The Antitumor Activity Of PBMCs In Patients With Endometrial Cancer Induced By IL21 And IL12

OBJECTIVE:To observe the role of IL21 and IL12 for inducing the antitumor activity of peripheral blood mononuclear cells (PBMCs) in patients with endometrial cancer, and detect the possible related mechanisms.METHODS:Endometrial cancer patients (n=14) were included in the study from Qilu Hospital of Shandong University from March 2010 to July 2010. PBMCs were separated from peripheral blood obtained from endometrial cancer patients by Ficoll density gradient centrifugation in vitro, and suspended in RPMI 1640 medium with 20% FBS, IL2 (5 ng/ml),100 U/ml penicillin and 100μg/ml streptomycin as control group. And IL-2-stimulated PBMCs were cocultured with anti-human IL21 antibody (2μg/ml), IL21 (50 ng/ml) alone, IL12 (5 ng/ml) alone and IL21 (50 ng/ml) plus IL12 (5 ng/ml) as study groups, respectively. The above-mentioned PBMCs were incubated in 6-well plates (5×106 cells/well) and 96-well plates (1×104 cells/well) for 72 h at 37℃,5% CO2. PBMCs stimulated for 72 h served as effector cells, and Ishikawa cells served as target cells (E/T: 100/1), and cell cytotoxicity was detected by LDH released methods.106 cells were collected to detect Treg and Th17 cell proportion according to the manufacture's instructions. Cells were first extracellularly stained, then fixed and permeabilized, finally intracellularly stained. PBMCs were stimulated for 4.5 h with PMA (25 ng/ml), ionomycin (1μg/ml) and monensin (1.7μg/ml) to detect Th17 cells before staining. Flow cytometry was performed to analyze CD4+ CD25+FOXP3+T regulatory (Treg) cell and CD4+IL17A+T-helper (Th17) cell proportion. After PBMCs were stimulated for 72 h in 96-well plate, 10μl CCK-8 solution was added into every well and blank controls including triplicate wells. The cells were incubated for 4h at 37℃,5% CO2. Then cell proliferation was measured by CCK-8 assay. 1×105 cells were collected in every group, and then cell apoptosis was measured by flow cytometry.RESULTS:Compared to control group, the cytotoxicity of PBMCs to Ishikawa cells was significantly elevated in IL21 group, IL12 group and the combined group. Moreover, IL-21 plus IL-12 significantly elevated PBMCs cytotoxicity in comparison to IL-21 alone and IL-12 alone (P all<0.01). Furthermore, our study showed that IL21 alone, IL12 alone and IL21 plus IL12 significantly decreased Treg cell proportion and decreased PBMCs apoptosis rate, respectively (P all<0.05). Finally, we showed that a different regulation was played by IL21 and anti-IL21 neutralizing antibody, which further verified the role of IL21. IL21 or IL12 had no significant effect on the differentiation of Th17 cells and the proliferation of PBMCs (P all>0.05).CONCLUSIONS:IL21 and IL12 could enhance the cytotoxicity of PBMCs in patients with endometrial cancer, which could be further strengthened with the combination of IL21 and IL12. One of the possible mechanisms was to inhibit Treg cell differentiation and PBMCs apoptosis, but not related to Th17 cell differentiation or PBMCs proliferation. Our study provided the evidence for the cytokine immunotherapy of endometrial cancer, but the safety and efficacy of IL21 in combination with IL12 needs to be verified in clinical trails.