A Simple and Robust Protocol for in vitro Differentiation of Mouse Non-pathogenic T Helper 17 Cells from CD4⁺ T Cells

Functional and mechanistic studies of CD4⁺ T cell lineages rely on robust methods of in vitro T cell polarization. Here, we report an optimized protocol for in vitro differentiation of a mouse non-pathogenic T helper 17 (T_H17) cell lineage. Most of the previously established protocols require irradiated splenocytes as artificial antigen presenting cells (APC) for TCR activation. The protocol described here employs plate-bound antibodies and a T_H17-polarizing cytokine cocktail to activate and differentiate naïve CD4⁺ T (T_nai) cells, reflecting a simple and robust protocol for in vitro T_H17n differentiation. Using T cells that are genetically engineered with an IL-17 reporter, this protocol may enable the rapid production of a pure population of IL17-expressing CD4⁺ T cells for system biology studies and high-throughput functional screening.