Protocol for the generation of a human-derived nasal epithelial model and induction of tissue-resident memory-like T cells in a co-culture system

Tissue-resident memory T cells (TRMs) are a distinct subset of T lymphocytes that permanently localize in non-lymphoid tissues and mediate rapid, localized immune responses. Here, we present a protocol to study human TRMs using an in vitro model system that induces TRM-like T cells. We describe steps for generating the 3D nasal epithelial model and inducing TRMs via co-culture with autologous immune cells. We then detail procedures to analyze TRMs using flow cytometry, single-cell RNA sequencing, and cytokine release. • Instructions for generating a 3D nasal epithelial model • Steps to induce tissue-resident memory-like T cells • Guidance on analyzing tissue-resident memory-like T cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Tissue-resident memory T cells (TRMs) are a distinct subset of T lymphocytes that permanently localize in non-lymphoid tissues and mediate rapid, localized immune responses. Here, we present a protocol to study human TRMs using an in vitro model system that induces TRM-like T cells. We describe steps for generating the 3D nasal epithelial model and inducing TRMs via co-culture with autologous immune cells. We then detail procedures to analyze TRMs using flow cytometry, single-cell RNA sequencing, and cytokine release.