Mouse Naive CD4+ T Cell Isolation and In vitro Differentiation into T Cell Subsets

Antigen inexperienced (naive) CD4(+) T cells undergo expansion and differentiation to effector subsets at the time of T cell receptor (TCR) recognition of cognate antigen presented on MHC class II. The cytokine signals present in the environment at the time of TCR activation are a major factor in determining the effector fate of a naive CD4(+) T cell. Although the cytokine environment during naive T cell activation may be complex and involve both redundant and opposing signals in vivo, the addition of various cytokine combinations during naive CD4(+) T cell activation in vitro can readily promote the establishment of effector T helper lineages with hallmark cytokine and transcription factor expression. Such differentiation experiments are commonly used as a first step for the evaluation of targets believed to promote or inhibit the development of certain CD4(+) T helper subsets. The addition of mediators, such as signaling agonists, antagonists, or other cytokines, during the differentiation process can also be used to study the influence of a particular target on T cell differentiation. Here, we describe a basic protocol for the isolation of naive T cells from mouse and the subsequent steps necessary for polarizing naive cells to various T helper effector lineages in vitro.