Adoptive cell therapy with genetically modified regulatory T cells (Tregs) is under clinical investigation for the treatment of transplant rejection and various autoimmune conditions. A limitation of modelling this approach in mice is the lack of optimized protocols for expanding and transducing mouse Tregs. Here we describe a protocol for purifying, expanding and retrovirally transducing mouse Tregs with a vector encoding a chimeric antigen receptor as a model transgene. We found that isolation of Tregs from C57Bl/6J Foxp3^EGFP mice solely based on eGFP expression resulted in sufficiently pure cells; co-sorting of CD25^hi cells was not essential. Although expansion with rapamycin reduced Treg expansion, it promoted maximal in vitro suppressive activity. Retroviral transduction of Tregs following 2 days of stimulation with anti-CD3/CD28 beads achieved a transduction efficiency of ~40% and did not impair their suppressive capacity. When injected into a conventional T cell (Tconv)-transfer-induced colitis model, transduced Tregs inhibited colitis progression at ratios as low as 1 Treg to 100 Tconvs, and maintained Foxp3 and transgene expression throughout an 8-week period. This method facilitates the study of transduced Tregs in animal models and will enable the study of genetically engineered Treg therapy for a variety of inflammatory diseases.

使用转基因调节性 T 细胞 (Treg) 的过继细胞疗法目前正在临床研究中，用于治疗移植排斥和各种自身免疫性疾病。在小鼠中建立这种方法的一个局限性是缺乏用于扩展和转导小鼠 Tregs 的优化方案。在这里，我们描述了一种使用编码嵌合抗原受体的载体作为模型转基因来纯化、扩展和逆转录病毒转导小鼠 Tregs 的方案。我们发现仅基于 eGFP 表达从 C57Bl/6J Foxp3 ^{E GFP}小鼠中分离 Tregs 会产生足够纯的细胞； CD25 ^hi细胞的共分选不是必需的。尽管用雷帕霉素进行扩增会降低 Treg 的扩增，但它会促进最大的体外抑制活性。用抗 CD3/CD28 珠刺激 2 天后，Treg 的逆转录病毒转导达到了约 40% 的转导效率，并且没有损害其抑制能力。当注射到传统 T 细胞 (Tconv) 转移诱导的结肠炎模型中时，转导的 Tregs 以低至 1 个 Treg 对 100 个 Tconv 的比率抑制结肠炎进展，并在整个 8 周内维持 Foxp3 和转基因表达。该方法有助于在动物模型中研究转导的Treg，并将使基因工程Treg治疗多种炎症性疾病的研究成为可能。