# 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

## TL;DR

This paper provides a detailed protocol for creating a 3D nasal epithelial model and generating tissue-resident memory-like T cells in a lab setting.

## Contribution

The novel contribution is a reproducible in vitro method to induce and study human TRM-like T cells using nasal epithelial co-cultures.

## Key findings

- A 3D nasal epithelial model can be generated for co-culture experiments.
- TRM-like T cells can be induced via co-culture with autologous immune cells.
- Flow cytometry and RNA sequencing can effectively analyze TRM-like T cell characteristics.

## Abstract

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

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.