Optimizing mesenchymal stromal cell-facilitated ex vivo expansion of human regulatory T cells
Ou, Qifeng
Ou, Qifeng
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Publication Date
2025-06-25
Type
doctoral thesis
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Abstract
Regulatory T cells (Treg) are essential for suppressing immune overactivity. Clinical trials have confirmed the safety and potential efficacy of ex vivo-expanded Treg for immune-mediated diseases, yet challenges remain in achieving consistent Treg yield, purity, and cytokine profiles. This thesis project aimed to evaluate the potential to enhance culture expansion of primary human total Treg (CD4+/CD25+/CD127lo) and Treg subpopulations through coculture with human umbilical cord-derived mesenchymal stromal cells (hUC-MSC).
In 14-21 day anti-CD3/anti-CD28-, interleukin-2- and rapamycin-containing cultures, FACS-purified total human Treg underwent 4-fold greater expansion following hUC-MSC coculture. The suppressive function of hUC-MSC-expanded Treg on effector T cell proliferation was maintained and correlated with increased expression of HLA-DR, CD39, and ICOS. Enhanced yields were also noted in hUC-MSC-facilitated ex vivo expansion of FACS-purified Treg subpopulations [CD45RA+ (naïve), CD45RA-HLA-DR+ and CD45RA-HLA-DR- (non-naive)]. Notably, the magnitude of expansion was substantially greater in non-naïve Treg than in naïve Treg. In non-naïve Treg, hUC-MSC coculture also led to increased percentages of FoxP3⁺ and Helios⁺ cells. Additionally, coculture with hUC-MSC upregulated HLA-DR expression across all Treg subtypes, while preserving their suppressive potency and maintaining IL-10 and IFN-y production (ELISA). Using intracellular flow cytometry, IFN-y-producing cells were found to be FoxP3+/- but predominantly Helios⁻ and present in ex vivo-expanded CD45RA⁻CD39⁻ Treg subsets. PMA/ionomycin stimulation followed by magnetic depletion effectively reduced these IFN-y-producing cells. Although stimulation led to substantial FoxP3 loss, coculture with hUC-MSC mitigated this effect. Additionally, freshly isolated CD39⁺ Treg displayed stable CD39 and FoxP3 expression during expansion, showed increased yield with hUC-MSC coculture, and were notably more frequent in blood of patients with diabetic kidney disease than in healthy volunteers.
Collectively, these findings demonstrate that coculture with clinical-grade hUC-MSC significantly enhances ex vivo yield and phenotypic stability of total Treg and Treg subpopulations while preserving their suppressive potency. The findings have potential to facilitate identification, functional characterization and manufacturing of Treg subpopulations with distinct therapeutic benefits.
Publisher
University of Galway
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Attribution-NonCommercial-NoDerivatives 4.0 International