The last decade has seen a significant increase of cell therapy protocols using effector T cells (Teffs) in particular, but also, more recently, non-engineered and expanded polyclonal regulatory T cells (Tregs) to control pathological immune responses such as cancer, autoimmune diseases, or transplantation rejection. However, limitations, such as stability, migration, and specificity of the cell products, have been seen. Thus, genetic engineering of these cell subsets is expected to provide the next generation of T cell therapy products. Lentiviral vectors are commonly used to modify Teffs; however, Tregs are more sensitive to mechanical stress and require specific culture conditions. Also, there is a lack of reproducible and efficient protocols to expand and genetically modify Tregs without affecting their growth and function. Due to smaller number of cells and poorer viability upon culture in vitro, mouse Tregs are more difficult to transduce and amplify in vitro than human Tregs. Here we propose a step-by-step protocol to produce both human and mouse genetically modified CD8+ and CD4+ Tregs in sufficient amounts to assess their therapeutic efficacy in humanized immunocompromised mouse models and murine models of disease and to establish pre-clinical proofs of concept. We report, for the first time, an efficient and reproducible method to isolate Tregs from human blood or mouse spleen, transduce with a lentiviral vector, and culture, in parallel, CD8+ and CD4+ Tregs while preserving their function. Beyond chimeric antigen receptor (CAR)-Treg cell therapy, this protocol will promote the development of potential new engineered T cell therapies to treat autoimmune diseases and transplantation rejection.
Keywords: CAR; Tregs; expansion; genetic engineering; lentiviral vector; transduction.
© 2020 The Author(s).