Method for generation and ex vivo expansion of genetically edited mouse Tregs

Regulatory T cells (Tregs) play a crucial role in the immune system, and their dysfunction can lead to the development of autoimmune conditions. In cancer, tumors frequently hijack the immunosuppressive function of Tregs to evade immune responses. Due to their central role in key pathological processes, Tregs have gained increasing attention as promising targets for various clinical applications. However, their relative scarcity (∼5% to 10% of CD4+ T cells) and instability presents a technical challenge for research and therapeutic development. In congenic animal models used to investigate autologous cell transfer-based therapies, this challenge is even greater, as Treg donor animals may only be able to provide cells to a small number of recipient mice. Here, we present an optimized protocol for ex vivo editing and expansion of mouse Tregs. Because a recent study demonstrated the anticancer potential of SRC-3 KO mouse Tregs, we use them here as a case study.