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Recent findings showing that T<jats:sub>RM<\/jats:sub> expand <jats:italic>in vivo<\/jats:italic> after repeated antigenic stimulation indicate that these memory T cells are not terminally differentiated. This suggests an opportunity for <jats:italic>in vitro<\/jats:italic> T<jats:sub>RM<\/jats:sub> expansion to apply in an immunotherapy setting. However, it has also been shown that T<jats:sub>RM<\/jats:sub> may not maintain their identity and form circulating memory T cells after <jats:italic>in vivo<\/jats:italic> restimulation. Therefore, we set out to determine how T<jats:sub>RM<\/jats:sub> respond to antigenic activation in culture. Using <jats:italic>Listeria monocytogenes<\/jats:italic> and <jats:italic>LCMV<\/jats:italic> infection models, we found that T<jats:sub>RM<\/jats:sub> from the intraepithelial compartment of the small intestine expand <jats:italic>in vitro<\/jats:italic> after antigenic stimulation and subsequent resting in homeostatic cytokines. A large fraction of the expanded T<jats:sub>RM<\/jats:sub> retained their phenotype, including the expression of key T<jats:sub>RM<\/jats:sub> markers CD69 and CD103 (ITGAE). The optimal culture of T<jats:sub>RM<\/jats:sub> required low O<jats:sub>2<\/jats:sub> pressure to maintain the expression of these and other T<jats:sub>RM<\/jats:sub>\u2010associated molecules. Expanded T<jats:sub>RM<\/jats:sub> 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<jats:sub>RM<\/jats:sub> profile retention, including expression of T<jats:sub>RM<\/jats:sub>\u2010related transcription factors, tissue retention factors, adhesion molecules, and enzymes involved in fatty acid metabolism. Collectively, our data indicate that limiting oxygen conditions supports <jats:italic>in vitro<\/jats:italic> expansion of T<jats:sub>RM<\/jats:sub> cells that maintain their T<jats:sub>RM<\/jats:sub> phenotype, at least in part, suggesting an opportunity for therapeutic strategies that require <jats:italic>in vitro<\/jats:italic> expansion of T<jats:sub>RM<\/jats:sub>.<\/jats:p>","DOI":"10.1002\/eji.202350873","type":"journal-article","created":{"date-parts":[[2024,3,19]],"date-time":"2024-03-19T13:11:48Z","timestamp":1710853908000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Intestinal tissue\u2010resident memory T cells maintain distinct identity from circulating memory T cells after <i>in vitro<\/i> restimulation"],"prefix":"10.1002","volume":"54","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9571-000X","authenticated-orcid":false,"given":"Ammarina","family":"Beumer\u2010Chuwonpad","sequence":"first","affiliation":[{"name":"Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory Amsterdam UMC, University of Amsterdam  the Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Felix M.","family":"Behr","sequence":"additional","affiliation":[{"name":"Department of Hematopoiesis, Sanquin Research and Landsteiner Laboratory Amsterdam UMC, University of Amsterdam  the Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Floris P.J.","family":"van Alphen","sequence":"additional","affiliation":[{"name":"Department of Research Facilities Sanquin Research and Laboratory Services  Amsterdam the Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0985-0856","authenticated-orcid":false,"given":"Natasja A. 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