulation caused a more than two folds increase in expression of IL-21R from 1.88% to 4.25%. We further affirm that JAK/STAT is actively involved in IL-21 signalling. STAT3 and STAT5b could be potential signalling mechanisms taking part in IL-21 enhanced cytotoxic potential of CIK cells.
Using this information we have concluded that increased expression of perforin, Granzyme B, FasL, IFN-γ and TNF-α plays a significant role in IL-21 enhanced cytotoxic potential of CIK cells and STAT-3 and STAT-5b signaling pathway are involved in the processes. Our data indicate that IL-21 is a potent enhancer of antitumor function of CIK cells. As CIK cells and IL-21 have both been shown to increase patient survival or tumor free periods in certain hematological malignancies using them in conjunction might be therapeutically more beneficial.

Disclosures:
No relevant conflicts of interest to declare.