体外扩增的Treg细胞抑制NK细胞介导的抗肿瘤免疫

In vitro expanded regulatory T cells suppress NK cells-mediated anti-tumor immunity

目的研究CD4+CD25+调节性T细胞(regulatory T cells,Tregs)对NK细胞肿瘤杀伤力的影响及Treg细胞介导的抗肿瘤免疫抑制的机制;初步探讨过继输注NK细胞逆转Treg细胞介导的抗肿瘤免疫抑制的作用。方法免疫磁珠分离法(MACS)分离得小鼠脾脏Treg细胞及NK细胞,用流式细胞术检测其纯度。以CD3/CD28单克隆抗体磁珠和重组小鼠白介素2(rm IL-2)联合刺激体外扩增Treg细胞,重组小鼠白介素15(rm IL-15)、rm IL-2以及氢化可的松联合刺激体外扩增NK细胞。将扩增后Treg细胞及NK细胞按不同比例混合淋巴细胞培养,MTT比色法检测NK细胞的杀伤活性。将B16-F10小鼠黑色瘤细胞输注至Balb/c小鼠体内建立肺移植瘤模型[1],将荷瘤小鼠分为4组:A组单独接种B16-F10小鼠黑色瘤细胞;B组接种Treg细胞+B16-F10黑色素瘤细胞;C组接种B16-F10黑色素瘤细胞+NK细胞;D组接种Treg细胞+B16-F10黑色素瘤细胞+NK细胞。MTT比色法测定各实验组小鼠脾脏NK细胞的杀伤活性,并比较不同处理组小鼠肺部肿瘤结节数目。结果体外扩增后的Treg细胞对新鲜分选及扩增后的NK细胞活性均具有明显抑制作用(P<0.05),且抑制作用呈剂量依赖关系;A组荷瘤小鼠NK细胞活性低于正常小鼠,且B组荷瘤小鼠NK细胞活性较A组进一步降低(P<0.05);D组荷瘤小鼠NK细胞活性高于A组和B组荷瘤小鼠,但仍低于正常小鼠组(P<0.05)。B组荷瘤小鼠肺部移植瘤数目(105.33±10.97)较A组明显增多(17±4.58)(P<0.01);C组荷瘤小鼠肺部移植瘤数目(2.00±1.00)较A组(17±4.58)明显减少(P=0.037);D组荷瘤小鼠肺移植瘤数目(79.00±8.54)较B组明显降低(105.33±10.97)(P=0.030),但仍高于A组荷瘤小鼠(17±4.58)(P<0.001)。结论体内种植肿瘤会抑制机体NK细胞活性;输注体外扩增Treg细胞能够通过抑制NK细胞发挥抗肿瘤免疫抑制;过继输注体外扩增NK细胞能够部分逆转Treg细胞介导的抗肿瘤免疫抑制。

Adoptive transfer of in vitro expanded CD4＋CD25＋regulatory T cells（Tregs） promoting transplantation tolerance is well documented in animal models and it is also been reported that Tregs can promote progression of cancer through their ability to suppress the antitumor immunity. We hypothesized that combined infusion of ex-vivo expanded NK cells and Tregs could reduce the inhibition of anti-tumor immunity by Tregs. Tregs and NK cells were separated by magnetic activated cell sorting（MACS）. Both the purity of fresh sorted Tregs and NK cells were more than 90% measured by flow cytometry. Tregs were expanded by anti-CD3/CD28-coated microbeads and recombinant murine interleukin 2（rm IL-2） in vitro for 14 days, and the purity of expanded Tregs was more than90%. Fresh sorted NK cells were cultured in vitro in the presence of rm IL-2, recombinant murine interleukin 15（rm IL-15） and hydrocortisone for 15 days, and the purity of expanded NK cells was more than 80%. 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. We 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. Then, we set up the animal model as we previous reported, briefly, Balb/c mice were firstly（day-1） intravenously injected with 1 ×10^7expanded Tregs and 24 hours later, they were inoculated intra venously with 5 ×10^5B16-F10 tumor cells（day 0）, followed by three subsequent inoculations of 5×10^6expanded NK cells at 4 days intervals（day 1,5, 9）. Fourteen days after, mice were sacrificed and tumor nodules in lung were counted. The number of tumor nodules in tumor-bearing mice pretreated with 1×10^7expanded 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 ×10^6expanded NK cells（P =0.030）. The number of tumor nodules in tumor-bearing mice injected with 5×105B16-F10 tumor cells followed by three subsequent inoculations of 5 ×10^6expanded NK cells was decrease to（2 ±1） compared with the control group（17 ±4.58）（P =0.037）. In conclusion,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.