A method for expansion and retroviral transduction of mouse regulatory T cells

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 C57B1/6J Faxp3(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 similar to 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.

Automated summary

This study describes a new method for purifying, expanding, and retrovirally transducing mouse Tregs, and confirms the feasibility of the method in experiments. By using this method, it is possible to study transduced Tregs in animal models, providing a new avenue for research on genetically engineered Treg therapy for various inflammatory diseases.