Protocol to isolate and enrich mouse splenic naive CD4⁺ T cells for in vitro CD4⁺CD8αα⁺ cell induction

Recent studies have shown that CD4⁺CD8αα⁺ T cells are induced in the hypoxic environment of the small intestinal epithelium. Herein, we describe a protocol for CD4⁺CD8αα⁺ T cell induction from freshly isolated naive CD4⁺ T cells, including procedures for the isolation and enrichment of mouse splenic T cells. In addition, we present an approach that can induce more CD4⁺CD8αα⁺ T cells by artificially creating a hypoxic environment in vitro. For complete details on the use and execution of this protocol, please refer to Harada et al. (2022).