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

Resident memory T (T) 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 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 expansion to apply in an immunotherapy setting. However, it has also been shown that T may not maintain their identity and form circulating memory T cells after in vivo restimulation. Therefore, we set out to determine how T respond to antigenic activation in culture. Using Listeria monocytogenes and LCMV infection models, we found that T 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 retained their phenotype, including the expression of key T markers CD69 and CD103 (ITGAE). The optimal culture of T required low O pressure to maintain the expression of these and other T-associated molecules. Expanded T 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 profile retention, including expression of T-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 cells that maintain their T phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require in vitro expansion of T.