Th138 - Rapamycin Maintains FOXP3 Expression and Modulates Metabolic Function in Expanded Regulatory T Cells

Anna Ligezka, PhD – Mayo Clinic; Sameena Nikhat, PhD – Mayo Clinic; Feda Hamdan, PharmD, PhD – Mayo Clinic; Marion Curtis, PhD – Mayo Clinic; Mohsen Khosravi Maharlooei, MD – Mayo Clinic

Abstract Text:

Introduction:
Cell-based therapies employing regulatory T cells (Tregs) offer promise for reinstating immune tolerance in transplantation and autoimmune disorders. In vitro expansion of Tregs, however, is challenging due to the loss of the Treg master transcription factor FOXP3 during long-term culture. This study investigates the mTOR inhibitor rapamycin's potential for preserving FOXP3 expression during Treg expansion, alongside its downstream metabolic effects.

Methods:
Tregs from an 83-year-old donor underwent 3-week in vitro expansion with or without rapamycin. Multi-color flow cytometry tracked cellular phenotype, while Seahorse assays assessed metabolism, and Treg suppression assays measured functionality. Additionally, in vivo experiments were performed to test rapamycin-treated Tregs' capacity to mitigate disease onset in a GVHD mouse model.

Results:
Rapamycin-treated Tregs exhibited significantly higher FOXP3 expression beyond 2 weeks of expansion (p < 0.0001), indicating enhanced Treg stability compared to untreated cells. Moreover, these cells displayed altered mitochondrial function, demonstrated by elevated oxygen consumption rate (p=0.042), reduced proton leak (p=0.0063), and improved respiratory coupling efficiency (p=0.0097). Differential expression of activation and senescence markers further suggested rapamycin's potential for modulating crucial factors influencing Treg stability in vitro.

Discussion:
These findings shed light on phenotypic attributes of rapamycin-treated expanded Tregs, indicating that it may play a role in sustaining Treg stability during prolonged culture. Further, the observed correlations between rapamycin treatment and other cellular functions offer valuable insights into its utility for generating cells for clinical cell-based therapies.