IL-21 Enhances Anti-Leukemia Effect By Acting On Both CD3+CD56+ CIK Cells and Regulatory T Cells Derived From Umbilical Cord Blood In Vitro
Authors: Meng Juanxia , Zhao Mingfeng , Chai Xiao , Xiao Xia , Mu Juan , Li Qing , Deng Qi , Li Yuming 
Published: October 2019

Abstract
Cytokine-induced killer (CIK) cells are the effective kind of immunocytes participated in biotherapy of tumors and CD3+CD56+ cells display a major role in cytolytic activity against tumors. Recombinant human interleukin-2 (rhIL-2) is one of the most necessary cytokines during induction of CIK cells, however, some extra kinds of cells eapecially regulatory T (Treg) cells may expand during the induction of CIK cells because of the presence of IL-2. As Treg cells may exert a negative effect on function of CIK cells through cell-to-cell contact and some cytokines secreted by Treg cells, so what we need to consider is how to reduce Treg cells in the culture system of CIK cells. Some studies have confirmed that IL-21, which is belonged to a subset of cytokines where the receptors share the common cytokine receptor γ chain, could express an inhibitory influence on proliferation in Treg cells and our previous study has also confirmed its enhancement on proliferation and function of CIK cells. Here we hypothesize that IL-21 not only promotes the proliferation and function of CD3+CD56+ CIK cells, but also depressed Treg cells in the culure system of CIK cells and then result in the enhanced anti-leukemia effect.
In this study, we first detected whether Treg cells existed in the culture system of CIK cells. Firstly, CIK cells were obaind from umbilical cord blood mononuclear cells (CBMCs) by the sequential addition of interferon-γ, anti-CD3 antibody , and rh-IL-2. Then the immunophenotype of Treg cells was detected by the flow cytometry through CD4-FITC and Foxp3-PE antibodies double staining. It was found that a certain proportion of Treg cells at about (10.24±1.42)% consisted in the culture system of CIK cells.
The second step in this study was to explore the effect of IL-21 acting on CD3+CD56+ CIK cells. Firstly, CD3+CD56+ CIK cells were separated from the culture cells mentioned above by positive selection using Fluorescence-activated Cell Sorter. Cells were then stimulated with IL-21 for a defined period of time and subjected to CCK-8 and LDH assays to measure cellular viability and cytotoxicity against to leukemia cell line—K562 cells, respectively. The CCK-8 assay results showed that OD values in the cells without IL-21 stimulation was 1.08 which was much lower than that in the cells with IL-21 stimulaiton at 1.45 (P<0.01), thus IL-21 could significantly enhance in vitro proliferation of CD3+CD56+ CIK cells. And after 72 hours of stimulation by IL-21, cytotoxicity of CD3+CD56+ CIK cells against K562 cells at an Effector:Target ratio of 20:1 was observed to be (52.99±1.26)%, while the rate in the cells without IL-21 stimulation was (29.31±0.58)% , which was much lower than the cells with IL-21 stimulation (P<0.01).
Next, we investigated the effect of IL-21 on both Treg and CIK cells in the culture system. The culture system of CIK cells was established as mentioned above, and then part of these cells were stimulated with IL-21 for 72 hours. Immunophenotype of Treg and CIK cells was detected by flow cytometry. It was found that cells stimulated by IL-21 showed a decreased proportion of CD4+Foxp3+ Treg cells at (1.48±0.06)% compared with the cells without IL-21 stimulation at (10.24±1.42)% (P<0.01). The CD3+CD56+ cells occupied a certain proportion at (39.80±1.80)% in the cells stimulated with IL-21, which was much higher than that in the cells without IL-21 stimulation at (20.46±1.11)% ( P<0.01). Then cytotoxicity of CIK cells was detected by LDH assay and the results showed that IL-21 could extremely strengthen the cytotoxicity of CIK cells to K562 cells, and the cytotoxity index in the cells with IL-21 stimulation at (52.99±1.26)% , which was much higher than that in the cells without IL-21 stimulation at (29.31±0.58)% ( P<0.01).
In conclusion, our findings indicate that IL-21 could promote the proliferative and cytotoxic activity of CD3+CD56+ CIK cells, meanwhile it also could suppress the viability of Treg cells in the CIK cells culture system. So a conclusion can be summarized that IL-21 could enhance anti-leukemia effect not only by promoting CD3+CD56+ CIK cells but also by depressing Treg cells derived from umbilical cord blood in vitro.

Disclosures:
No relevant conflicts of interest to declare.