Zhang Huan-xin, Chen Chong, Zeng Ling-yu, Zhang Yin, Xu Kai-lin
The Affiliated Hospital of Xuzhou Medical College, Xuzhou 221002, China.
Zhonghua Xue Ye Xue Za Zhi. 2011 Dec;32(12):825-9.
To generate engineering Th17 cells from mice CD4(+)CD25(-) naïve T cells, and to evaluate whether the phenotypes or functions of these engineering cells were similar to natural Th17 cells.
Recombinant lentivirus carrying mouse RORγt (pXZ9-RORγt) and mock control pXZ9 were generated by co-transfected three-plasmids into 293FT packing cells. CD4(+)CD25(-) naïve T cells were purified from mice spleens by magnetic activated cell sorting, and stimulated by anti-CD3ε, anti-CD28 mAb plus IL-2. The stimulated cells were further infected by pXZ9-RORγt or pXZ9 virus with or without polarization by TGF-β plus IL-6 and divided into five groups: pXZ9-RORγt (group A), pXZ9 + TGF-β + IL-6 (group B), pXZ9-RORγt + TGF-β + IL-6 (group C), pXZ9 (group D) and control (group E). Production efficiency of engineering Th17 cells was referred as the percentage of IL-17A producing cells. Cytokine production profiles of these cells were assayed by realtime RT-PCR and cells function was evaluated by susceptibility of mouse experimental autoimmune encephalomyelitis (EAE).
(1) High-title lentivirus was prepared and was succeeded to transduce CD4(+)CD25(-) naïve T cells. Forced expression of RORγt (group A) resulted in (40.25 ± 5.46)% CD4(+)CD25(-) naïve T cells converted into engineering Th17 cells and the convert efficiency increased to (60.59 ± 8.15)% in addition of TGF-β and IL-6 (group C), or decreased to (14.36 ± 5.27)% when presence of TGF-β and IL-6 only (group B). (2) IL-17A, IL-17F and IL-21 production of pXZ9-RORγt infected cells combined with TGF-β and IL-6 were most similar to natural Th17 cells while cells over expression of RORγt alone showed deficiency in IL-21 production. (3) Both pXZ9-RORγt infected cells, TGF-β and IL-6 polarized cells and polarized of RORγt transduced cells could promote the susceptibility to mouse EAE in C57BL6 mice models.
High yield of engineering Th17 cells was prepared from CD4(+)CD25(-) naïve T cells by over expression RORγt plus TGF-β and IL-6 polarization. These engineering Th17 cells were similar to the natural Th17 cells in phenotypes and functional identification.
从小鼠CD4(+)CD25(-)初始T细胞中生成工程化Th17细胞，并评估这些工程化细胞的表型和功能是否与天然Th17细胞相似。
通过将三种质粒共转染到293FT包装细胞中，构建携带小鼠RORγt的重组慢病毒（pXZ9-RORγt）和空载对照pXZ9。通过磁珠激活细胞分选从小鼠脾脏中纯化CD4(+)CD25(-)初始T细胞，并用抗CD3ε、抗CD28单克隆抗体加IL-2刺激。将刺激后的细胞用pXZ9-RORγt或pXZ9病毒进一步感染，在有或无TGF-β加IL-6极化的情况下分为五组：pXZ9-RORγt（A组）、pXZ9 + TGF-β + IL-6（B组）、pXZ9-RORγt + TGF-β + IL-6（C组）、pXZ9（D组）和对照组（E组）。工程化Th-17细胞的产生效率以产生IL-17A的细胞百分比表示。通过实时RT-PCR检测这些细胞的细胞因子产生谱，并通过小鼠实验性自身免疫性脑脊髓炎（EAE）的易感性评估细胞功能。
（1）制备了高滴度慢病毒并成功转导CD4(+)CD25(-)初始T细胞。RORγt的强制表达（A组）使（40.25±5.46）%的CD4(+)CD25(-)初始T细胞转化为工程化Th17细胞，在添加TGF-β和IL-6时转化效率提高到（60.59±8.15）%（C组），而仅存在TGF-β和IL-6时转化效率降至（14.36±5.27）%（B组）。（2）pXZ9-RORγt感染的细胞与TGF-β和IL-6联合时IL-17A、IL-17F和IL-21的产生与天然Th17细胞最相似，而单独过表达RORγt的细胞在IL-21产生方面存在缺陷。（3）pXZ9-RORγt感染的细胞、TGF-β和IL-6极化的细胞以及RORγt转导细胞的极化在C57BL6小鼠模型中均可促进对小鼠EAE的易感性。
通过过表达RORγt加TGF-β和IL-6极化，从CD4(+)CD25(-)初始T细胞中制备了高产率的工程化Th17细胞。这些工程化Th17细胞在表型和功能鉴定上与天然Th17细胞相似。
