Recent reports suggest that tissue resident memory T-cells (TRM) may contribute to antitumor immunity. Specifically, the accumulation of CD103+ tumor-infiltrating lymphocytes (TILRM) in a wide array of solid tumors including breast, lung, ovarian, and melanoma is associated with favorable prognosis. However, relatively little is known regarding TRM differentiation and endogenous TRM are difficult to isolate and culture, impeding their basic study and translational applications such as adoptive cellular therapy (ACT). Thus, a means to generate TRM in vitro would be desirable. TGF-β is known to be critical in establishing TRM populations, and attempts to identify other factors have focused on cytokines. Oxygen tension is another factor that distinguishes the circulation from peripheral tissues; thus, we hypothesized that hypoxia may contribute to a TGF-β1-induced TRM phenotype in human CD8+ T cells. Naive CD8+ T cells from human peripheral blood were activated in normal cell culture conditions (20% O2) or hypoxia (2% O2) and then cultured in the presence of rhTGF-β1. We demonstrate that differentiation of human CD8+ T cells in hypoxia and TGF-β1 in vitro led to the development of a TRM phenotype, characterized by an increase in CD69+CD103+ cells expressing hallmarks of human TRM: CD49a, CD101, and PD-1, as determined by flow cytometric analysis. Hypoxia and TGF-β1 synergized to produce a significantly larger population of TRM phenotype cells than the additive effects of either condition alone. RNA sequencing analysis confirmed that cells differentiated in atmospheric oxygen, hypoxia alone, or hypoxia with TGF-β1 had distinct transcriptional profiles and gene set enrichment analysis (GSEA) showed enrichment for TRM and TILRM gene signatures in CD69+CD103+ cells generated in hypoxia and TGF-β1. We then extended this method to generate MART-1 (melanoma antigen recognized by T-cells 1)-specific in vitro induced TRM by stimulation with autologous dendritic cells in hypoxia and TGF-β1. Hypoxia and TGF-β1 can also be used in a modified rapid expansion protocol (REP) to induce TRM phenotype in antigen-specific T-cells generated via other means, highlighting this method’s potential application to multiple existing ACT modalities. There is great interest in leveraging resident phenotype cells for immunotherapy; however, current approaches focus on using vaccination to induce these cells in vivo as there is no reliable method to generate these cells in vitro. We demonstrate, for the first time, recapitulation of TRM phenotype in vitro from human peripheral blood-derived T cells, enabling a facile means of generating significant numbers of CD8+ TRM phenotype cells for basic research and translational applications such as ACT.

Citation Format: Farah Hasan, Yulun Chiu, Rebecca M. Shaw, Junmei Wang, Cassian Yee. In vitro induction of human tissue resident memory phenotype T-cells for use in adoptive cellular therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A64.