In Vitro Expanded Regulatory T Cells Suppress NK Cells Mediated Anti-Tumor Immunity

Objective Adoptive transfer of in vitro expanded CD4+CD25+ regulatory T cells (Tregs) promote transplantation tolerance is well documented in animal models and it has also been reported that Tregs can promote progression of cancer through their ability to suppress the antitumor immunity. While, NK cells have the ability to suppress the growth of malignant tumor and do not evoke immunological rejection. So, we do the experiment to explore if combined infusion of ex-vivo expanded NK cells and Tregs could reduce the the inhibition of anti-tumor immunity by Tregs.Methods Tregs and NK cells were separated by magnetic activated cell sorting (MACS). The purity of Tregs and NK cells were measured by flow cytometry. Tregs were expanded by anti-CD3/CD28-coated microbeads and recombinant murine interleukin 2 (rmIL-2) in vitro for 14 days. Fresh sorted NK cells were cultured in vitro in the presence of rmIL-2, recombinant murine interleukin 15 (rmIL-15) and hydrocortisone for 15 days. Thereafter, the suppressive activity of expanded Tregs to NK cells was tested by the mixed lymphocyte reaction (MLR) and the cytotoxicity of NK cells to K562 tumor cells was evaluated by MTT assays. Then, we set up the animal model as we previous reported, briefly, Balb/c mice were intravenously injected with 1×107 expanded Tregs at day-1, and 24 hours later, they were inoculated intravenously with 5×105 B16-F10 tumor cells (day0), followed by three subsequent inoculations of 5×106 expanded NK cells at 4 days intervals (day 1,5,9). After 14 days, mice were sacrificed and tumor nodules in lung were counted.Results Both the purity of fresh sorted Tregs and NK cells were more than 90%. The purity of expanded Tregs was more than 90% and the purity of expanded NK cells was more than 80%. We also found that expanded NK cells exhibited better cytotoxicity to K562 tumor cells compared with fresh sorted.The in vitro expanded Treg cells could suppress the cytotoxicity of NK cells both before and after expansion in vitro. The suppressive activity of Tregs was in a dose depended manner when the ratio of Tregs/NK cells was increased from 1:10 to 2:1. The number of rumor nodules in tumor-bearing mice pretreated with 1×107 expanded Tregs was significantly elevated from (17±4.58) to (105.33±10.97) compared with the control group (p<0.001), and was significantly decreased to (79±8.54) when followed by three subsequent injection of 5×106 expanded NK cells (p=0.030). The number of tumor nodules in tumor-bearing mice injected with 5×105 B16-F10 tumor cells followed by three subsequent inoculations of 5x106 expanded NK cells was decrease to (2±1) compared with the control group (17±4.58) (p=0.037).Conclusions Injection of tumor cells can suppress anti-tumor immunity in vivo. Adoptive transfer of expanded Tregs can suppress anti-tumor immunity and the inhibition of NK cells tumor killing ability maybe one of the mechanism. Infusion of ex-vivo expanded NK cells can partially reverse the inhibition of anti-tumor immunity by regulatory T cells.