Protocol to assess cell-intrinsic regulatory mechanisms using an ex vivo murine T cell polarization and co-culture system

This protocol describes an ex vivo cell culture system for simultaneously generating a mixture of CD4(+) T helper lineages, including T helper 17 (Th17), RORγt(+) Treg, and conventional Treg (cTreg), in proportions representative of those found in mucosal tissues in vivo. When combined with a co-culture approach, this system allows a more rapid assessment of a candidate molecule’s T cell-intrinsic and -extrinsic functions over the traditional bone marrow chimera and co-transfer approaches. For complete details on the use and execution of this protocol, please refer to Ma et al. (2022).