Yang Hui, Ren Ying, Zhu Ping, Chen Li-Na, Lv Ming-Hua, Fan Chun-Mei, Wang Yan-Hong
Department of Clinical Immunology, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2012 Apr;28(4):377-80.
To investigate whether monocytes activated with lipopolysaccharide(LPS) have an effect on Th17 cell differentiation in humans, CD4(+) T cell and CD14(+) monocytes activated with LPS were treated in the absence or presence of anti-CD3 mAb with various concentrations at different time points.
Purification of CD4(+) T cell and CD14(+) monocytes were performed by magnetic cell sorting and cultured together. Cultures were stimulated with LPS alone or anti-CD3 mAb alone or LPS plus anti-CD3 mAb for 3 days. In the anti-CD3 mAb stimulation cells were added different concentrations of LPS. Cells were activated under LPS/anti-CD3 costimulation for 3, 6, or 10 days. The percentage of IL-17(+) T cells and INF-γ(+) T was determined by flow cytometry.
LPS or anti-CD3 mAb alone induced only very low levels of IL-17(+) T cells, (1.30 ± 0.19)%, (1.10 ± 0.21)%, respectively. The percentage was substantially higher in the LPS and anti-CD3 mAb costimulationa as much as(2.01 ± 0.46)%. In the presence of 0.1 μg/mL, 1 μg/mL, 10 μg/mL LPS, the proportion of Th17 reached to (1.92 ± 0.21)%, (1.30 ± 0.37)%, (1.01 ± 0.25)%. Low-concentration LPS (0.1 μg/mL) stimulation favored Th17 differentiation. The highest proportion of IL-17(+) T cells was found at day 3(2.13 ± 0.32)%, with levels declining at day 6 and day 10, while, Th1 at day 6(17.45 ± 3.04)%, declining at day 10.
Low-concentration LPS stimulation plus anti-CD3 mAb in short term support optimal Th17 generation. Nevertheless, this model closely mimics the environment of rheumatoid arthritis in vivo and proposes an effective model for the generation of human Th17 cells.
研究经脂多糖（LPS）激活的单核细胞是否对人类Th17细胞分化有影响，在不同时间点，用不同浓度的抗CD3单克隆抗体处理经LPS激活的CD4(+) T细胞和CD14(+)单核细胞。
通过磁珠细胞分选法纯化CD4(+) T细胞和CD14(+)单核细胞，并共同培养。培养物分别用单独的LPS、单独的抗CD3单克隆抗体或LPS加抗CD3单克隆抗体刺激3天。在抗CD3单克隆抗体刺激的细胞中加入不同浓度的LPS。细胞在LPS/抗CD3共刺激下激活3、6或10天。通过流式细胞术测定IL-17(+) T细胞和INF-γ(+) T细胞的百分比。
单独的LPS或抗CD3单克隆抗体仅诱导出极低水平的IL-17(+) T细胞，分别为(1.30±0.19)%、(1.10±0.21)%。在LPS和抗CD3单克隆抗体共刺激下，该百分比显著更高，高达(2.01±0.46)%。在存在0.1μg/mL、1μg/mL、10μg/mL LPS的情况下，Th17的比例分别达到(1.92±0.21)%、(1.30±0.37)%、(1.01±0.25)%。低浓度LPS（0.1μg/mL）刺激有利于Th17分化。在第3天发现IL-17(+) T细胞的比例最高，为(2.13±0.32)%，在第6天和第10天水平下降，而Th1在第6天为(17.45±3.04)%，在第10天下降。
短期低浓度LPS刺激加抗CD3单克隆抗体支持最佳的Th17生成。然而，该模型紧密模拟了类风湿关节炎的体内环境，并提出了一种生成人类Th17细胞的有效模型。
