Intestinal tissue-resident memory T cells maintain distinct identity from circulating memory T cells after in vitro restimulation

Resident memory T (T_RM) cells have been recently established as an important subset of memory T cells that provide early and essential protection against reinfection in the absence of circulating memory T cells. Recent findings showing that T_RM expand in vivo after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for in vitro T_RM expansion to apply in an immunotherapy setting. However, it has also been shown that T_RM may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how T_RM respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that T_RM from the intraepithelial compartment of the small intestine expand in vitro after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded T_RM retained their phenotype, including the expression of key T_RM markers CD69 and CD103 (ITGAE). The optimal culture of T_RM required low O₂ pressure to maintain the expression of these and other T_RM-associated molecules. Expanded T_RM retained their effector capacity to produce cytokines after restimulation, but did not acquire a highly glycolytic profile indicative of effector T cells. The proteomic analysis confirmed T_RM profile retention, including expression of T_RM-related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports in vitro expansion of T_RM cells that maintain their T_RM phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of T_RM.